Program & Abstracts

Alfred Medical Research and Education Precinct (AMREP),

The Alfred Hospital,

Melbourne, Australia

14th & 15th October 2013

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Program at a Glance

DAY 1

08:00 - 09:15 Registration

09:15 - 09:30 Welcome and Housekeeping

09:30 - 10:30 Plenary Session 1

10:30 - 11:00 Morning Tea

11:00 - 12:30 Session 1: Epidemiology & Field-based Research I

12:30 - 14:00 Lunch & Poster Session (P1-24)

14:00 - 15:30 Session 2: Malaria in Pregnancy

15:30 - 16:00 Afternoon Tea

16:00 - 17:30 Session 3: Molecular and Cellular Parasitolgy 18:15 Conference Dinner: College Lawn

DAY 2

09:00 - 10:45 Session 4: Host/Parasite Interactions & Remodelling

10:45 - 11:15 Morning Tea

11:15 - 12:45 Session 5: Epidemiology & Field-based Research II

12:45 - 14:15 Lunch & Poster Session (P25-48)

14:15 - 15:45 Session 6: Drug Resistance & Drug Discovery

15:45 - 16:15 Afternoon Tea

16:15 - 17:30 Plenary Session 2 17:30 Awards Ceremony

18:00 Come Together at Village

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Welcome Dear Malaria in Melbourne 2013 Delegate, Welcome to Malaria in Melbourne 2013, the latest instalment of this successful Conference Series, to be held over the next two days at the Alfred Medical Research and Education Precinct (AMREP), The Alfred Hospital, Melbourne, Australia. Australia is a major international hub for malaria research, with more than fifty research groups across the country investigating different approaches to finding solutions to this global public health issue. Malaria in Melbourne is a unique forum that brings together the Australian malaria research community to present their latest data and facilitate the development of collaborative links between institutes, working towards our common strategic goal of controlling, eliminating and ultimately eradicating malaria. The Malaria in Melbourne 2013 program reflects the wide range of disciplines covered by the malaria research community, ranging from basic science through to clinical/epidemiological studies and translational research. This year our plenary speakers reflect the malaria communities research goal with Dr Peter Ryan and Professor Mike Toole who have both made major contributions to the global control of infectious diseases. The Malaria in Melbourne Committee comprises early career malaria researchers representing a number of Medical Research and Academic Institutions. We all warmly welcome you to this meeting and hope you enjoy the scientific and social program!

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Program DAY 1 08:00 - 09:15 Registration

09:15 - 09:30 Welcome & Housekeeping

09:30 - 10:30 Plenary Session 1

Sponsored by: Malaria Nexus Chair: Freya Fowkes

PL1 Using Wolbachia infections to control dengue transmission

Peter Ryan – Faculty of Science, Monash University

10:30 - 11:00 Morning Tea

11:00 - 12:30 Session 1: Epidemiology & Field-based research I

Sponsored by: Medicines for Malaria Venture Chairs: Celine Barnadas and Leanne Robinson

T1 Can the physical examination facilitate the triage of adults with

falciparum malaria? Josh Hanson – Menzies School of Health Research, Darwin

T2 Contribution of P. vivax hypnozoites to the burden of malaria infection and disease in children from Papua New Guinea Leanne Robinson - Infection and Immunity Division, WEHI

T3 Prevalence and effects of Plasmodium parasites in pediatric anaemia in Mozambique Ariel Achtman – Infection and Immunity Division, WEHI

T4 Sympatric populations of Plasmodium vivax and Plasmodium falciparum in Papua New Guinea show different patterns of differentiation and genetic structure Charlie Jennison – Infection and Immunity Division, WEHI; Department of Medical Biology, University of Melbourne

T5 Quantifying invasion inhibitory antibodies to AMA1 in human populations using transgenic Plasmodium falciparum: Implications for vaccine design Silvia Teguh - Department of Biochemistry and Molecular Biology, Bio21 Institute, University of Melbourne; Department of Medicinal Chemistry, Monash Institute of Pharmaceutical Science

12:30 - 14:00 Lunch & Poster Session (numbers P1-24)

   

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14:00 - 15:30 Session 2: Malaria in Pregnancy Sponsored by: Burnet Institute Chairs: Louise Randall and Philippe Boeuf

T6 Evaluating rapid diagnostic tests for suspected malaria in pregnancy

Alex Umbers – Department of Medicine, University of Melbourne T7 Antibodies to malaria in pregnancy and birth outcomes: a longitudinal

study in PNG Alistair Mclean – Burnet Institute

T8 The effect of interactions between iron deficiency and malaria in pregnancy on adverse birth outcomes Kerryn Moore – Centre for Molecular, Environmental, Genetic and Analytical Epidemiology, University of Melbourne; Burnet Institute

T9 Placental mTOR signaling and foetal growth restriction on placental malaria Kris Dimasuay - Department of Medicine, University of Melbourne

T10 The materno-foetal transfer of antimalarial antibodies Asha Herten-Crabb – Burnet Institute

15:30 - 16:00 Afternoon Tea

16:00 - 17:30 Session 3: Molecular and Cellular Parasitology Sponsored by: Australian Society of Parasitology Chairs: Nicholas Proellocks and Catherine Nie

T11 Diverse cellular sources of CXCL10 modulate different processes involved in the development of severe malaria Lisa Ioannidis - Infection and Immunity Division, WEHI

T12 Carbonic anhydrase, a non-essential enzyme in rodent malaria Sarah Morton - School of Botany, University of Melbourne

T13 Identification of gene regulatory elements in the malaria parasite Plasmodium falciparum Jingyi Tang – Department of Medicine, University of Melbourne

T14 High-throughput analysis of the regulation of alternative splicing in Toxoplasma gondii Lee Yeoh – Department of Biochemistry and Molecular Biology, Bio21 Institute, University of Melbourne; School of Botany, University of Melbourne

T15 Coronin is a regulator of actin filament bundling at the cell cortex of the motile Plasmodium and Toxoplasma parasite cell Maya Olshina – Infection and Immunity Division, WEHI; Department of Medical Biology, University of Melbourne

18:15 Conference Dinner

Sponsored by: Beckman Coulter College Lawn Map in program

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DAY 2

09:00 - 10:45 Session 4: Host/Parasite Interactions & Remodeling Sponsored by: CXS Chairs: Ming Kalanon and Xavier Sisquella

T16 PfMSPDBL1 and PfMSPDBL2 – novel members of the major Merozoite

Surface Protein 1 complex in Plasmodium falciparum Clara Lin - Infection and Immunity Division, WEHI

T17 Phosphorylation of αSnap is required for secretory organelle biogenesis in Toxoplasma gondii Rebecca Stewart - Infection and Immunity Division, WEHI

T18

Elucidating protein transport in P. falciparum: the role of chaperones Sarah Charnaud – Burnet Institute

T19

Characterising a novel component of the malaria translocon Scott Chisholm – School of Medicine, Deakin University

T20

Apical complex protein RNG2 implicates the apical polar ring in the regulated secretion of micronemes Nicholas Katris – School of Botany, University of Melbourne

T21

Characterisation of a lysine-rich membrane-associated PHISTb protein of P. falciparum Nicholas Proellocks – Department of Microbiology, Monash University

10:45 - 11:15 Morning Tea

11:15 - 12:45 Session 5: Epidemiology & Field-based research II

Sponsored by: School of Medicine, Deakin University Chairs: Alicia Arnott and Sophie Zaloumis

T22 Impaired red cell deformability in knowlesi malaria in proportion to

disease severity Bridget Barber – Menzies School of Health Research, Darwin

T23 Opsonising antibodies to P. falciparum merozoites associated with immunity to clinical malaria Danika Hill - Infection and Immunity Division, WEHI; Department of Medical Biology, University of Melbourne

T24 Identifying key targets of antibodies to P. falciparum-infected erythrocytes using genetically-modified parasites with disrupted surface antigen expression Jo-Anne Chan – Burnet Institute

T25 The role of binding inhibitory antibodies for Plasmodium falciparum erythrocyte binding antigen (EBA175) in protecting children from symptomatic malaria Vashti Irani – Centre for Biomedical Research, Burnet Institute

T26 High blood-stage parasitaemia and age are the main determinants of Plasmodium falciparum and P. vivax gametocyte prevalence Christian Koepfli - Infection and Immunity Division, WEHI; Department of Medical Biology, University of Melbourne

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12:45 - 14:15 Lunch & Poster Session (numbers P25-48)

14:15 - 15:45 Session 6: Drug Resistance and Drug Discovery

Sponsored by: International Journal of Parasitology: Drugs and Drug Resistance Chairs: Danny Wilson and Belinda Morahan

T27 Immunity to malaria and the assessment of emerging artemisinin

resistance in Thailand Rosanna Powell – Burnet Institute

T28 Transition state mimetic inhibitors of Plasmepsin V from Plasmodium falciparum and Plasmodium vivax Brad Sleebs - Infection and Immunity Division, WEHI; University of Melbourne

T29 High-throughput screening reveals different chemosensitivities of Plasmodium falciparum developing gametocytes and asexual blood stages Leonardo Lucantoni - Discovery Biology, Eskitis Institute for Drug Discovery, Griffith University

T30 High-throughput screening for antimalarial drugs using metabolomic approaches Charlie Chua – Department of Biochemistry and Molecular Biology, Bio21 Institute, University of Melbourne

T31 Profiling compound stage of effect against Plasmodium falciparum asexual stages Sandra Duffy – Discovery Biology, Eskitis Institute for Drug Discovery, Griffith University

15:45 - 16:15 Afternoon Tea 16:15 - 17:00 Plenary Session 2

Sponsored by: Monash University Chair: Matthew Dixon

PL2 Closing Address

Mike Toole – Burnet Institute

17:00 Awards Ceremony

Sponsored by: WEHI and International Journal for Parasitology 17:30 Come Together at Village

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Plenary Lecture 1: Using Wolbachia infections to control dengue transmission Dr Peter Ryan Program Manager, Eliminate Dengue, School of Biological Sciences, Monash University Dengue fever is an emerging insect-transmitted disease causing an estimated 390 million human infections each year. Currently there are no effective treatments beyond prevention measures that focus on reducing population abundance of the main mosquito vector, Aedes aegypti. These strategies are failing to reduce dengue incidence in tropical communities and there is an urgent need for effective alternatives. The Eliminate Dengue Program (www.eliminatedengue.com) is examining the potential use of inherited bacterial symbionts of insects known as Wolbachia as a novel method to interfere with dengue transmission. This work is now moving from basic bench studies into open field trials in Australia, Vietnam and Indonesia. The presentation will give an overview of Wolbachia-mosquito-pathogen interactions as well as results from open field trials involving releases of Wolbachia infected Aedes aegypti mosquitoes.

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Plenary Lecture 2: Closing Address Professor Michael Toole AM, Deputy Director, Burnet Institute. Prof. Mike Toole has spent over 40 years working on a range of international health areas such as communicable disease control (including HIV and malaria), maternal and child health and nutrition, and public health in emergency settings. Working for eight years in Thailand, two years in Somalia, and ten years at the CDC in Atlanta, Prof. Toole has undertaken projects in more than 30 countries in five continents. A founding member of Medecins sans Frontieres Australia, Prof. Toole is also a member of the Global Fund Technical Review Panel, the Board of the Three Diseases Fund for Burma, and the Independent Monitoring Board of the Global Polio Eradication Initiative. In 2013 Professor Mike Toole was awarded a Member of the Order of Australia for significant service to international health, particularly through leadership in medical research.

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Session 1: Epidemiology & Field-based research I T1 Can the physical examination facilitate the triage of adults with falciparum malaria? Josh HANSON

Menzies School of Health Research, Darwin Mahidol-Oxford-Research Unit, Bangkok, Thailand Up to a third of adults with severe falciparum malaria will die from their disease, the majority within 48 hours of their admission to hospital. To improve outcomes, clinicians must optimise supportive care during this critical early phase of a patient’s hospitalisation; a key component of this initial care is the rapid identification of patients at greatest risk. However, most patients with severe malaria are managed in the resource-poor setting, where laboratory and radiology services are rarely available promptly. Accordingly decisions about patient management must frequently be made by clinical evaluation alone. We retrospectively analysed 4 studies of 1801 adults with severe falciparum malaria to determine whether simple clinical findings present were helpful in patient triage. If present on admission, shock, anuria, abnormal glucometry, an increased respiratory rate, a decreased Glasgow Coma Score and the absence of fever were independently predictive of death. The variables were incorporated into a simple clinical algorithm. When applied to the 1801 patients in the four studies, the algorithm’s positive predictive value (PPV) for survival to 48 hours was 99.4 (95% confidence interval (CI) 97.7 – 99.9) and to discharge was 96.9% (95% CI 94.3 – 98.5). In the 712 patients receiving artesunate, the algorithm’s PPV for survival to 48 hours was 100% (95% CI 97.3 - 100) and to discharge was 98.5% (95% CI 94.8 - 99.8). This simple clinical algorithm, calculable on admission by junior staff at the bedside in minutes, can be safely used to triage adults with falciparum malaria in settings with limited intensive care services. If patients are classified as low risk by this clinical algorithm they may be safely be managed initially on a general ward while senior clinical review and laboratory data are awaited.

T2 Contribution of P. vivax hypnozoites to the burden of malaria infection and disease in children from Papua New Guinea Leanne Robinson1,2, Inoni Betuela1, Benson Kiniboro1, Nandao Tarongka1, Hector Morris1, Mariabeth Silkey3, Andreea Waltmann2, Rahel Wampfler3, Natalie Hoffman3, Cristian Koepfli1, Celine Barnadas1, Peter Siba1, Louis Schofield2, Ingrid Felger3 and Ivo Mueller1,2,4 1PNG Institute of Medical Research, Madang & Maprik, Papua New Guinea; 2Walter & Eliza Hall Institute, Parkville, Victoria, Australia; 3Swiss Tropical & Public Health, Basel, Switzerland; 4Centre de Recerca en Salut Internacional de Barcelona (CRESIB), Barcelona, Spain In Papua New Guinea (PNG), Plasmodium falciparum and P. vivax are co-endemic, with studies in East Sepik Province demonstrating a higher prevalence of P. vivax than P. falciparum in young children. P. vivax can develop into hypnozoites that become dorment in the liver for long periods of time and contribute to relapse malaria. Routinely administered anti-malarials, chloroquine and artemisinin combination therapies, act against the blood-stage of the parasite and do not eliminate P. vivax hypnozoites in the liver, resulting in a high rate of relapse. Currently, primaquine is the only licensed drug effective against liver-stage parasites, however due to several limiting factors, this drug is not in widespread use in PNG. To investigate the contribution of long-lasting liver-stage hypnozoites to the burden of malarial infection and disease, a treatment to re-infection study was conducted in children aged 5-10 years in the Maprik District of East Sepik Province. Briefly, 524 children were randomised to receive chloroquine (3d)/artemether-lumefantrine (3d) and primaquine (20d; 0.5mg/kg); or chloroquine (3d)/artemether-lumefantrine (3d) and placebo (20d). The children were actively monitored for re-infection every fortnight for 8 months and passive surveillance measures were also implemented. The primary aim was to compare the effect of combined liver/blood-stage anti-malarial treatment with blood-stage anti-malarial treatment only on subsequent rates of P. vivax infection and illness. Preliminary analysis has revealed that 244 children (48.1%) became re-infected with P. vivax during follow-up. Of these, only 66 occurred in the 249 children that received primaquine (26.5% with recurrent parasitamia) compared to 178 in those 258 that did not (69.0%, p < 0.0001). The majority of these recurrent infections (82%) occurred within 12 weeks of treatment suggesting rapid activation of hypnozoites. Failure to adequately treat P. vivax infections with effective anti-hypnozoite drugs will severely impede attempts to control P. vivax in PNG.

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T3 Prevalence and effects of Plasmodium parasites in pediatric anaemia in Mozambique Ariel H. Achtman1, Connie Li Wai Suen1, Ruth Aguilar3, Cinta Moraleda4, Ariel Magallon-Tejada1, Regina Joice6, Pau Cisteró1, Charity W. Law2, Hendrik Falk3, Montserrat Renom4, Sandra Pilat1, Lazaro Mussacate5, Augusto Nhabomba5, Eusebio Macete5, Pedro L. Alonso4, Gordon K. Smyth2, Matthias Marti6, Alfredo G. Mayor1, Ivo Mueller1, Clara Menéndez4, Louis Schofield1

1Division of Infection and Immunity and 2Division of Bioinformatics and 3Division of Chemical Biology, The Walter and Eliza Hall Institute, Melbourne, 4CRESIB, Barcelona, Spain and 5CISM, Manhiça, Mozambique, 6Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, USA

The complex aetiology and mechanisms of pathogenesis of anaemia in human populations must be understood to design effective public health interventions. We elucidated the contribution of Plasmodium parasites to anaemia in Mozambican children by determining i) the prevalence of sexual and asexual parasites in the bone marrow, ii) the association of Plasmodium parasites with mechanisms of red cell production failure. Microscopic analysis of 290 bone marrow smears from anaemic children revealed parasites in 42% of samples. All asexual stages were present, including trrophozoites in 71% and schizonts in 23% of parasitaemic bone marrows. Gametocyte prevalence was assessed by microscopy and quantitative polymerase chain reaction (qPCR) in 174 of these bone marrows. Transcripts of the P. falciparum housekeeping gene PF08_0085 were detected in 136 bone marrows, of which 98% were qPCR-positive for at least one gametocyte stage marker (PF14_0748, PF13_0247, PF10_0303), indicating an unexpectedly high transmission potential. The predominance of immature gametocytes in bone marrow and mature gametocytes in peripheral blood was confirmed with these qPCRs (p<0.001). To assess red cell production failure, erythropoiesis was quantified using a combination of classic and novel methods. We performed uni- and multivariate statistical testing to identify risk factors associated with erythropoiesis that is particularly low or high for the degree of anaemia. Contrary to expectations, infection with Plasmodium falciparum (multivariate results: p=0.03 to 0.009) was associated with high levels of erythropoiesis, as were haemolysis (p=0.03 to 0.007), shifts in the bone marrow balance of myeloid cells and lymphocytes (p=0.04 to <0.001) and high EPO levels relative to the degree of anemia (p=0.02 to <0.001). High C-reactive protein levels (p=0.009 to <0.001) and bacteraemia (p=0.06 to <0.001) were most highly associated with low erythropoiesis. Dyserythropoieis was associated with the presence of bone marrow haemozoin and high levels of parasitized erythrocytes in the bone marrow. T4 Sympatric populations of Plasmodium vivax and Plasmodium falciparum in Papua New Guinea show different patterns of differentiation and genetic structure Charlie Jennison1,2, Celine Barnadas1,2,3, Alicia Arnott4, Cristian Koepfli1,2, Livingstone Tavul3, Peter M. Siba3, John C. Reeder4, Ingrid Felger5, Ivo Mueller1,2,6, Alyssa Barry1,2

1Division of Infection and Immunity Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia, 2Department of Medical Biology, University of Melbourne, Melbourne, Australia, 3Papua New Guinea Institute of Medical Research, Goroka, Papua New Guinea, 4Burnet Institute, Melbourne, Australia, 5Swiss Tropical and Public Health Institute, Basel, Switzerland, 6El Centro de Investigación en Salud Internacional de Barcelona (CRESIB), Barcelona, Spain. Outside of Africa, Papua New Guinea (PNG) has one of the highest endemicities of malaria. Since the cessation of the insecticide spraying in the late 1970s, P. falciparum has been the predominant parasite species. Here, we investigate sympatric populations of P. falciparum and P. vivax from the North Coast of PNG (East Sepik and Madang), where there is intense perennial malaria transmission (prevalence 31% and 24% respectively). Methods: Samples were collected in 2005/6 from asymptomatic individuals residing in 14 villages clustered into four catchment areas in the East Sepik (n=1 catchment) and Madang (n=3) Provinces. Highly polymorphic markers were used to identify monoclonal infections. A total of 226 P. vivax monoclonal infections were genotyped at 11 microsatellite markers. Population genetic analyses were conducted to estimate levels of genetic diversity and population structure. The P. vivax data were also compared to previously published data from 320 P. falciparum isolates genotyped at 10 microsatellite markers. Results: On the basis of microsatellite markers, the diversity of both malaria parasite species was high, with expected heterozygosity (He) values of 0.79 and 0.82 for the P. falciparum and P. vivax

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populations respectively. Population genetic analyses identified more differentiation between P. falciparum populations (FST = 0.050-0.140) than for those of P. vivax (FST = 0.008-0.023) and while Bayesian cluster analyses identified four genetically distinct populations for P. falciparum, only one highly diverse population was observed for P. vivax. Conclusions: On the North Coast of PNG, despite a higher prevalence of infection, P. falciparum is less diverse and more genetically structured than P. vivax. The high diversity and limited population structure of P. vivax is consistent with previous reports and suggest that P. vivax will be more resilient to control efforts than P. falciparum in PNG. These data act as a baseline against which recently intensified control efforts can be assessed to evaluate their impact on parasite populations. T5 Novel conjugated quinoline-indoles compromise Plasmodium falciparum mitochondrial function and show promising antimalarial activity  Silvia C. Teguh a,c, Craig A. Hutton b, Nectarios Klonis a, Leonardo Lucantoni d, Sandra Duffy d, Vicky M. Avery d, Jonathan B. Baell c, Leann Tilley 1. a Department of Biochemistry and Molecular Biology, and b School of Chemistry, Bio21 Institute, University of Melbourne, VIC 3010.; c Department of Medicinal Chemistry, Monash Institute of Pharmaceutical Science, VIC 3052.; d Discovery Biology, Eskitis Institute for Drug Discovery, Griffith University, QLD 4111.

A class of conjugated quinoline-indole compounds (1, 2) has been identified with promising activity against Plasmodium falciparum1. Structure-Activity Relationship investigations revealed that some compounds from the set (particularly those with quaternary quinolinium structure, 1) show higher activity than chloroquine (CQ) against the CQ-resistant K1 strain (IC50 of 0.1 – 0.2 µM c.f. 0.3 µM of CQ). Non-quaternerised (i.e. not permanently charged) 4-aminoquinolines (2) retained significant potency against a CQ-sensitive 3D7 strain, and have the tendency to display better oral bioavailability, despite their moderate activity against the K1 strain (IC50 of 0.3 – 0.4 µM). CQ exerts its activity by inhibiting the conversion of the toxic haemoglobin breakdown product, haem, to hemozoin - an activity that can be monitored in vitro by inhibition of the formation of its synthetic version known as β-haematin2. Accordingly inhibition of hemoglobin digestion with a cysteine protease inhibitor antagonizes CQ action. By contrast the quinoline-indoles show only weak activity as inhibitors of β-haematin formation and their activity is only weakly antagonized by the haemoglobinase inhibitor. The compounds appear to dissipate mitochondrial potential as an early event in their antimalarial action, and therefore may exert their activity by compromising Plasmodium mitochondrial function. Interestingly, we observed a structural relationship between our compounds and the anti-cancer and anthelmintic compound, pyrvinium pamoate, which has also been proposed to exert its action via compromising mitochondrial function.

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Session 2: Malaria in Pregnancy T6 Evaluating rapid diagnostic tests for suspected malaria in pregnancy Alex Umbers 1, Anna Rosanas-Urgell 2,3, Holger Unger 1, Leanne Robinson 3, 4, Maria Ome 3, Regina Wangnapi 3, Inoni Betuela 3, Peter Siba 3, Ivo Muller 4, 5, Stephen Rogerson1. 1 Department of Medicine, University of Melbourne, Australia, 2Institute of Tropical Medicine, Antwerp, Belguim, 3 Papua New Guinea Institute for Medical Research, Goroka, Papua New Guinea, 4 Infection and Immunity Division, Walter and Eliza Hall Institute, Melbourne, Australia, 5 CRESIB, Barcelona, Spain. Background: Detection of peripheral malaria in pregnancy before delivery is often complicated by low or absent venous blood parasitaemia. Several malaria detection strategies exist, including light microscopy (LM), polymerase chain reaction (PCR), and rapid diagnostic tests (RDTs). While the World Health Organisation recommends RDTs for the confirmation of malaria, few studies (mainly in Africa) have evaluated RDTs’ performance in the context of pregnancy, or in transmission settings where P. falciparum and P. vivax co-exist, such as Papua New Guinea (PNG). Objective: We evaluated RDT performance (sensitivity and specificity) compared with LM and quantitative PCR for the confirmation of suspected malaria in pregnant women. We determined the clinical characteristics of women who met testing criteria. Method: RDT test criteria included febrile illness, anaemia, or other malaria symptoms. Women were tested using CareSTART™ (Access Bio) combination RDT, which distinguishes P. falciparum infections from non-falciparum infection. RDT positive cases were treated with Artemether-lumifantrine. Results: Of 1649 suspected malaria cases screened by RDT, 11.8% were positive for malaria. Among positive cases, P. falciparum single infections accounted for 34% of total infections, 41% were mixed infections and 25% were non-falciparum infections. Preliminary performance analysis of 779 screening events against confirmed parasitemia (by LM) demonstrate that CareSTART™ RDT has low sensitivity (51%) and high specificity (93%) for the detection of parasitemia during pregnancy. Compared with women who did not ever meet criteria for RDT screening, suspected malaria cases tended to have greater risk of adverse outcomes including a low birth weight infant (P=.02) and lower haemoglobin levels at delivery (P=.02). Outcomes: Future analysis on a expanded sample size, as compared with light microscopy and PCR by malaria species, and refined by testing criteria will indicate the clinical value of RDTs to confirm suspected malaria in pregnancy in PNG. The risk of adverse birth outcomes associated with the occurrence of a false negative RDT result (and therefore untreated malaria) will also be presented. T7 Antibodies to Malaria in Pregnancy and Birth Outcomes: A Longitudinal Study in PNG Alistair McLean1,2*, Freya Fowkes1,2,3, Danielle Stanisic 4, Francesca Baiwog4, Peter Siba 4, Ivo Mueller

4, Stephen Rogerson 4 and James Beeson 1,5,61Burnet Institute, 2Melbourne School of Population Health, University of Melbourne, 3DEPM, Monash University, 4Papua New Guinea Institute of Medical Research, PNG, 5Department of Medicine, University of Melbourne, 6Department of Microbiology, Monash University

Malaria in pregnancy is associated with poor birth outcomes such as low birth weight and preterm delivery. The ability of Plasmodium falciparum to infect the placenta through the action of VAR2CSA is thought to be an important virulence determinant that contributes to poor birth outcomes. AIM: To determine if VAR2CSA-DBL5 specific antibodies are associated with improved birth outcomes in a cohort of pregnant women. METHODS: Levels of VAR2CSA-DBL5 antibody at enrolment and delivery were tested by ELISA in a group of 350 pregnant women (31.4% with P. falciparum infection at enrolment) attending prenatal care at Alexishafen Health Centre, Madang, PNG who had been recruited to a longitudinal study of malaria and pregnancy. Clinical and demographic data were obtained at enrolment and delivery visits to the health centre. Univariate and multivariate analyses were undertaken to assess associations between antibody levels and birth outcomes. RESULTS: Women infected at enrolment with high levels of VAR2CSA-DBL5 IgG were less likely to give birth to a low birth weight baby (p=0.020), less likely to deliver preterm (p=0.062) and less likely to have an active placental infection at delivery (p<0.001) than those infected women with low levels of VAR2CSA-DBL5 IgG. These associations were not observed in women who were uninfected at enrolment.

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CONCLUSION: High antibody levels against VAR2CSA-DBL5 mid-pregnancy are significantly associated with improved birth outcomes amongst women with P. falciparum infection at enrolment. The association followed a temporal relationship whereby antibodies precede birth outcomes, strengthening the case for a causal relationship between VAR2CSA-DBL5 antibodies and protection against negative birth outcomes. These results suggest that VAR2CSA-DBL5 antibodies have a protective role against adverse birth outcomes, establishing further support for VAR2CSA-DBL5 as a vaccine target to protect pregnant women and their babies against malaria in pregnancy. T8 The effect of interactions between iron deficiency and malaria in pregnancy on adverse birth outcomes. Kerryn Moore1,2, Freya Fowkes2, Freya Langham2, Francesca Baiwog3, Julie Simpson1, Danielle Stanisic5, Christopher King6, Ivo Mueller3, Peter Siba3, Stephen Rogerson4, James Beeson2. 1. Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, University of Melbourne, Parkville, Victoria, Australia; 2. The Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Victoria, Australia; 3. Papua New Guinea Institute of Medical Research, Goroka, Papua New Guinea.; 4. Department of Medicine, University of Melbourne, Parkville, Victoria, Australia.; 5. Department of Parasitology, New York University, New York, NY, USA; 6. Centre for Global Health and Diseases, Case Western Reserve University and Department of Veteran’s Affairs Medical Centre, Cleveland, OH, USA. The World Health Organization recommends maternal iron supplementation and malaria prophylaxis to improve birth outcomes. However, there are concerns that iron supplementation can increase malaria risk, and iron deficiency has been shown to protect against malaria. It is unknown how iron deficiency and malaria interact to influence defined birth outcomes such as low birthweight and preterm birth. We determined malariametric and iron deficiency parameters (ferritin, CRP, transferrin receptor) in 470 pregnant women attending antenatal clinics in a malaria-endemic region of Papua New Guinea at enrolment (mean 25 weeks gestation). Women were followed to delivery and birth outcomes were recorded. The prevalence of iron deficiency was high, ranging from 61% to 99% depending on iron marker and definition used. We found a significant inverse association between ferritin (a marker of body iron stores) and birthweight, which was modified by gravidity; in primigravidae, birthweight decreased by 109 grams (p <0.001) for every two-fold increase in ferritin, whereas there was no association in multigravidae (p = 0.650). Primigravidae with iron deficiency had babies that were on average 284 grams heavier (p = 0.006) than babies or iron replete primigravidae, whereas there was no association in multigravidae (p = 0.984). Similar associations (in magnitude and direction) were observed between iron deficiency and odds of LBW, but did not reach statistical significance. There was no association between ferritin and odds of preterm birth (p = 0.712). This study provides evidence that iron deficient women are giving birth to heavier babies than their iron replete counterparts in a malaria-endemic area. Results question the use of antenatal iron supplementation during pregnancy for the prevention of adverse birth outcomes in malaria-endemic regions. Further research is needed to understand the mechanisms behind the protective effects of iron deficiency on adverse birth outcomes. T9 Placental mTOR signalling and foetal growth restriction in placental malaria Kris Genelyn Dimasuay1, Thomas B. Jansson2, Jocelyn Glazier3, Stephen J. Rogerson1,4, Philippe Boeuf1,4 1The University of Melbourne, Department of Medicine (RMH), Parkville, Victoria, Australia, 2 Department of Obstetrics & Gynecology, University of Texas Health Science Centre at San Antonio, Texas, USA, 3 Maternal and Foetal Health Research Centre, University of Manchester, St. Mary’s Hospital, Manchester, United Kingdom, 4 Victorian Infectious Diseases Service, Royal Melbourne Hospital, Parkville, Victoria, Australia, Placental malaria can cause foetal growth restriction, especially when the infection triggers a local inflammation termed intervillositis. Foetal growth restriction in placental malaria was recently shown to be associated with placental functional impairment, especially transplacental amino acid transport capacity. The mechanistic target of rapamycin (mTOR) pathway is actively involved in the regulation of amino acid uptake by the placenta in response to various environmental cues.

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We hypothesized that the impaired amino acid transport capacity observed in placental malaria infection with intervillositis is mediated by a down-regulation of placental mTOR signalling activity, contributing to poor foetal growth. Participants were selected from a cohort of Malawian pregnant women. Term villous tissue biopsies from uninfected placentas and from infected placentas with and without intervillositis (n=4/group) were used. We assessed mTOR signalling activity by quantifying the expression levels of downstream effectors of the mTOR pathway namely ribosomal protein S6 (rpS6), Phospho-rpS6 (Ser235/236), eukaryotic initiation factor 4E binding protein 1 (4EBP1), Phospho-4EBP1 (Thr70), Phospho-4EBP1 (Thr37/46) using western blot. We will present an analysis of the association between mTOR pathway activation profiles, placental histology and birth weight. Identifying mTOR as a central regulator of foetal growth in placental malaria would pave the way for innovative intervention strategies, including supplementation with nutrients that specifically activate mTOR signalling, to improve foetal growth and pregnancy outcomes in women with malaria. T10 The Materno-Foetal Transfer of Antimalarial Antibodies Asha Herten-Crabb1, Sarah Charnaud1, Rose McGready4,5,6, Nadia J Cross1,2, Julie A Simpson3, Andrew Guy1, Kurt Lackovic2, Jacher Viladpai-nguen4, David Narum8, Takafumi Tsuboi9, Robin F Anders10, François Nosten4,5,6, James G Beeson1,2, Freya JI Fowkes1,2,3 1Macfarlane Burnet Institute of Medical Research, 85 Commercial Road, Melbourne, Victoria, Australia ; 2 Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia ; 3 Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, Melbourne School of Population Health, University of Melbourne, Victoria, Australia ; 4 Shoklo Malaria Research Unit, Mae Sot, Tak, Thailand ; 5 Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand ; 6 Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, CCVTM, Oxford UK ; 7 Department of Medical Biology, University of Melbourne, Victoria, Australia ; 8 Malaria Vaccine Development Branch, National Institute of Allergy and Infectious Diseases/National Institutes of Health, Rockville, Maryland, USA ; 9 Cell-Free Science and Technology Research Center, Ehime University, Ehime, Japan ; 10 Department of Biochemistry, La Trobe University, Victoria, Australia Infants display immunity to malarial infection in the first 6 months of life. The general hypothesis for this protection is materno-foetal antimalarial antibody transfer during pregnancy, however data on materno-foetal antibody transfer is limited. We performed a nested case-control study of 166 pregnant women (57 Plasmodium-infected cases and 109 uninfected control subjects) in north-western Thailand. Antibody levels to P. falciparum and P. vivax merozoite and the pregnancy-specific PfVAR2CSA antigen were determined at delivery in mother, baby and umbilical cord. The association between the timing and number of infections, malaria chemoprophylaxis and gravidity with maternal antibody transfer was also assessed. Analysis showed that maternal antibodies were transferred to the infant and this was dependent on Plasmodium spp. infection during pregnancy. P. falciparum and P. vivax antibody levels remained equivalent from mother to infant in control subjects, but decreased in species-specific cases. Chloroquine prophylaxis successfully prevented P. vivax infection, leading to a decrease in antibody transfer of vivax antigens between chloroquine- and placebo-allocated subjects. Infants born to multigravid women who had experienced P. falciparum infection during pregnancy had higher levels of PfVAR2CSA compared to primigravid infected women. These findings are important for our understanding of materno-foetal transfer and the development of malaria vaccines administered during pregnancy for the protection of neonates.

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Session 3: Molecular and Cellular Parasitology T11 Diverse cellular sources of CXCL10 modulate different processes involved in the development of severe malaria Lisa Ioannidis1, Victoria Ryg-Cornejo1, Chris Chiu1, Catherine Q. Nie2 and Diana S. Hansen1 1The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia and 2Burnet Institute, 85 Commercial Road, Melbourne, Victoria 3004, Australia Cerebral malaria (CM) is one of the most severe clinical manifestations of Plasmodium falcipaum infection. This syndrome is thought to result from the sequestration of parasitized red blood cells in the brain microvasculature and a strong inflammatory response to infection. Although the role of cytokines in CM has been widely investigated, the role of chemokines remains to be fully elucidated. Case control studies identified the CXCR3 chemokine C-X-C motif chemokine 10 (CXCL10) as a biomarker of CM severity. Consistent with this, we have previously shown that CXCL10 neutralization or genetic deletion alleviates brain intravascular inflammation and protects malaria-infected mice from CM. Malaria-stimulated brain endothelial cells were also shown to produce CXCL10, suggesting that these cells mediate leukocyte recruitment to the site of parasite sequestration. In addition to organ-specific effects, here we found that a lack of CXCL10 during infection reduces parasite biomass, suggesting that this chemokine compromises the induction of protective immunity. To identify the cellular sources of CXCL10 involved in these processes, wild-type and CXCL10-/- mice were irradiated and reconstituted with bone marrow from either wild-type or CXCL10-/- mice. Chimeric mice were infected with luciferase-expressing P. berghei ANKA and parasite biomass was assessed. Chimeras that were unable to express CXCL10 in hematopoietic-derived cells had significantly lower whole body and brain parasite densities than wild-type controls. Conversely, chimeras that could produce CXCL10 in hematopoietic-derived cells had higher whole body biomass and exacerbated brain parasite sequestration compared to knock-out chimeras, indicating that hematopoietic-derived sources of CXCL10 have a detrimental role in controlling parasite growth. Our results suggest that the cells that produce CXCL10 in response to malaria differ in the brain and lymphoid organs and that they control different processes in these two sites: leukocyte recruitment in the brain and parasite biomass in the spleen. T12 Carbonic anhydrase, a non-essential enzyme in rodent malaria Sarah Morton, C. Dean Goodman, Vanessa Mollard, Geoffrey I. McFadden

School of Botany, University of Melbourne, VIC 3010. Carbonic anhydrase catalyses the reversible hydration of carbon dioxide (CO2) to generate bicarbonate (HCO3

-). The HCO3- is substrate for various essential metabolic processes including

pyrimidine biosynthesis, fatty acid biosynthesis and nitrogen metabolism, and carbonic anhydrases are ubiquitous across the tree of life. Carbonic anhydrase inhibiting drugs are safe and effective in treating human diseases such as glaucoma and cancer and have had some success as antimalarials. In Plasmodium, a single carbonic anhydrase is presumed to supply HCO3

- for essential metabolic functions located in both the cytoplasm and apicoplast. To understand how this single enzyme might fulfil these metabolic roles in the malaria parasite, we localised the enzyme at different life cycle stages by epitope tagging the carbonic anhydrase of the rodent malaria parasite Plasmodium berghei. We also generated a carbonic anhydrase knockout in P. berghei and assayed knockout parasites for viability across the life cycle. Unexpectedly, the knockout line shows no detectable loss of viability in blood, mosquito or liver stages compared to wild type parasites; no obvious growth defects or phenotypes have been observed in the mutant. Our results raise questions as to how the parasite acquires HCO3

- in order to feed its metabolism and cast doubt on the suitability of carbonic anhydrase as a malaria drug target. T13 Identification of gene regulatory elements in the malaria parasite Plasmodium falciparum Jingyi Tang, Michaela Petter, Michael Duffy Department of Medicine (RMH), the University of Melbourne Epigenetic control of malaria parasite gene expression regulates three critical processes in malaria pathogenesis, i.e. i) erythrocyte invasion, ii) immune evasion through antigenic variation and iii)

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adhesion of parasite infected erythrocytes to small blood vessels. Modification of histones and exchange of histone variants are distinct mechanisms in epigenetic control in the Plasmodium genome. Recent studies including the human ENCODE project indicate that histone modifications at enhancers precede and control changes in gene expression and that histone variants are associated with regulatory elements within dynamic chromatin. To identify sequence elements important for gene expression in P. falciparum, chromatin is collected from parasites at ring, trophozoite and schizont stages and DNA sequences enriched in H3K4me1, H3K18ac, H3K27ac and H2A.Z will be identified by chromatin immunoprecipitation.

T14 High-throughput analysis of the regulation of alternative splicing in Toxoplasma gondii Lee M. Yeoh1,2, Christopher D. Goodman2, Nathan E. Hall3,4, Giel G. van Dooren5, Geoffrey I. McFadden2 and Stuart A. Ralph1 1Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia. 2School of Botany, The University of Melbourne, Parkville, Victoria, Australia. 3Life Sciences Computation Centre, Victorian Life Sciences Computation Initiative, The University of Melbourne, Parkville, Victoria, Australia. 4La Trobe University, Bundoora, Victoria, Australia. 5Research School of Biology, The Australian National University, Acton, ACT, Australia.

Single genes are frequently capable of generating multiple mRNAs, coding for alternative protein isoforms, via a process known as alternative splicing. These transcripts generally arise when the spliceosome binds to alternative 5' or 3' splice sites of introns. This phenomenon is widespread in metazoans, and observed in over 90% of human genes. Recent data suggest it may also be common in Apicomplexa. These parasites have small genomes, and economy of DNA appears to be evolutionarily favoured in this phylum. Thus, alternative splicing might play an even more important role for these organisms than metazoans. We are investigating the mechanism of alternative splicing in Plasmodium spp. and T. gondii, and have localised four homologues of ASF/SF2 (alternative-splicing factor/splicing factor 2) to a subnuclear compartment in T. gondii. In addition, we have conditionally over-expressed a homologue in T. gondii using the ddFKBP-Shld1 system. Induced overexpression is deleterious to growth, and perturbation of some alternatively-spliced genes was detected by qRT-PCR. We also performed high-throughput RNA-seq on our mutant, to determine what splicing is regulated by this homologue. Current RNA-seq algorithms are poorly suited for apicomplexans, which have more compact genomes than metazoans. Hence, we complemented existing tools by writing new programs that address this deficiency. Alternative splicing was perturbed in 50% of genes in our mutant, and we are working to determine which pathways are represented, and to investigate the biological consequences of perturbation.

T15 Coronin is a regulator of actin filament bundling at the cell cortex of the motile Plasmodium and Toxoplasma parasite cell

Maya A. Olshina*1,2, Fiona Angrisano1,2, Danushka S. Marapana1, David T. Riglar1,2, Wilson Wong1,2, Lachlan Whitehead1, Bruno Catimel3, Andrew Holmes4, Simone Lepper5, Friedrich Frischknecht5, David R. Kovar6, Jake Baum1,2 1 The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia. 2 Department of Medical Biology, University of Melbourne, Australia. 3 Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Royal Melbourne Hospital, Melbourne, Australia. 4 School of Chemistry and Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, Australia. 5 Department of Infectious Diseases, University of Heidelberg Medical School, Heidelberg, Germany. 6 The University of Chicago, Department of Molecular Genetics and Cell Biology, Department of Biochemistry and Molecular Biology, Chicago, USA. Malaria parasite gliding motility is mediated by an actin myosin motor anchored within the parasite pellicle, which provides the force for motility via direct interaction with actin filaments. Due to the dynamic nature of actin, tightly controlled regulation is necessary to direct the formation and disassembly of filaments in an appropriate spatio-temporal manner. Malaria parasites possess a markedly reduced repertoire of actin regulators, among which only a few are predicted to interact with actin filaments – formins, ADF/cofilins, capping protein and coronin. In other eukaryotic systems,

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coronin is involved in filament branching, filament bundling and agonism/antagonism of ADF/cofilin activity. Here we sought to determine the function of malaria parasite coronin using in vivo observations from Plasmodium falciparum, the deadliest malaria parasite infecting humans and the related apicomplexan parasite Toxoplasma gondii, as well as in vitro observations using recombinant PfCoronin. C terminally HA-tagged coronin localises in a restricted distribution at the periphery of the blood stage malaria parasite and Toxoplasma tachyzoites. Alpha-toxin treatment of the motile tachyzoites demonstrates that this localisation is consistent with the supra-alveolar space – the compartment lying between inner membrane complex and plasma membrane which houses the actin-myosin motor. Detergent extraction of tagged coronin is consistent with it having a strong membrane association. This is backed up by in vitro biochemical studies with recombinant PfCoronin, that demonstrate a high affinity of the regulator to PI(4,5)P2. Sedimentation analysis shows PfCoronin to be a true filament binding protein, while Transmission Electron Microscopy and TIRF microscopy both demonstrate that PfCoronin bundles actin filaments. Taken together, these data identify malaria coronin as a potentially key regulator of actin filament recruitment and bundling at the cell cortex of motile malaria and related apicomplexan parasites.

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Session 4: Host/Parasite Interactions & Remodeling T16 PfMSPDBL1 and PfMSPDBL2 – novel members of the major Merozoite Surface Protein 1 complex in Plasmodium falciparum

Clara S. Lin1, Hermann Bujard2, Christian Epp2, Jeff Babon1, Alessandro D. Uboldi1, Peter E Czabotar1, Anthony N Hodder 1 and Alan F. Cowman1 1The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia, 2Heidelberg University, Heidelberg, Germany The invasion of Plasmodium falciparum into host erythrocytes during the parasites asexual blood stage is a critical step in the perpetuation of symptomatic infection in the human host. Merozoites, the invasive form of the parasite, express a glycosylphosphatidylinositol-anchored 190 kDa Merozoite Surface Protein 1 (MSP1) on the surface. This major protein on the surface of the parasite exists in a larger form that includes other merozoite surface proteins. Prior to invasion, a proteolytic event occurs to release the MSP1 complex, leaving only a 10 kDa C-terminus on the surface of the parasite. Despite MSP1 being a vaccine candidate, the mechanism through which this major protein mediates invasion remains unknown.

In an attempt to understand how merozoite surface proteins interact with each other and with MSP1, we expressed two novel proteins, PfMSPDBL1 and PfMSPDBL2 that were first identified in a family of proteins on the same locus on chromosome 10. These two merozoite surface proteins appear to be important for parasite survival. More importantly, these peripheral binding surface proteins have a Duffy Binding Like (DBL) domain involved in binding to human erythrocytes.

We have used both parasite-derived and recombinant proteins to show that both PfMSPDBL1 and PfMSPDBL2 are involved in the MSP1 complex. Importantly, we have also been able to show that PfMSPDBL1 and PfMSPDBL2 bind to human erythrocytes with specificity, allowing the MSP1 complex to directly interact with erythrocyte receptors. Overall, these findings will advance the overall understanding of the structure-function studies of MSP1, MSPDBL1 and MSPDBL2, which will aid in the understanding of them as potential vaccine candidates T17 Phosphorylation of αSnap is required for secretory organelle biogenesis in Toxoplasma gondii Rebecca J. Stewart1,2*, Clare H. Bradin1,2, Hong J. Wu1,2, Chaitali Dekiwadia3, Stuart A. Ralph3, Jake Baum1,2, Chris Tonkin1,2 1The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia; 2Department of Medical Biology, University of Melbourne, Melbourne, Australia; 3Department of Biochemistry and Medical Biology, Bio21 Molecular Science and Biotechnology Institute The phylum Apicomplexa encompasses a number of important human parasitic pathogens. Of specific note are Plasmodium spp., causative agent of Malaria and responsible for up to 1 million deaths per year, and Toxoplasma gondii, the most ubiquitous apicomplexan infecting approximately 30% of the worlds population. Apicomplexan parasites are obligate intracellular pathogens that absolutely require invasion into host cell for survival and as such have evolved specialized invasion machinery. During replication, these parasites utilize the secretory pathway for the de novo synthesis of the apical invasion organelles, as well as of inner membrane complex (IMC) and plasma membrane (PM) biogenesis. As a part of the secretory pathway, vesicle trafficking and membrane fusion events are mediated by a suite of proteins that are highly conserved throughout eukaryotes. Membrane specific tethering and priming brings vesicle and target membranes in close proximity allowing trans interactions between corresponding vesicle (v)-SNAREs and target (t)-SNAREs. Further protein interactions and membrane modifications lead to fusion of membranes and release of vesicle contents resulting in cis-pairing of v-SNAREs and t-SNAREs in the same membrane. αSoluble NSF Attachment Protein (αSNAP) is required for recycling of cis-SNARE complexes, through recruitment of ATPase N-ethylmaleimide sensitive factor (NSF), to recharge the cellular pool of SNAREs for further trafficking events. Utilising T.gondii, modulation of key phosphorylation sites on αSNAP allows us to interfere with endogenous αSNAP processes with a regulated dominant negative system. We have shown that ablation of normal αSNAP functions leads to severely disrupted secretory systems lethal to the parasite. Cytokenesis, de novo apical organelle, PM and IMC biogenesis are all disordered while secretory-independent mechanisms, such as karyokenesis are not directly affected.

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Understanding of molecular trafficking mechanisms in Apicomplexa, especially those related to the unique invasion organelles, can aid our understanding of these parasites and inform future drug and vaccine designs. T18 Elucidating protein transport in P. falciparum: the role of chaperones Sarah C Charnaud 1,2, Mauro F Azevedo1, Hayley E Bullen1, Simone Kuelzer3, Jude M Przyborski3, Brendan S Crabb1,4,5, Paul R Gilson1,5 1Burnet Institute, Melbourne; 2 Department of Medicine, Monash University; 3Parasitology, Philipps University, Marburg, Germany; 4Department of Microbiology and Immunology, University of Melbourne; 5Department of Immunology, Monash University Plasmodium falciparum resides in enucleated erythrocytes, devoid of trafficking machinery. In order to survive inside this host the parasite must import nutrients, and evade the immune system. These processes require proteins to be exported out of the parasite, crossing the plasma membrane (PM) and the parasitophorous vacuole space (PV) and membrane (PVM), into the host cytoplasm and on to where the proteins are required. A protein translocon that facilitates transport across the PVM has been identified, termed PTEX, which consists of 5 major protein components, including the AAA+ ATPase HSP101 predicted to provide the energy for protein translocation. Protein members of this class often interact transiently with other chaperone components, including HSP70/HSP40 systems that have been well characterized in yeast and bacteria. Previously it was assumed that refolding of proteins post translocation within the host cytosol was performed by host HSP70s. We have shown that parasite derived HSP70x has potential to interact with exported proteins in the PV and within the erythrocyte, such as the importance virulence protein PfEMP1. We probe potential parasite derived interacting partners, using localization and biochemical techniques. We present data on the change in localization of this chaperone over the course of the erythrocytic cycle, which hints at multiple chaperone partners. We have used multiple methods to knockout the gene, and to knock down the level of protein expression in an inducible manner. We will discuss the phenotype of parasites lacking this chaperone, and its implications in export and pathogenicity of P. falciparum. These data will help elucidate the working mechanism of protein export in the parasite and provide new opportunities for drug development.

T19 Characterising a novel component of the malaria translocon

Scott Chisholm1, Ming Kalanon1, Brendan Elsworth2, Paul Gilson2, Brendan Crabb2, Tania de Koning-Ward1 1School of Medicine, Deakin University; 2Burnet Institute Malaria parasites extensively remodel their erythrocytic host by exporting hundreds of its own proteins across the parasitophorous vacuole membrane (PVM) into the host cell. This process is essential for the parasite to acquire the nutrients required for survival, and also aids in preventing clearance of the parasite through the spleen. The mechanism by which the parasite exports its proteins has not been conclusively demonstrated, however there is growing evidence to suggest that a protein translocon at the PVM, termed PTEX (Plasmodium translocon of exported proteins), is acting as a gateway for exported proteins to pass through. One component of this translocon, PTEX88 has no conserved domains that provide insight into its function and reagents to aid in its characterisation have only recently been generated. Early experiments using these reagents produced data consistent with PTEX88 being a true component of PTEX, demonstrating co-localisation and interactions with known PTEX components. Further biochemical characterisation of PTEX88 has shown that PTEX88 has a peripheral membrane association, consistent with other PTEX components. Functional analysis of proteins through generation of knockouts is difficult in Plasmodium as it contains a haploid genome and thus deletion of essential genes yields a lethal phenotype. Therefore, attempts to elucidate the function of PTEX88 have relied on systems whereby expression of PTEX88 can be externally regulated to allow a controlled knockout or knockdown, thus enabling functional analysis. Using a new inducible system recently described for P. falciparum that utilizes GlmS riboswitch, we have been able to degrade PTEX88 mRNA such that PTEX88 expression is downregulated by ~60%. We are now characterizing the phenotypes of the inducible PTEX88 knockout in an attempt to establish the function of this protein.

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T20 Apical complex protein RNG2 implicates the apical polar ring in the regulated secretion of micronemes Nicholas Katris1, Giel van Dooren2, Ross Waller1 1 School of Botany, University of Melbourne, Melbourne, Australia ; 2 Research School of Biology, Australian National University (ANU), Canberra, Australia Apicomplexan parasites such as Plasmodium sp. and Toxoplasma gondii share a unique structure called the apical complex. The apical complex is the site of release of secretory vesicles, such as micronemes and rhoptries, which contain invasion factors that facilitate entry into host cells. The apical complex is also an important structure for internal daughter assembly as it is the earliest structure of daughter cell buds to form. Despite its biological importance, few proteins of this structure have been identified or functionally characterized. We’ve exploited a catalogue of the related ciliate pellicle proteome to identify novel apicomplexan pellicle proteins. One such protein is RNG2, which localizes to the posterior apical ring in the apical complex of T. gondii tachyzoites, as well as the developing internal daughter cells. In T. gondii, RNG2 first appears after centrosome duplication making it one of the earliest markers for the developing daughter cells. To analyse the function of RNG2, a tetracycline inducible RNG2 knock-down cell line was generated, and found to have a severe growth defect when RNG2 expression was shut down. Interestingly, there was no defect in the rate of daughter cell replication. There was, however, a defect in invasion, specifically in microneme secretion and motility, which are required for host cell attachment and entry. These results provide novel insights into the function of the apical complex in Apicomplexa and implicate the apical polar rings in the regulation of microneme secretion. T21 Characterisation of a lysine-rich membrane-associated PHISTb protein of P. falciparum Nicholas I. Proellocks1, Susann Herrmann1, Donna W. Buckingham1, Eric Hanssen2, Emma Hodges1, Brendan Elsworth1, Belinda J. Morahan1, Ross L. Coppel1 and Brian M. Cooke1 1 Department of Microbiology, Monash University. 2 Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne The genomes of Plasmodium parasites contain a large gene family that encode proteins with a PHIST domain that are predicted to be exported into the red blood cell (RBC) cytosol. These proteins can be subdivided into 3 groups based on sequence similarity, with the PHISTb-domain family containing proteins only present in P. falciparum. We have characterised a PHISTb protein encoded by PFE1605w which we have termed Lysine-rich membrane-associated PHIST (LMP). LMP is expressed in the mature stages of the parasites RBC life cycle and is exported into the infected RBC (iRBC) where it associates with the RBC membrane skeleton. Deletion of PFE1605w from the genome of P. falciparum parasites resulted in a significant reduction in the adhesive properties of the iRBC, while trafficking of PfEMP1 and knob formation remained unaltered. Importantly, the adhesive properties of iRBCs was restored back to wild-type levels when knock-out parasites were complimented with an episomal copy of PFE1605w, confirming that the altered adhesive phenotype was due directly to the action of LMP. Our results strongly implicate an important role for LMP in the pathogenesis of P. falciparum malaria infection.

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Session 5: Epidemiology & Field-based research II T22 Impaired red cell deformability in knowlesi malaria in proportion to disease severity Barber BE,1,2 Anstey NM,1,3 Grigg M,1,2 Parameswaran U, 1,2 William T,2 Dondorp AM, 4 Yeo TW 1,3 1. Menzies School of Health Research, Darwin, Northern Territory, Australia; 2. Infectious Diseases Unit, Queen Elizabeth Hospital, Kota Kinabalu, Sabah, Malaysia; 3. Royal Darwin Hospital, Darwin, Northern Territory, Australia ; 4. Mahidol-Oxford Tropical Medicine Research Unit, Bangkok, Thailand Plasmodium knowlesi is a common cause of human in Malaysian Borneo and can cause severe and fatal disease. However, little is known about the pathogenesis of disease. In severe falciparum malaria, sequestration of parasitized red cells results from cytoadherence to host endothelium and decreased red blood cell deformability (RBC-D), leading to microvascular obstruction, tissue hypoxia and organ dysfunction. In knowlesi malaria microvascular accumulation of parasitized cells also occurs, however the mechanisms are unknown. Reduced deformability with sludging of P. knowlesi-infected red cells has been demonstrated in rhesus macaques, but has not been studied in humans. Using ektacytometry we measured RBC-D in adults with severe (n=21) and non-severe (n=61) knowlesi malaria and severe (n=8) and non-severe (n=82) falciparum malaria; and 15 healthy controls. At a shear stress of 30 Pascals, RBC-D was reduced in patients with severe (elongation index [EI]=0.496, IQR 0.456-0.528) and non-severe (EI=0.551, IQR 0.494-0.569) knowlesi malaria compared to controls (EI=0.583, IQR 0.576-0.590; p<0.0001 for both comparisons), and reduced in severe compared to non-severe knowlesi malaria (p=0.002). RBC-D was similar among patients with severe (EI=0.510, IQR 0.496-0.539) and non-severe falciparum malaria (EI=0.516, IQR 0.475-0.558), but was reduced in both groups compared to controls (p=0.0045 and p<0.0001 respectively). RBC-D did not differ significantly between patients with severe knowlesi and severe falciparum malaria. Among patients with knowlesi, but not falciparum malaria, RBC-D was inversely correlated with parasite count (spearman’s correlation coefficient =-0.37, p=0.0006). Among patients with severe knowlesi malaria, reduced RBC-D may contribute to microvascular sludging, microvascular accumulation of parasitized red cells and impaired organ perfusion. T23 Opsonising antibodies to P.falciparum merozoites associated with immunity to clinical malaria. Danika L Hill 1,2 , Emily M Eriksson 1,2, Connie S N Li Wai Suen 1,2, Leanne J Robinson 1,3, Peter M Siba 3, Ivo Mueller 1,2,4, Diana S Hansen 1, Louis Schofield 1,2 1 Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia, 2 Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia, 3 Vector Borne Disease Unit, Papua New Guinea Institute of Medical Research, Goroka, Eastern Highlands Province, Papua New Guinea, 4 Barcelona Center for International Health, University of Barcelona, Barcelona, Spain Naturally acquired humoral immunity to the malarial parasite Plasmodium falciparum can protect against disease, although the precise mechanisms remain unclear. Although antibody levels can be measured by ELISA, few studies have investigated functional antibody assays in relation to clinical outcomes. In this study we applied a recently developed functional assay of antibody-mediated opsonisation of merozoites, to plasma samples from a longitudinal cohort study conducted in a malaria endemic region of Papua New Guinea (PNG). Phagocytic activity was quantified by flow cytometry using a standardized and high-throughput protocol, and was subsequently evaluated for association with protection from clinical malaria and high-density parasitemia. Opsonising antibody responses were found to: i) increase with age, ii) be enhanced by concurrent infection, and iii) correlate with protection from clinical episodes and high-density parasitemia. Stronger protective associations were observed in individuals with no detectable parasitemia at baseline. This study presents the first evidence for merozoite phagocytosis as a correlate of acquired immunity and clinical protection against P. falciparum malaria. T24 Identifying key targets of antibodies to P. falciparum-infected erythrocytes using genetically-modified parasites with disrupted surface antigen expression Jo-Anne Chan1, Katherine Howell2, Freya J.I. Fowkes1, Joanne Chesson2, Danielle Stanisic3, Christine Langer1, Ivo Mueller2, Michaela Petter4, Michael Duffy4, Alex Maier7, Peter M. Siba6, Alan F. Cowman2, Peter Bull5, Kevin Marsh5, James G. Beeson1;

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1Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, VIC, Australia; 2The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; 3Griffith University, Nathan, QLD, Australia; 4Department of Medicine, University of Melbourne, Parkville, VIC, Australia; 5Centre for Geographic Medicine Research, Kenya Medical Research Institute, Kilifi, Kenya; 6Papua New Guinea Institute of Medical Research, Madang, PNG; 7The Australian National University, Acton, ACT, Australia Effective clinical immunity that protects against symptomatic malaria in humans develops gradually after repeated exposure to Plasmodium falciparum. During intra-erythrocytic development, P. falciparum expresses novel antigens on the surface of infected erythrocytes, including PfEMP1, RIFIN, STEVOR, and SURFIN. Antibodies to surface antigens are typically variant-specific and appear to play an important role in contributing to protective immunity in humans. However, the significance of different surface antigens as targets of acquired immunity remains unclear. In our study, we used an innovative approach to evaluate the importance of surface antigens as antibody targets. This was achieved using genetically-modified P. falciparum lines with disrupted parasite protein trafficking, achieved by deletion of the skeletal-binding protein 1 (SBP1-knockout), and P. falciparum lines with inhibited expression of PfEMP1 (PfEMP1-knock-down). Currently, only PfEMP1 is known to be trafficked via the SBP1-pathway to the infected erythrocyte surface. We used high-throughput flow cytometry-based assays to measure antibody reactivity to the infected erythrocyte surface and opsonic phagocytosis assays using samples from cohort studies in Papua New Guinea and Kenya, including children, adults, and pregnant women. Comparison between the parental and genetically-modified parasites allowed us to quantify the proportion of antibodies targeting specific antigens on the infected erythrocyte surface. We found very little antibody response to SBP1-knockout parasites and markedly reduced antibody response to PfEMP1-knockdown parasites. Our results from studies using multiple parasite lines in two geographically different populations suggest that the major surface antigens targeted by human antibodies are dependent on SBP1 for trafficking to the infected erythrocyte surface and are consistent with PfEMP1 being the dominant target of acquired antibodies. These findings enhance our understanding of acquired immunity to human malaria and have significant implications for vaccine development. T25 The role of binding inhibitory antibodies for Plasmodium falciparum erythrocyte binding antigen (EBA175) in protecting children from symptomatic malaria Vashti Irani 1, 2, 3, Paul Ramsland1, 2, Rosemary Ffrench1, 2, Ivo Mueller 4,5, Peter Siba6, James Beeson1,

3, Jack Richards1, 3. 1Centre for Biomedical Research, Burnet Institute, Melbourne, VIC, Australia, 2Department of Immunology, Monash University, Melbourne, VIC, Australia, 3Department of Medicine, Melbourne University, Melbourne, VIC, Australia, 4 Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia, 5Barcelona Centre for International Health Research, Barcelona, Spain, 6

Papua New Guinea Institute of Medical Research, Madang, PNG, The blood stage of the malaria life cycle is associated with symptomatic disease. Importantly, individuals living in malaria endemic areas develop natural immunity that protects them from symptomatic disease. Immunity develops with time and cumulative parasite exposure, and the antibody response against blood stage parasites plays a major role. Some of these antibodies are known to target merozoite antigens, and are able to inhibit erythrocyte invasion. Multiple merozoite antigens are targeted, including P. falciparum vaccine candidate, EBA175. The presence of high levels of antibodies to EBA175 is associated with clinical protection, and it is believed that these antibodies function by inhibiting the binding of EBA175 to its erythrocyte receptor, glycophorin A. To advance our understanding of the immunological mechanisms that mediate immunity, there is a need for the development of a broader array of functional assays, especially for use in clinical studies. To address this issue, we developed immunoassays to specifically detect antibodies that inhibit the EBA175: glycophorin A interaction. These assays were tested in a cohort of 206 Papua New Guinean children who were treated at enrolment to clear parasitaemia, and prospectively observed for six months to detect reinfection and clinical disease. Approximately half the children were able to inhibit the binding of EBA175 to glycophorin A. The presence of binding inhibitory antibodies correlated with IgG levels to EBA175 Region II by ELISA. It was also evident that high antibody levels were required to mediate this functional effect. It appeared that previous cumulative exposure was important for developing these antibodies and concurrent infection with parasites boosted this response. Importantly, binding inhibitory antibodies were associated with protection from symptomatic

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disease. This study provides insight into the development of functional antibodies to EBA175, and improves our understanding of the functional role of binding inhibitory antibodies in naturally acquired and vaccine induced immunity.

T26 High blood-stage parasitemia and age are the main determinants of Plasmodium falciparum and P. vivax gametocyte prevalence Cristian Koepfli1,2*, Leanne J. Robinson1,2,3*, Patricia Rarau3, Naomi Sambale3, Rahel Wampfler4, Nicolas Senn1,3 Inoni Betuela3, Peter Siba3, Ingrid Felger4, Ivo Mueller1,2,5 1 Walter & Eliza Hall Institute, 1G Royal Parade, Parkville, VIC 3052, Australia; 2 Department of Medical Biology, University of Melbourne, Parkville, VIC 3010, Australia; 3 Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea; 4 Swiss Tropical and Public Health Institute, Basel, Switzerland; 5 Barcelona Centre for International Health Research, Barcelona, Spain. * contributed equally Understanding human-to-mosquito transmission is crucial to control malaria. To assess factors associated with gametocyte carriage 2121 blood samples from participants of all ages were collected in a cross-sectional survey in 2010 in Papua New Guinea. Plasmodium species were detected by light microscopy and qPCR and gametocytes by reverse-transcriptase PCR of markers pvs25 and pfs25. Prevalence by light microscopy and PCR was 5.0% and 12.8% for P. vivax and 5.1% and 17.3% for P. falciparum. Gametocytes were detected in 48% of P. vivax and 60% of P. falciparum positive samples. For both species, gametocytes prevalence was higher in samples with high blood-stage parasite densities, but age trends were observed independent from blood-stage parasitemia. In individuals with clinical malaria at time of sampling, gametocytes were detected in 25/ 25 P. falciparum positive and 4/6 P. vivax positive samples. Gametocyte densities (measured as Pvs25/Pfs25 transcripts per µL blood) were highest in children <3 years and dropped 10-fold thereafter. Plasmodium vivax gametocytes are believed to be short-lived and appear before the onset of clinical disease while mature P. falciparum gametocytes become apparent only 7-15 days after first appearance of blood-stage parasites but persist longer. Despite these factors differences in gametocyte carriage between the two species were moderate, and the main factor determining the detectability of gametocytes is blood-stage parasite density. As gametocyte densities drop fast in the first years of life possibly the potential for transmission is highest in young children.

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Session 6: Drug Resistance and Drug Discovery T27 Immunity to malaria and the assessment of emerging artemisinin resistance in Thailand Rosanna Powell, Alistair McLean, James G Beeson, Julie A Simpson, Francois Nosten, Freya JI Fowkes

Burnet Institute, Melbourne Australia; Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, University of Melbourne, Australia Shokolo Malaria Research Unit, Mae Sot, Thailand Resistance to artemisinin derivatives was initially reported in western Cambodia and has now spread to Thailand, Vietnam and Myanmar. The spread of artemisinin resistance will have profound consequences for morbidity and mortality associated with malaria globally, as there are currently no new antimalarials available that could replace artemisinin. As the genetic mechanism for artemisinin resistance is unknown, resistance is detected by examining parasite clearance curves in therapeutic efficacy trials. However, naturally acquired immunity to malaria will also influence parasite clearance curves. In malaria endemic areas, immunity to malaria can develop after repeated exposure and can reduce clinical symptoms and high-density parasitaemia in the blood. We hypothesise that naturally acquired host immunity may impede the assessment of parasite clearance rates after drug treatment because it increases the probability of parasite clearance independently of antimalarial drugs. The objective of our study was to examine the interaction between host immunity and parasite clearance on the Thai-Myanmar border. A recent longitudinal study at the Thai-Myanmar border showed that slow-clearing infections after artemisinin treatment have increased from 0.6% in 2001 to 20% in 2010. We determined antibody levels to MSP1-42, AMA1 and EBA140 in 1146 dried blood spot samples from this longitudinal study (2007-2011) and related levels to parasite clearance rates. We found that immunity influenced parasite clearance rates after artemisinin treatment. Immunity is an important confounder in the assessment of emerging artemisinin resistance and will impact on resistance monitoring and surveillance efforts. T28 Transition state mimetic inhibitors of Plasmepsin V from Plasmodium falciparum and Plasmodium vivax Brad E. Sleebs,a,b Michelle Gazdik,a,b Kurt Lackovic,a,b Matthew T. O’Neill,a,b Pravin Rajasekaran,a,b Sash Lopaticki,a,b Kym Lowes,a,b Brian J. Smith,c Alan F. Cowmana,b and Justin A. Boddey.a,b aThe Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Australia; bThe University of Melbourne, Parkville 3010, Australia; cLa Trobe University, Bundoora 3086, Australia Malaria parasites survive inside human red blood cells by exporting hundreds of proteins into the erythrocyte to remodel it. Approximately 450 proteins are exported by trafficking through the parasite Endoplasmic Reticulum (ER) and possess an N-terminal export motif, termed the Plasmodium export element (PEXEL), which targets the proteins to the erythrocyte. In order for proteins to be correctly exported, the PEXEL motif (RxLxQ/E/D) must be processed by an ER-resident aspartic acid protease called Plasmepsin V (PMV). If processing of PEXEL proteins does not take place they are not exported and accumulate inside the parasite. Plasmepsin V is conserved in all Plasmodium spp., including the most virulent human parasites P. falciparum and P. vivax, and is essential for blood-stage parasite survival. Plasmepsin V is therefore considered to be a prime target for therapeutic intervention of malaria. Here, we presented the design and synthesis of transition state mimetics of the native PEXEL substrate for use as chemical inhibitors to study Plasmepsin V. The mimetics display potent inhibition of Plasmepsin V from both P. falciparum and P. vivax with negligible off target activity against human proteases (BACE-1 and Cathepsin D), confirming that the export pathway is conserved in Plasmodium species and that Plasmepsin V has an exquisitely refined substrate specificity. For the first time we demonstrate that the treatment of P. falciparum parasites in culture with a small molecule mimetic causes a reduction in PEXEL processing and parasite death, reinforcing Plasmepsin V as an important antimalarial target.

T29 High-Throughput Screening Reveals Different Chemosensitivities of Plasmodium falciparum Developing Gametocytes and Asexual Blood Stages Leonardo Lucantoni1, Sandra Duffy1, Sophie H. Adjalley2, David A. Fidock2,3, Vicky M. Avery1

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1 Discovery Biology, Eskitis Institute for Drug Discovery, Griffith University, Nathan, QLD 4111, Australia; 2 Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY 10032, USA; 3 Division of Infectious Diseases, Department of Medicine, Columbia University Medical Center, New York, NY 10032, USA Plasmodium falciparum gametocytes, which develop in the human host over 8–12 days, are the most accessible developmental stage for the development of new transmission blocking components for combination therapy. Investigations on the drug sensitivity of developing gametocytes, as well as screening methods for identifying inhibitors of early gametocytogenesis, however, remain scarce. We have developed a luciferase-based high-throughput screening (HTS) assay using tightly synchronous stage I - III gametocytes from a GFP-luciferase recombinant P. falciparum line. The assay proved reproducible and suitable for the screening of large compound libraries, with an average % coefficient of variance (CV) ≤ 5%, an average signal to noise ratio (S/N) > 30 and a Z’ ∼0.8. The assay was used to evaluate the early-stage gametocytocidal activity of two collections of known asexual blood stage active compounds, namely the Medicines for Malaria Venture ‘Malaria Box’ (400 compounds), and the GNF-Novartis ‘Malaria Box’ (3835 compounds). Only 135 (34% of the total) and 797 (21%) compounds from the MMV and the GNF-Novartis Malaria Boxes, respectively, were found to inhibit early gametocytogenesis by at least 50% at the highest respective screening concentrations of 5.0 and 2.6 µM respectively. The dose-response testing of all the MMV Malaria Box screening hits and of 151 hits from the GNF-Novartis library identified 20 and 71 promising early stage gametocytocidal compounds, respectively, with IC50 values below 1.0 µM. The gametocytocidal potency of the hits was compared to their asexual blood stage activity on 3D7, as previously obtained in our lab, and was found to be significantly lower. Our findings highlight the need for screening efforts directed specifically against early gametocytogenesis, and indicate the importance of the experimental verification of early stage gametocytocidal activity in the development of new antimalarial candidates for combination therapy. T30 High throughput screening for antimalarial drugs using metabolomic approaches Hwa Huat Chua, James MacRae, Stuart Ralph & Malcolm McConville Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria 3010, Australia Malaria is a serious human parasitic disease caused by protozoan parasites of the genus Plasmodium. These obligate intracellular parasites have evolved to survive inside human red blood cells, accessing the host’s nutrient while evading immune defense responses. Over the past few decades, resistance has developed to several existing antimalarial drug, which has raised a major concern to the public health. High cost of manufacturing and distribution of antimalarial drugs contributes to an economic burden for poor countries. Because of this, and since there is no successful vaccine against malaria available to date, the requirement to discover new antimalarial drugs is becoming increasingly urgent. This project is focused on the development of a high throughput screening approach in order to discover novel antimalarial drugs. Here, metabolomic techniques will be employed to verify or elucidate the modes of action of drugs currently used of known or unknown action. In particular, stable isotope incorporation, metabolic foot-printing, and multivariate analyses will be used in order to discern perturbations in specific biochemical and physical pathways. Development of a high throughput procedure will potentially allow swift and efficient evaluation of other drug leads. T31 Profiling compound stage of effect against Plasmodium falciparum asexual stages Sandra Duffy, Vicky, M Avery

Discovery Biology, Eskitis Institute for Drug Discovery, Griffith University. An important aspect for determining the mechanism of action of antimalarial compounds against the asexual forms of Plasmodium falciparum is the identification of the parasite asexual stages at which the compound exerts its effect. Here we describe the stage specific profiling of a range of currently used antimalarial compounds as well as compounds with well documented targets and mechanisms of action. A new protocol for the isolation of high parasitemia, early ring stage parasites ranging from 0 to 1hour in age is described. The compounds are tested in 384 well plate IC50 format against a range of parasite ages over a total of one parasite life cycle, from ring through schizont and back to ring.

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Compound activity is determined by DNA staining and high content image analysis in a high throughput mode. Using DNA staining intensity and size, the age of the parasite at which growth is first arrested is also identified. The IC50 shift from one parasite age to the next is calculated along with the parasite age at which the parasite growth was arrested. This profiling can be completed on 50-100 compounds simultaneously.  

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Poster Presentations Monday: P1-P24 P1 Conditional knockdown of the PEXEL protease: Dissecting protein export in P.falciparum Danushka S Marapana1, Justin A Boddey1, Philip J.Shaw2 and Alan F Cowman1

1 The Walter & Eliza Hall Institute of Medical Research; 2 National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand

The intra-erythrocytic stage of P. falciparum leads to the disease symptoms of malaria in humans. Infection of erythrocytes by the parasite leads to extensive remodelling of the host cell. The parasite exports a diverse range of proteins into the host cell cytoplasm. These proteins mediate extensive host cell remodelling allowing the parasite to scavenge extracellular nutrients and to evade the host immune system. Exported proteins divide into two classes depending on whether or not they carry an N-terminal export motif known as the Plasmodium Export Element (PEXEL). Currently, more than 450 proteins are known to carry this export barcode. Upon export into the host cell cytoplasm, PEXEL-containing proteins alter properties of the erythrocyte cytoskeleton, create a cytoadherence complex on the underside of the erythrocyte membrane and are also involved with the trafficking and display of the major virulence factor of P. falciparum known as PfEMP1. Export of this protein class is dependent on initial recognition and subsequent proteolytic cleavage within the PEXEL motif by an aspartyl protease within the parasite endoplasmic reticulum. This protease, Plasmepsin V (PMV), is essential for parasite survival and has proved refractory to conventional gene knockout methods. In order to better understand PMV mediated protein export, we have created a transgenic P. falciparum parasite line in which PMV is under conditional regulation. We assess the consequences of PMV knockdown in parasites on the PEXEL export pathway and also on export and presentation of the major virulence factor PfEMP1. Defects in proteolytic cleavage and trafficking of transgenic fluorescent PEXEL-protein constructs will also be investigated within this framework of PMV knockdown. By this study we hope to address the influence of PMV on overall protein export and virulence of P. falciparum. P2 The Plasmodium translocon of exported proteins (PTEX) component thioredoxin-2 is important for maintaining normal blood-stage growth. Kathryn Matthews1, Ming Kalanon1, Scott Chisholm1, Angelika Sturm2, Christopher D Goodman2, Matthew W Dixon3, Paul Sanders4, Thomas Nebl5, Fiona Fraser1, Silvia Haase1, Geoffrey I McFadden2, Paul Gilson4, Brendan S Crabb4, Tania F de Koning-Ward1. 1School of Medicine, Deakin University, Waurn Ponds, Victoria, 3216, Australia; 2School of Botany, University of Melbourne, Melbourne, Victoria, 3010, Australia; 3Department of Biochemistry and Molecular Biology, Bio21 Institute, Melbourne, Victoria 3010, Australia; 4Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Victoria 3004, Australia; 5Infection and Immunity Division, The Walter and Eliza Hall Institute, Melbourne, Vic 3052, Australia Central to Plasmodium parasites growth and survival is the remodeling of the vertebrate host cell. This remodelling involves the translocation of several hundred parasite proteins across an encasing parasitophorous vacuole membrane (PVM) into the host cell cytosol via a putative export machinery termed PTEX. Previously PTEX150, HSP101 and EXP2 have been shown to be bona fide members of PTEX. To investigate the parasites mechanism of protein export and to elucidate the functional role of PTEX in pathogenesis, we have utilised the rodent malaria species P. berghei for which an in vivo model of infection exists. Here we validate that PTEX88 and TRX2 are indeed members of this complex and despite being amenable to genetic tagging, four of the five components could not be genetically deleted, indicating proteins encoded by these genes are likely to be essential for P. berghei survival. In contrast, TRX2, a putative thioredoxin-like protein, could be deleted, with TRX2 knockout parasites displaying growth and virulence-associated phenotypes. Thus, whilst not essential for parasite survival, TRX2 may regulate normal PTEX function. Importantly, the generation of TRX2 knockout parasites allows further dissection of PTEX and it’s role in parasite protein export.

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P3 The exomembrane system of Plasmodium falciparum and its role in the trafficking of virulence proteins.

Steven Batinovic1,2, Paul J. McMillan1,2, Matthew W.A. Dixon1,2, Joseph D. Smith3,4 and Leann Tilley1,2

1Biochemistry and Molecular Biology, Bio21 Institute, The University of Melbourne, Parkville, VIC, Australia; 2ARC Centre of Excellence for Coherent X-ray Science, The University of Melbourne, Parkville, VIC, Australia; 3Seattle Biomedical Research Institute, Seattle, WA 98109, USA; 4Department of Global Health, University of Washington, Seattle, WA 98105, USA

Plasmodium falciparum is the most virulent human malaria parasite, accounting for the overwhelming majority of malaria-related deaths each year. P. falciparum parasites invade red blood cells (RBCs) and extensively modify the structure and morphology of their host cell by generating and exporting a range of parasite proteins. One key parasite protein, P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1), is trafficked to the surface of infected RBCs where it mediates adhesion to the vascular endothelium of the human host. This study has investigated multiple facets of the complex protein trafficking pathway undertaken by PfEMP1 prior to presentation on the surface of infected RBCs. We have examined the trafficking of PfEMP1-GFP chimera in very early stage parasites to show PfEMP1 molecules accumulate in novel discrete foci at the parasite surface prior to further export to small parasite-generated exomembranous structures in the cytoplasm of the host RBC, known as Maurer’s clefts. In an effort to further elucidate the role of the parasite exomembrane system, we have used tightly synchronised parasite-infected RBCs to investigate the development and organisation of Maurer’s clefts and small membranous tether structures, or simply 'tethers', over the first 24 hours of the parasite lifecycle. Here we demonstrate that tethers are generated much earlier than previously thought and appear to latch onto the Maurer’s cleft structures. While initially mobile and freely moving throughout the host RBC cytoplasm, these Maurer’s cleft-tether complexes eventually dock onto the host RBC membrane by 20-24 hours post-invasion. Finally, we have explored a potential role of host actin remodelling in the final-step trafficking of PfEMP1 to the RBC surface by showing reduction in PfEMP1 surface presentation following treatment with drugs affecting actin polymerisation and stability. P4 Why is gluconeogenic fructose-1,6-bisphosphatase activity essential for Toxoplasma gondii tachyzoites in the presence of glucose? Martin Blume1*, Mordechay Gerlic2, Brunda Nijagal3, Seth Masters2 and Malcolm McConville1

1 Bio21 Institute, Department of Biochemistry, The University of Melbourne, Australia; 2 Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia; 3 Metabolomics Australia, Melbourne, Australia We have recently shown that intracellular Toxoplasma gondii tachyzoites utilize both glucose and glutamine as major carbon sources. While glucose catabolism presents a major source of ATP synthesis, its disruption has little effect on parasite growth and virulence in mice, indicating that these parasites can switch to a gluconeogenic mode of metabolism. To characterize the significance of gluconeogenesis under normal growth conditions, we have investigated the role of two putative fructose-1,6-bisphosphatases, TgFBP1 and TgFBP2. Only TgFBP2, but not TgFBP1 functionally complemented an E. coli FBP mutant and was shown to be essential for tachyzoite growth in fibroblasts and virulence in mice when expressed under anhydrotetracycline (atc)-inducible control. Interestingly, epitope-tagged TgFBP2-HA is not restricted to the cytosol but was also located in proximity of centrioles and the early inner membrane complex during daughter cell formation. The growth phenotype of the conditional TgFBP2 mutant was complemented by expression of unrelated Leishmania major and E. coli FBPs but not with an enzymatically inactivated TgFBP2. Mass spectrometry-based stable isotope-resolved metabolic flux analyses of intracellular tachyzoites confirmed the gluconeogenic function of TgFBP2 under starvation conditions. Remarkably, they indicated that TgFBP2/1 are also involved in a futile cycle with fructose-6-phosphate kinase under glucose replete conditions. Current data suggests that this futile cycle may be important in regulating sugar phosphate levels and metabolic fluxes into key pathways, such as dolichol-oligosaccharide and GPI biosynthesis and the pentose phosphate pathway. These data demonstrate multiple roles for TgFBPs that are essential for parasite growth in the presence and absence of exogenous glucose.

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P5 Influence of nutrient supplementation on naturally acquired antibody responses to malaria during pregnancy and association with pregnancy outcomes Upeksha P Chandrasiri1, Jack Richards2, James Beeson2, Philippe Boeuf1, Per Ashorn3, Ken Maleta4, Stephen J Rogerson1 1Department of Medicine, The University of Melbourne, Australia, 2Burnet Institute, Melbourne, Australia, 3University of Tampere, Finland, 4University of Malawi, College of Medicine, Blantyre, Malawi Malaria infection and under nutrition frequently coexist in malaria endemic regions where pregnant women and children under the age of five bear the greatest burden. Nutrition is known to modulate malaria morbidity and mortality; however, the interaction between suboptimal nutrition and malaria immunity remains poorly understood. We investigated the influence of a maternal lipid based nutrient supplementation on acquisition of antibodies to malaria during pregnancy and association with pregnancy outcomes. Naturally acquired antibody levels were measured against pregnancy-specific and non-pregnancy-specific P.falciparum variant surface antigens (VSA) and merozoite antigens (MSP-1, MSP-2, MSP-3, Rh2A9 and EBA-175, recognized as potential vaccine candidates) at <20 and 36 gestation weeks (gw), in a cohort of pregnant women currently enrolled in a nutrient supplementation trial in Malawi, Africa. Preliminary results show that total immunoglobulin G (IgG) and functional antibodies to pregnancy-specific VSA significantly increased during pregnancy (p<0.0001), and were positively correlated with maternal haemoglobin levels (p=0.0071), suggesting a role in protection against malaria-induced anaemia. Antibodies to merozoite antigens and non-pregnancy-specific VSA however declined or fluctuated, possibly due to the lack of exposure to malaria over pregnancy, and were not associated with pregnancy outcomes. Antibody levels were not associated with birth weight or were affected by the use of insecticide treated bed nets. HIV infection (13% prevalence) was associated with lower levels of antibodies to MSP-2, pregnancy and non-pregnancy-specific VSA agreeing with previous findings. Further research is currently underway investigating the association between the type of nutrient supplementation and malaria antibody levels in pregnant women and their infants. If nutrient supplementations are found to have a positive effect on malaria immunity and fetal and infant growth, they could be an important complement to conventional malaria prevention as a cost effective method of improving clinical outcomes in vulnerable groups. P6 Non-VAR2CSA-mediated placental adhesion of malaria-infected erythrocytes. Christelle Buffet1, Wina Hasang1, 2, Charanya Lakshmanan1, Stephen Rogerson1, 2, Philippe Boeuf1, 2

1 Department of Medicine, (RMH), University of Melbourne; 2 Victorian Infectious Diseases Service, RMH In malaria-endemic areas, pregnant women are more susceptible to the development of severe disease. The unborn child is also affected and often born underweight. Malaria in pregnancy can lead to placental malaria, characterized by the accumulation of malaria-infected erythrocytes in the placenta, supported by the interaction between VAR2CSA (a pregnancy-specific PfEMP1 variant) and chondroitin sulphate A (CSA), a receptor expressed on the placental syncytiotrophoblast layer. With increasing of parity, women have been shown to develop protective anti-VAR2CSA antibodies, suggesting that VAR2CSA is a potential vaccine candidate. However, some domains of non-VAR2CSA PfEMP1 have been demonstrated to bind to CSA in vitro. Hence, we hypothesized that the adhesion of malaria-infected erythrocytes to the placental syncytiotrophoblast could be supported by interactions other than VAR2CSA/CSA. Using a novel flow cytometry-based adhesion assay, we selected a var2csa knockout (KO) parasite lines for the adhesion to placental syncytiotrophoblast vesicles. The characterization of this selected var2csa KO parasite line in terms of immune recognition and adhesion characteristics will be presented. Identification of interactions other than VAR2CSA/CSA could help in the design of a fully protective vaccine against placental malaria. P7 The Materno-Foetal Transfer of Antimalarial Antibodies Asha Herten-Crabb1, Sarah Charnaud1, Rose McGready4,5,6, Nadia J Cross1,2, Julie A Simpson3, Andrew Guy1, Kurt Lackovic2, Jacher Viladpai-nguen4, David Narum8, Takafumi Tsuboi9, Robin F Anders10, François Nosten4,5,6, James G Beeson1,2, Freya JI Fowkes1,2,3

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1Macfarlane Burnet Institute of Medical Research, 85 Commercial Road, Melbourne, Victoria, Australia ; 2 Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia ; 3 Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, Melbourne School of Population Health, University of Melbourne, Victoria, Australia ; 4 Shoklo Malaria Research Unit, Mae Sot, Tak, Thailand ; 5 Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand ; 6 Centre for Tropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford, CCVTM, Oxford UK ; 7 Department of Medical Biology, University of Melbourne, Victoria, Australia ; 8 Malaria Vaccine Development Branch, National Institute of Allergy and Infectious Diseases/National Institutes of Health, Rockville, Maryland, USA ; 9 Cell-Free Science and Technology Research Center, Ehime University, Ehime, Japan ; 10 Department of Biochemistry, La Trobe University, Victoria, Australia Infants display immunity to malarial infection in the first 6 months of life. The general hypothesis for this protection is materno-foetal antimalarial antibody transfer during pregnancy, however data on materno-foetal antibody transfer is limited. We performed a nested case-control study of 166 pregnant women (57 Plasmodium-infected cases and 109 uninfected control subjects) in north-western Thailand. Antibody levels to P. falciparum and P. vivax merozoite and the pregnancy-specific PfVAR2CSA antigen were determined at delivery in mother, baby and umbilical cord. The association between the timing and number of infections, malaria chemoprophylaxis and gravidity with maternal antibody transfer was also assessed. Analysis showed that maternal antibodies were transferred to the infant and this was dependent on Plasmodium spp. infection during pregnancy. P. falciparum and P. vivax antibody levels remained equivalent from mother to infant in control subjects, but decreased in species-specific cases. Chloroquine prophylaxis successfully prevented P. vivax infection, leading to a decrease in antibody transfer of vivax antigens between chloroquine- and placebo-allocated subjects. Infants born to multigravid women who had experienced P. falciparum infection during pregnancy had higher levels of PfVAR2CSA compared to primigravid infected women. These findings are important for our understanding of materno-foetal transfer and the development of malaria vaccines administered during pregnancy for the protection of neonates.

P8 Host Cell Effectors in Toxoplasma Michael J Coffey and Christopher J Tonkin The Division of Infection and Immunity; The Walter and Eliza Hall Institute of Medical Research Toxoplasma gondii is an obligate intracellular parasite closely related to Plasmodium spp. Acute Toxoplasmosis is caused by tissue destruction by the rapidly growing tachyzoite however in response to immune pressure parasites differentiate into cyst forming bradyzoites, which initiate lifelong chronic infection. The first Toxoplasma ‘effector’ molecules have recently been described and interestingly these are secreted into host cells during invasion; in contrast to Plasmodium spp. which export many proteins following infection. Several of the identified Toxoplasma effectors represent members of a large group of polymorphic secreted kinases, known as the rhoptry kinases (ROPKs). To study the function of other ROPK family members, we have tagged candidate ROPKs at their endogenous locus with a haemagluttinin-beta lactamase (HA-BLA) tag to observe localisation within parasites and assess whether these proteins are secreted into host cells. Protein localization within parasites was observed by probing for HA while secretion of proteins into host cells was monitored using the highly sensitive BLA/GeneBLAzer reporter system, as used to study the secretion of bacterial effectors. We have targeted 20 possible Toxoplasma effectors at their endogenous loci and confirmed tagging by western blot. Further, we have successfully detected export of a known rhoptry protein (toxofilin) that is secreted into host cells using both microscopy and FACS analyses. We will present the results of my Honours year outlining which of these ROPKs are exported into host cells and show our attempts to localise these proteins in encysted bradyzoite parasites which chronically infect humans for life. Given that these ROPKs are potentially exported beyond the cyst wall and into the host cell these kinases may represent promising drug targets to clear chronic infection, present in approximately 30% of human populations. P9 Characterisation of the Malaria Parasite Export Complex B Elsworth, MF Azevedo, CQ Nie, BS Crabb, PR Gilson The Macfarlane Burnet Institute for Medical Research and Public health, Melbourne, Victoria, Australia

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Plasmodium parasites that cause malaria spend part of their life cycle within human host hepatocytes and red blood cells (RBCs) where they extensively remodel the infected cells through the export of hundreds of parasite proteins. These proteins play many roles including altering the infected RBC (iRBC) membrane permeability to acquire nutrients required for survival, increased pathogenesis and host immune evasion. Proteins are secreted from the parasite into the parasitophorous vacuole, surrounding the parasite within the infected cell. They must then pass across the parasitophorous vacuole membrane (PVM) into the RBC cytoplasm. It has previously been shown that a protein complex, the Plasmodium Translocon of EXported proteins (PTEX), is found on the PVM and is most likely the protein translocating channel or translocon responsible for this export process. Here we show that we are able to inducibly knock-down the levels of PTEX150, a core component of the translocon, within parasites. Even a relatively low level of protein knockdown leads to a significant decrease in the ability of the parasite to grow, eventually leading to death. This demonstrates the importance of this complex to the malaria parasite and validates it as a viable drug target. We are currently investigating the ability of these PTEX knockdown parasite lines to export proteins into the host RBC to validate its role as a translocon using a number of techniques, including the use exported luciferase reporter proteins.

P10 Antibodies that promote phagocytosis of P. falciparum merozoites are associated with protection against malaria and can be induced by human vaccination Gaoqian Feng1*, Faith Osier2*, Michelle J. Boyle1, Jack S. Richards1, Nadia Cross1, Christine Langer1 Fiona J. McCallum3, James McCarthy4, Robin Anders5, Kevin Marsh2, James G. Beeson1; 1 Centre for immunology, The Burnet Institute, Melbourne, Australia; 2 KEMRI Centre for Geographic Medicine Research, Kenya; 3Army Malaria Institute, Brisbane, Australia; 4 Queensland Institute of Medical Research (QIMR), University of Queensland, Brisbane, Australia; 5 Department of Biochemistry, La Trobe University, Melbourne, Australia * Equal contribution Malaria illness develops during the blood-stage infection when the merozoite form of the parasite infect human erythrocyte and replicate inside them. Merozoite antigens are important targets of antibodies that are thought to be the key mediators of protective immunity, and merozoite antigens have long been regarded as promising vaccine candidates. The protective mechanisms of antibodies against merozoite antigens are not well understood, but may include opsonization of merozoites for phagocytic clearance. Currently, there is a lack of assays to measure functional antibody activity in studies of acquired and vaccine-induced immunity in humans. To address these questions, we developed a high-throughput assay to quantify antibody-mediated phagocytosis using a human monocyte cell line and purified P. falciparum merozoites. We used this assay to assess the opsonic activity of antibodies in cohort studies of children and adults in Kenya, and a phase 1 vaccine trial of a major merozoite surface antigen. We found that antibodies that promote opsonic phagocytosis were acquired with increasing age and exposure to malaria, and broadly correlated with IgG levels to merozoite antigens measured by ELISA. Importantly, high levels of opsonic phagocytosis activity among children were prospectively associated with a decreased risk of malaria, suggesting a role in protection. Human immunization with recombinant merozoite surface protein 2 (MSP2), an abundant protein on the merozoite surface, generated cytophilic antibodies, IgG1 and IgG3, that bound the merozoite surface and promoted opsonic phagocytosis. Our findings suggest that opsonic phagocytosis of merozoites may be an important mechanism by which antibodies to merozoite antigens contribute to protective immunity to malaria and and support the further development of merozoite surface proteins as potential malaria vaccine antigens. Furthermore, these studies have established a high-throughput assay to measure the functional activity of antibodies to merozoites to investigate acquired and vaccine-induced human immunity.) P11 Attempts to place Plasmodium vivax into continuous culture Wasan Forsyth1*, Ramin Mazhari1*, Wanlapa Roobsoong2*, Nattawan Rachapaew2*, Amandine Carmagnac1*, Joseph Manitta3, Kelly Rogers1, Damon Eisen4, Jetsumon Prachumsri2*, John Adams5, Krystal Evans1*, Louis Schofield1*

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1- Walter and Eliza Hall Institute for Medical Research, Parkville, Victoria, Australia; 2- Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand; 3- Victorian Infectious Diseases Reference Laboratory, Melbourne, Victoria, Australia; 4- Victorian Infectious Diseases Service, Royal Melbourne Hospital, Parkville, Victoria, Australia; 5- Department of Global Health, College of Public Health, University of South Florida, 3720 Spectrum Boulevard, Suite 304, Tampa, FL 33612, USA *The in vitro Blood-stage P. vivax culture consortium was funded by the Bill and Melinda Gates Foundation. Introduction: Plasmodium vivax (P. vivax) infects 80-350 million people annually, causing significant morbidity and occasional mortality. Investigation of P. vivax biology is severely hampered by lack of continuous in vitro culture. Culture of P. vivax is difficult because this parasite exhibits a very strong preference for reticulocytes, which are hard to source. To address this problem we used CD34+ haematopoietic stem cells to produce cultured red blood cells (CRBCs, >90% reticulocytes) in vitro. CRBCs were used to support P. falciparum and P. vivax invasion and growth. Live imaging was used to track P. vivax development. Methods: CD34+ cells were isolated using MACS anti-CD34+ beads and induced to proliferate and differentiate. Day 18 nucleated cells were removed by filtration. The filtered CRBCs were >90% reticulocytes by new methylene blue staining. CRBCs were used with D10-GFP P. falciparum parasites in 96 well format growth assays. Flow cytometry was used to determine the percentage of GFP positive cells at 0 and 48 hours. Infected blood was obtained from either: 1) patients presenting to Melbourne health centres who had contracted malaria overseas, or 2) from patients presenting at rural clinics in Kanchanaburi province, Thailand. Infected blood was placed into culture in a variety of culture conditions with CRBCs. P. vivax isolates were placed into culture and parasite development followed using DIC live cell imaging. Results and Discussion: We were able to robustly and reproducibly generate reticulocytes at >109 scale/week. These reticulocytes were capable of supporting invasion and growth of P. falciparum. P. vivax isolates typically showed dramatic collapses in parasitaemia when placed into culture, but some isolates have been cultured for >90 days at very low parasite densities. Live imaging of individual P. vivax parasites over the course of a life cycle may prove informative in the analysis of P. vivax viability in vitro. P12 The design of small molecule inhibitors of Plasmepsin V for intervention against malaria Michelle Gazdik1,2, Brad Sleebs1,2, Alan Cowman1,2 and Justin Boddey1,2 1Chemical Biology/Infection and Immunity Division, The Walter and Eliza Hall Institute, Parkville, Victoria, 3052, Australia; 2Department of Medical Biology, The University of Melbourne, Parkville, Victoria, 3010, Australia

The human malaria parasite Plasmodium falciparum is responsible for the most severe form of malaria. Central to the capacity of this organism to grow inside red blood cells and thrive inside the blood-stream is its ability to export an estimated 450 proteins into the host cell erythrocyte. The proteins exported to the erythrocyte possess a conserved N-terminal export motif termed the Plasmodium export element (PEXEL). In order for proteins to be exported, the PEXEL motif (RxLxE/Q/D) must be cleaved by an ER-resident aspartic protease called Plasmepsin V (PMV).1-4 PMV is one of ten malarial Plasmepsins and is expressed in the blood, gametocyte and liver stages of the parasite life cycle. It is hypothesized that a small inhibitor of PMV would block the export of essential proteins into the host cell, resulting in parasite death. Hence, PMV is considered a prime target for the development of new antimalarial therapies.

The goal of this project is to design small molecule inhibitors of PMV that block protein export in parasites. A common practice of targeting aspartic proteases involves the use of transition state (TS) mimetics. Statine is one of many TS mimetics successfully used to inhibit aspartyl proteases whereby it mimics the tetrahedral state of peptide-bond hydrolysis. A series of compounds were synthesised and the most potent compounds were shown to inhibit PMV with an IC50 of 20 nM. However, these compounds only exhibit moderate activity in parasite cultures. We aim to enhance the potency of the mimetics in parasite culture by improving membrane permeability and proteolytic stability. One strategy to improve these properties is via the N-methylation of the peptide backbone. Another

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strategy involves altering the P3 region of the mimetic, housing the polar guanidine moiety of arginine. The synthesis of analogues and the outcome of these strategies are discussed. P13 Monitoring the Conformational Change of Plasmodium falciparum AMA1 upon ligand binding using 19F-NMR spectrometry Xiaopeng Ge1,3, Christopher MacRaild2, Robin F. Anders1, Raymond S. Norton2, Michael Foley1 1Biochemistry, La Trobe University, Bundoora, Australia; 2Monash institute of Pharmaceutical Sciences, Monash University, Royal Parade, Parkville, Australia The hydrophobic pocket on apical membrane antigen 1 (AMA1) is the binding site for RON2, and this interaction leads to tight junction formation, which is essential for invasion of erythrocytes by malaria merozoites. Crystal structures of complexes of AMA1 with a RON2 peptide and AMA1 with R1, a competing peptide from a phage display library, showed that ligand binding induces a major conformational change in AMA1. Peptides and antibodies that block the interaction of AMA1 with RON2 also inhibit merozoite invasion. Consequently, new anti-malarial drugs might be developed that act by inhibiting the AMA1-RON2 interaction, either by binding tightly in the hydrophobic pocket or by preventing the conformational change that allows access of RON2 to the pocket. To assess the mode of action of small molecules that inhibit the AMA1-RON2 interaction, we have developed a method using site-specific 19F-NMR to detect the conformational change of AMA1 induced by ligand binding. Two forms of AMA1 (domains I+II) were prepared for these experiments: (i) wild-type AMA1 with all four Trp residues replaced by 5F-Trp, and (ii) F367W AMA1 with an additional 5F-Trp in the domain II loop of AMA1, which is the region of the protein that undergoes the conformational change. A comparison of the 19F-NMR spectra of these two proteins enabled the resonance for the 5F-Trp inserted into the domain II loop to be identified. When the mutated form of AMA1 was complexed with either the RON2 or R1 peptide the resonance was significantly sharper with an approximately two-fold increase in intensity. These resonance changes are consistent with the domain II loop becoming more disordered and the results suggest 19F-NMR may be a useful method for identifying small molecules that bind into the hydrophobic pocket of AMA1. P14 Defining the structural and antigenic properties of EBA-175 RIII-V Andrew J Guy1,2, James G Beeson1,3, Christine Langer1, Paul R Sanders1, Peter M Siba4, Ivo Mueller4,5, Matthew Stellato6, Marie-Isabel Aguilar6, Jack S Richards1,3, Paul A Ramsland1,2

1Centre for Biomedical Research, Burnet Institute; 2Department of Immunology, Monash University; 3Department of Microbiology, Monash University; 4Papua New Guinea Institute of Medical Research; 5Infection and Immunity Division, Walter and Eliza Hall Institute; 6Department of Biochemistry and Molecular Biology, Monash University Erythrocyte binding antigen 175 (EBA-175) is a P. falciparum merozoite protein antigen with a functionally important role in erythrocyte invasion, and contains a poorly characterised domain known as region III-V (RIII-V). Previous studies have demonstrated that antibodies against EBA-175 RIII-V can inhibit parasite invasion, and that high levels of antibodies against RIII-V are associated with protection from symptomatic malaria. In silico predictions identified RIII-V as being an intrinsically disordered protein domain; disordered proteins lack significant secondary or tertiary structure, but often have important physiological functions despite the lack of a defined three-dimensional structure. To verify the prediction of disorder for EBA-175 RIII-V we produced recombinant RIII-V protein and subjected it to biophysical analysis by circular dichroism (CD) and dynamic light scattering (DLS). EBA-175 RIII-V was found to adopt an extended conformation in solution by DLS, and have little secondary structure as measured by CD. To investigate the immunological consequences of protein disorder, linear epitope mapping of RIII-V was performed using an overlapping peptide array, testing human IgG interactions with peptides by ELISA. EBA-175 RIII-V was found to contain a large number of linear B-cell epitopes using sera from a malaria endemic area of Papua New Guinea. This work highlights the potential importance of disordered protein domains as targets of antibodies produced in the course of natural exposure to malaria parasites. P15 Biomarkers in early pregnancy as predictive markers of adverse outcomes in pregnancy-associated malaria Annjaleen Hansa, Dr. Louise Randall, Prof James Beeson and Prof Stephen Rogerson,

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Monash University, Clayton. University of Melbourne, Department of Medicine, Parkville. Burnet Institute, Alfred Hospital, Prahran. The placenta, as the maternal-fetal interface, is critical in delivering oxygen and nutrients to the growing fetus. Early adequate placentation is important, otherwise placental insufficiency can lead to intrauterine growth restriction, low birth weight (LBW) and small-for-gestational-age (SGA) babies, all of which are associated with placental malaria and conditions like pre-eclampsia. The relationship between malaria infection, placental insufficiency and the immune response is crucial to assess, especially during early pregnancy as this is when infection rates are most prevalent and the foundation for optimal placental development occurs. C-reactive protein (CRP) is a common clinical measure of infection and inflammation. Raised CRP levels in the mother’s blood during early pregnancy have been shown to have significant predictive value in determining LBW and SGA babies. Raised CRP levels are evident in later pregnancy-associated malaria. CRP may also have a role in dysregulated angiogenesis, being associated with levels of other inflammatory mediators such as C5a, IL-6 and TNF-α, which may prevent optimal placentation. CRP, the primary biomarker, was measured in a population of 1763 Melanesian women in early pregnancy with longitudinal data regarding delivery and birth outcomes. The main outcome measures are placental malaria, birth weight, haemoglobin levels and preterm birth. In addition, 100 malaria cases and 200 matched control cases will be tested for additional biomarkers (IL6, TNFα, IFNγ, PlGF and sFLT-1) in early pregnancy. We hypothesise that elevated CRP levels in early pregnancy, in combination with the other biomarkers, are predicative of placental malaria and fetal adverse outcomes. This study will provide novel insight into the impact of immune regulation during pregnancy-associated malaria and ideally present an easily measurable set of clinical biomarkers for use in the field.    P16 Towards a fine-scale map of the population structure of Plasmodium falciparum in Papua New Guinea G.L. Abby Harrison1, Celine Barnadas1, Livingstone Tavul2, Magnus Manske3,4, Olivo Miotto 4,5

Dominic Kwiatkowski 3,4, Inoni Betuela 2, Peter M. Siba 2, Ivo Mueller 1 Alyssa E. Barry1 1. Infection and Immunity Division, Walter and Eliza Hall Institute, Melbourne, Australia and Department of Medical Biology, University of Melbourne, Melbourne, Australia.; 2. Papua New Guinea Institute of Medical Research, Goroka, Papua New Guinea.; 3. Wellcome Trust Sanger Institute, Hinxton, CambridgeCB10 1SA, UK.; 4. MRC Centre for Genomics and Global Health, University of Oxford, Oxford OX3 7BN, UK. ; 5. Mahidol-Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok 10400, Thailand. We are working towards defining a high-resolution map of parasite population structure, networks, and migration patterns throughout Papua New Guinea(PNG). Recently we have shown that the population structure of Plasmodium falciparum on the north coast of PNG is fragmented. If this population structure is observed throughout PNG, then maps of population structure could identify isolated populations and routes of migration, as well as enable outbreak infections of unknown origin to be geographically positioned. Such knowledge will be valuable for the success of malaria elimination and control programmes. The first phase of our project aims to utilize genome-wide sequence data for parasites from three distinct areas in PNG, and to use this data to identify a panel of geographically informative single nucleotide polymorphisms (SNPs). Here we present the results for one of these areas, Madang Province. We show that a panel of candidate SNPs can distinguish between parasites from different areas within Madang. These SNPs show great promise for fine-scale mapping of P. falciparum in PNG. P17 Utilising novel compounds to study cAMP-dependent phosphorylation of Apical Membrane Antigen 1: a key event in red blood cell invasion by Plasmodium falciparum.

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Katherine Harvey1,2, Brittany Howard3, Melissa Buskes4, Nicole Ramsey5, Mauro Azevedo1, Philip Thompson3, David Manallack3, David Wilson4, Belinda Abbot4, Lonny Levin5, Jochen Buck5, Paul Gilson1,3 and Brendan Crabb1,2,3 1Burnet Institute, VIC, Australia. 2University of Melbourne, VIC, Australia. 3Monash University, VIC, Australia. 4La Trobe University, VIC, Australia. 5Cornell University, NY, United States of America. The clinical manifestations of malaria are attributable to infection of human red blood cells by Plasmodium falciparum parasites. Entry into host red blood cells by invasive “merozoites” entails a complex sequence of receptor ligand interactions and signal transduction events. Apical Membrane Antigen 1 (AMA1) is an essential membrane spanning ligand with a critical pre-invasion role. Previous work has shown that the cytoplasmic domain of AMA1 is phosphorylated at residue serine 610, which is important for efficient red blood cell invasion, however currently the specific role of phosphorylation remains unclear. Here we explore the opportunity to utilise novel compounds as functional probes for S610 phosphorylation by manipulating the putative constituents within the preceeding signal cascade. Protein kinase A, a 3’5’-cyclic AMP (cAMP) -dependent kinase, has been shown indirectly to phosphorylate S610 of AMA1. cAMP levels in parasites are regulated through the action of adenylyl cyclases, which catalyse the production of cAMP from ATP, and phosphodiesterases, which degrade cAMP into 5’-AMP. Both adenylyl cyclase β (ACβ) and phosphodiesterase β (PDEβ) are co-transcribed with PKA and AMA1 and are essential for blood stage infection thus are the most likely candidates to act within this signal cascade. We have tested 4 novel compounds designed to bind and block the PKA catalytic subunit and 3 compunds designed against ACβ, all of which should inhibit phosphorylation of S610, and one new phosphodiesterase inhibitor which may stimulate phosphorylation of S610. Here we aimed to (a) provide direct evidence that S610 is phosphorylated by PKA and (b) assess the potential of these compounds to target S610 phosphorylation. In addition to providing new biological insight into the mechanisms by which malaria parasites establish infection, this work may pave the way for the optimisation of these compounds as novel therapeutics against malaria. P18 A Novel Actin Cytoskeleton in Plasmodium falciparum Gametocytes Marion Hliscs1,2, Paul J. McMillan1,2,3, Coralie Millet1,2 Matthew W.A. Dixon1,2, Leann Tilley1,2 1Department of Biochemistry and Molecular Biology, 2Bio21 Molecular Science and Biotechnology Institute and 3Biological Optical Microscopy Platform, The University of Melbourne, Victoria Sexual differentiation of the malaria parasite into gametocytes, represent a bottleneck of malaria transmission from humans to mosquitoes. During maturation, P. falciparum gametocytes undergo remarkable morphologic changes to develop the unique falciform shape that leads to drastic distortion of the host red blood cell and possibly present one of the parasite sequestration strategies to prevent splenic clearance. We aim to understand the molecular mechanisms underlying this morphological transformation on scale of cytoskeletal elements. Here, we describe a novel actin-based skeleton in maturating P. falciparum gametocytes (stage II-IV) that locates underneath the inner membrane complex (IMC) juxtaposed to the tubulin cytoskeleton. Super resolution microscopy (3D-SIM) revealed that this actin is organized in long filamentous bundles spanning the parasite body as it elongates. In mature stage V gametocytes, actin bundles disassemble together with the tubulin skeleton, indicating (i) spatiotemporal distinct roles for actin during gametocyte maturation and (ii) an intimate crosstalk between both cytoskeletons during morphogenesis. To investigate the role of the actin skeleton during gametocyte development we performed drug assays using actin filament depolymerizing and stabilizing chemicals. Whereas actin stabilization was lethal for late stage gametocytes, actin destabilization did not inhibited morphologic transition between gametocyte stages. Interestingly, actin bundles were still detected after drug treatment by 3D-SIM, indicating a reduced susceptibility of gametocyte actin to “conventional” depolymerization methods. We will present initial data that support these novel and previously unrecognized roles for actin in gametocytes. P19 Transmission Blocking Drug Screening Assay Development Based on the ability of Plasmodium falciparum Gametocytes to cross a spleen-like device

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John P. Holleran1*, Julien Duez1,2*, Alioune Ndour2, Pierre Buffet2¤, Vicky M. Avery1¤

* and ¤ : Equal contributions 1Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Queensland; 2 Inserm-UPMC (Paris 6 University) UMRs945, Paris France In an effort to discover transmission blocking drugs for Plasmodium falciparum, we are currently developing a unique drug screening assay based on the ability of gametocytes to cross narrow slits. This approach utilizes microsphere filtering to mimic the mechanical challenges imposed on red blood cells in the human spleen. During gametocyte maturation from stages II through IV, gametocytes are rigid and non-deformable, thus are retained within the microsphere filter. However, stage V gametocytes undergo a switch to become highly deformable, thereby allowing passage through the filter. This microsphere filtration technology has been successfully translated to a 96 well plate format, suitable for drug screening. The filtration protocol has been streamlined through the use of large capacity reservoirs and a reduced number of washing step to improve throughput. Importantly, performance was validated using asexual blood stages as a model for gametocyte deformability. Ring stages were retained with modest retention (mean of 39%), while Trophozoites and Schizonts exhibited substantially higher retention (mean of 88%). Automated, high-content image quantification methods were developed using fluorescence detection to enable the rapid and accurate determination of gametocyte numbers up- and down-stream from filters. Using this screening platform, we plan to screen small molecule libraries for compounds which are capable of modulating the deformability of gametocytes. This novel approach to gametocyte drug screening has the potential to reveal compounds which increase the rigidity of late stage gametocytes thereby inducing splenic clearance and reducing transmission to the Anopheline mosquito. P20 The programmed death-1 receptor drives chronic malaria Joshua M. Horne-Debets1^, Rebecca Faleiro1^, Deshapriya S. Karunarathne1^, Xue, Q. Liu1, 2, Katie E. Lineburg1, Chek Meng Poh3,4, Gijsbert M. Grotenbreg4,5,6, Geoff R. Hill1, Kelli P.A. MacDonald 1, Michael F. Good1,2, Laurent Renia3,4, Rafi Ahmed7 and Arlene H. Sharpe8 ,Wykes, M.1. 1QIMR Berghofer Medical Research Institute, Australia; 2Griffith University, Australia; 3Singapore Immunology Network, Agency for Science, Technology and Research (A*STAR),; 4Department of Microbiology, 5Department of Biological Sciences, Faculty of Science, 6 National University of Singapore, Singapore; 7Emory Vaccine Center, Atlanta, USA; 8Harvard Medical School, Boston, USA. Malaria is a highly prevalent and devastating disease that can persist for years. Antibodies and CD4+ T cells are thought to protect against blood-stage infections by the causative parasite, Plasmodium. However, there has been considerable difficulty in developing a malaria-vaccine, highlighting our incomplete understanding of immunity against this disease. We used an experimental rodent malaria model, to show that programmed death-1 (PD-1) mediates a 95% reduction in numbers and functional capacity of parasite-specific CD8+ T cells during acute malaria, driving chronic disease. Furthermore, in contrast to widely held views, parasite-specific CD8+ T cells are required to control both acute and chronic blood-stage disease even when parasite-specific antibodies and CD4+ T cells are present. Our findings provide a molecular explanation for chronic malaria which will be relevant to future malaria-vaccine design and may need consideration when vaccine development for other infections is problematic. P21 Dual targeted tRNA synthetases as targets for cell-wide protein translation inhibition in Plasmodium falciparum. Katherine E. Jacksona, James S. Phama, Michelle Kweka, Nilushi S. De Silvaa, Stacey M. Allena, Jessica K. Holienb, Christopher D. Goodmanc, Geoffrey I. McFaddenc, Michael W. Parkerb, Lluis Ribas de Pouplanad,e and Stuart A. Ralpha. a Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria 3010, Australia. b St. Vincent's Institute of Medical Research, Victoria 3065, Australia. cSchool of Botany, The University of Melbourne, Victoria 3010, Australia. d ICREA, Passeig Lluís Companys 1, Barcelona 08010, Catalonia, Spain. e Institute for Research in Biomedicine, Carrer Baldiri Reixac 10, Barcelona 08028, Catalonia, Spain. Plasmodium falciparum has three translationally active compartments: the cytosol, the mitochondrion and a relic plastid called the apicoplast. The nuclear genome encodes 36 distinct copies of canonical

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aminoacyl-tRNA synthetases (aaRSs), insufficient to supply a unique enzyme for each amino acid in all three compartments. We have investigated the subcellular localisation of four tRNA synthetases (AlaRS, CysRS, GlyRS and ThrRS) and we show each of these enzymes are dually localised to the P. falciparum cytosol and the apicoplast. Additionally, we show a single aaRS can recognise and charge the tRNA encoded in the nucleus as well as the apicoplast genome. Our data indicate Plasmodium parasites overcome their deficit in aaRSs by dual-targeting enzymes that are able to charge organellar and eukaryotic tRNAs. We are investigating these unique aaRSs as targets for possible drugs, and have used an in silico docking approach to identify potential aaRS inhibitors that inhibit P. falciparum growth in culture. P22 Characterisation of a novel bromodomain protein of Plasmodium falciparum Gabrielle Josling1, Danny Wilson2, Michaela Petter1, Melvin Lim1, Till Voss3, Zbynek Bozdech4, Graham V. Brown1, Michael F. Duffy1 1 Department of Medicine, University of Melbourne. 2 The Walter and Eliza Hall Institute of Medical Research. 3 Department of Medical Parasitology and Infection Biology, Swiss Tropical Institute, University of Basel, Basel, Switzerland. 4 School of Biological Sciences, Nanyang Technological University, Singapore. Epigenetic regulation has been shown to be extremely important for crucial pathogenic processes such as cytoadhesion, immune evasion, and red blood cell invasion. Among the various epigenetic mechanisms that Plasmodium falciparum utilises, post-translational modification of histone tails is perhaps the best studied. Although several modifications have been identified, very little is known about the proteins that recognise these modifications. In light of this, we are characterising a bromodomain-containing protein we have called Bromodomain Protein 1 (BDP1). The protein contains a bromodomain, which is a domain that specifically recognises acetylated lysine residues in histone tails. Acetylated histones are generally associated with active genes, so BDP1 is potentially important in transcription activation. It also has an ankyrin domain; ankyrin domains are generally involved in protein-protein interactions. This combination of domains is unique to apicomplexan parasites, indicating that the protein may be involved in a novel epigenetic pathway. We have generated an HA-tagged parasite line in order to investigate BDP1. Partial knockdown of BDP1 using a destabilisation domain results in a dramatic reduction in growth due to a defect in invasion. Immunofluorescence assays revealed that BDP1 is primarily found in the nucleus, and also that BDP1 colocalises with histone modifications associated with active genes but not with those associated with repressed genes. We have also used this tagged line for chromatin immunoprecipitation investigate the genome-wide of BDP1 distribution. In addition, the recombinant bromodomain of BDP1 was shown to bind to histones, recognising histones 3 and 4 as well as the histone variant H2AZ. P23 Hijacking complement regulators – an evasion strategy for malaria parasites? Alexander T. Kennedy1, Christoph Q. Schmidt2, Alan F. Cowman1,3, Wai-Hong Tham1,3 1 The Walter and Eliza Hall Institute of Medical Research, 2 Institute of Pharmacology of Natural Products & Clinical Pharmacology, Ulm University, 3 Department of Medical Biology, The University of Melbourne. The complement system is the first line of defence against invading pathogens within the human body. Complement is a protein based component of the innate immune system that can either directly lyse pathogens by causing membrane attack complex formation, or lead to phagocytosis via opsonisation of the pathogen surface with C3b. Malaria parasites are exposed to the complement system when merozoites, the invasive form of the life cycle, are released from a spent erythrocyte into circulation. Merozoites have to evade the onslaught of complement activation in order to enter new red blood cells and perpetuate the blood stage infection. Here we investigate potential mechanisms malaria parasites employ to evade this key component of the immune system. We show that merozoites specifically recruit host complement regulators factor H and factor H-like 1 to their surface. When bound these regulators of complement retain their functional activity, which is to facilitate C3b inactivation via proteolytic cleavage, thus avoiding formation of the lytic membrane attack complex on malaria parasites. Together these studies show for the first time how merozoites may avoid complement-mediated attack in order to establish an effective infection.

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P24 Alterations of the cytoskeleton across blood stage development mediates survival of human malaria parasite Plasmodium falciparum Shannon I Kenny, Megan I Dearnley , Steven | Batinovic, Leann | Tilley and Matthew I Dixon1 Department of Biochemistry and Molecular Biology and the Bio21 Molecular Science and Biotechnology Institute, Centre of Excellence for Coherent X-ray Science, The University of Melbourne, Melbourne, VIC 3010, Australia

Survival of the human malaria parasite Plasmodium falciparum in the circulation of the host relies on its ability to drastically alter its red blood cell (RBC) host cell. This remodelling is mediated by the export of parasite derived proteins which interact with components of the RBC cytoskeleton and modify RBC behaviour. As a consequence of this remodelling the parasite must cytoadhere within the microvasculature of the host to avoid clearance by the spleen. In asexual stage parasites the formation of knob structures underneath the RBC membrane in addition to other exported parasite proteins drives a dramatic change in the RBC deformability. Using super resolution microscopy and long-term fluorescence microscopy we investigate the relationship between host cell cytoskeleton remodelling, and the assembly of knobs at the erythrocyte membrane. In particular the relationship between the major knob component, knob associated his-rich protein KAHRP and other exported proteins and structures. We functionally assess the RBC cytoskeleton remodelling processes across blood stage development, by employing ektocytometry and spleen mimic filtration assays. We show that the asexual stage parasites and early stage gametocytes employ different mechanisms to mediate their RBC deformability, with the early stage gametocyte exhibiting a highly undeformable RBC in the absence of all known cytoskeleton interacting proteins, showing that modulation of the host cell cytoskeleton itself contributes significantly to RBC rigidification. We show that these RBC modifications of the early stage gametocyte are reversed in the late stage gametocyte allowing parasite survival within the host and disease transmission.

Tuesday P25-48 P25 Plasmodium methionyl-tRNA synthetase as potential antimalarial target Erin E. Lim1*, Karen L. Dawson1, James S. Pham1, Claire Simons2, Stuart A. Ralph1

1. Department of Biochemistry and Molecular Biology, Bio21 Molecular Biology and biotechnology Institute, The University of Melbourne, Flemington Road, Parkville, Victoria 3010, Australia; 2. School of Pharmacy and Pharmaceutical Sciences, Cardiff University, King Edward VII Avenue, Cardiff CF10 3NB, United Kingdom. The widespread resistance of Plasmodium to existing antimalarials generates an urgent need to develop new antimalarial drugs. A crucial component of the protein translation machinery, the aminoacyl-tRNA synthetases, has been validated as promising antimalarial targets. We focus on one member of this family, methionyl-tRNA synthetase, which has been identified as potential drug target in both eukaryotic and prokaryotic pathogens including Trypanosoma brucei, Clostridium difficile and Staphylococcus aureus. There are two versions of MetRS in Plasmodium falciparum, one of them has a N-terminal bipartite leader sequence and is expected to be apicoplast targeted while the other version lacks obvious targeting sequences and is expected to be cytosolic. We have generated HA-tagged recombinants to study localisation and expression of these proteins in P. falciparum. A recombinant HIS-tagged PfMetRS was overexpressed in E.coli and purified and will be used for structural characterisation, and for activity and inhibitor assays. We are seeking novel inhibitors of the Plasmodium MetRS enzymes using in silico docking approaches, as well as testing analogues of known inhibitors of bacterial MetRSs. Several of these compounds kill P. falciparum grown in culture, and experiments to establish the targets of these compounds are underway. P26 Adhesin cytoplasmic tail phosphorylation: a role in P. falciparum invasion?

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Nicholas Lim, Wai-Hong Tham, Brendan R.E. Ansell, Sash Lopaticki, Dominique Dorin-Semblat, Christian Doerig, Alan F. Cowman The Walter and Eliza Hall Institute of Medical Research, Australia, Department of Medical Biology, University of Melbourne, Australia Inserm-EPFL Joint Research Unit, Switzerland, Department of Microbiology, Monash University, Australia Invasion into red blood cells is an essential step in the erythrocytic life cycle of the malaria parasite, Plasmodium falciparum. This process involves members of the parasite adhesin families, EBAs and PfRhs, interacting with erythrocytic surface receptors. These single-pass transmembrane parasite adhesins are present at the apical tip of invading merozoites and their extracellular domains are required for recognition of human erythrocytes. However, it is the cytoplasmic region that is in contact with the parasite’s cellular machinery which initiates the invasion event. Therefore, any signal generated upon binding must be communicated via the tail domain to other intracellular targets. Here we investigate the role of adhesin phosphorylation in the P. falciparum invasion process. Using in vitro kinase assays we show that the majority of Plasmodium adhesin cytoplasmic tails are phosphorylated. Mutational analyses of the adhesin cytoplasmic tails identified important residues involved in phosphorylation. This suggests that a common kinase, PfCK2, may be responsible for this post-translational modification. Using transgenic lines and the CR1-PfRh4 invasion pathway as a model, we attempt to understand the functional importance PfCK2 has on cytoplasmic tail phosphorylation as well as its role in the cellular processes of the parasite. P27 GLOBAL POPULATION STRUCTURE OF THE PLASMODIUM VIVAX VACCINE CANDIDIATES APICAL MEMBRANE ANTIGEN 1 (AMA-1) AND MEROZOITE SURFACE PROTEIN 1 (MSP-1) Alicia Arnott1, Ivo Mueller2, 3, Peter Siba4, John C. Reeder5, 6, Alyssa E. Barry3, 7 1 Centre for Immunology, Burnet Institute, Melbourne; 2 Barcelona Centre for International Health Research, Barcelona, Spain; 3 Division of Infection and Immunity, Walter and Eliza Hall Institute of Medical Research, Melbourne; 4 Papua New Guinea Institute for Medical Research, Goroka, Papua New Guinea; 5 Centre for Population Health, Burnet Institute, Melbourne; 6 Department of Epidemiology and Preventative Medicine, Monash University, Melbourne, Australia;7 Department of Medical Biology, University of Melbourne, Parkville, Australia; Despite 40% of the world’s population living at risk of Plasmodium vivax infection only two vaccine candidates targeting non blood-stage antigens have reached phase I clinical trials. The blood-stage antigens apical membrane antigen 1 (AMA-1) and merozoite surface protein 1 (MSP-1) represent promising vaccine candidates. These molecules are known to be highly polymorphic, however it remains unclear as to whether the extent and distribution of worldwide Pvama1 and Pvmsp1 genetic diversity might preclude vaccine coverage. The aim of this study was to investigate the population genetic structure of Pvama1 and Pvmsp1 in Papua New Guinea (PNG), and to compare the sequence data from PNG to published data from parasite populations of other P. vivax endemic areas worldwide. The complete Pvama1 ectodomain and Pvmsp1 C-terminal were amplified from 102 single-clone infections from PNG, and published Pvama1 and Pvmsp1 sequences were obtained from GenBank. Nucleotide and haplotype diversity was determined; signatures of balancing selection and non-synonymous single nucleotide polymorphisms (SNPs) with a minor allele frequency >10% were identified. Clusters of related haplotypes were identified using STRUCTURE and network analyses, and the geographical distribution of haplotypes investigated. Despite high levels of genetic diversity, these analyses were able to provide a valuable framework for the selection of a limited number of representative haplotypes that may cover a large proportion of this diversity. Immunological studies are needed to determine the breadth and magnitude of humoral immune responses induced by the selected haplotypes, and whether these responses are associated with clinical immunity to P. vivax infection. P28 Retrospective investigation of the selection of drug resistant Plasmodium vivax parasites in Papua New Guinea Celine Barnadas 1,2,3, Elisheba Malau 2,3, Raksmei Keo 2, Lincoln Timinao 1, Sarah Javati 1, Peter M Siba, 1, Timothy Davis 4, Ivo Mueller,2,3,5 1 Papua New Guinea Institute of Medical Research, Goroka, Papua New Guinea; 2 Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia; 3 University of Melbourne, Parkville,

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Victoria, Australia; 4 University of Western Australia, Perth, Australia; 5 Centre de Recerca en Salut Internacional de Barcelona, Barcelona, Spain;

Plasmodium vivax resistance to chloroquine (CQ) was first described in 1989 in Papua New Guinea (PNG). In 2000, sulfadoxine-pyrimethamine was added to CQ and amodiaquine (AQ) as the treatment of first choice for P. vivax malaria. In 2005, the treatment failure rate ranged from 0% in East Sepik to 29% in Madang Province, indicating that drug-resistant parasites had been selected and that levels of resistance were not homogeneous across the country. We therefore aimed to characterize the selection and spread of resistant P. vivax parasites in these two PNG populations. Genotyping of P. vivax dihydrofolate reductase gene (pvdhfr) and multidrug resistance 1 (pvmdr1) potentially associated with resistance to pyrimethamine and 4-aminoquinolines, respectively, was performed on archived samples collected between 1991 and 2010 in East Sepik and Madang Provinces. Polymorphisms in the flanking regions of these genes were characterized. Prevalence of wild-type pvdhfr isolates decreased from 95.6% in 1991-92 to 52.4% in 2006 (P<0.001) while the quadruple mutant increased from 2.0% to 16.7% (P=0.031). In 2005, the prevalence of the quadruple mutant pvdhfr genotypes was significantly higher in Madang than East Sepik (52.2% vs 16.7%, P=0.001). Similarly, the pvmdr1 976F mutation putatively associated with CQ resistance was significantly higher in Madang (71.6% vs 26.2%, P=0.001). Preliminary sequence analysis of pvdhfr indicates at least three distinct origins of the quadruple mutant pvdhfr isolates in PNG.

P29 The J-like domain of a type IV HSP40 of Plasmodium falciparum cannot be turned into a functional J-domain by insertion of an HPD motif Eva-Rachele Pesce1, Jessica C. Lawson2, Gregory L. Blatch1

1Victoria University, Melbourne 8001 Australia, 2Rhodes University, Grahamstown 6140 South Africa It is becoming more evident that molecular chaperones are important for the survival of Plasmodium falciparum in the human host. All HSP40s possess a conserved domain called J-domain, the main region involved in the interaction of HSP40s with HSP70s. A canonical J-domain contains the highly conserved HPD motif, which is crucial for the ATPase stimulation of HSP70 by an HSP40. P. falciparum expresses several members of the HSP40 chaperone family that can be classified into four types (I-IV) according to their domain organisation. Of particular interest because of their uniqueness are the type IV HSP40s. These proteins contain a J-like domain in which the HPD is not conserved. It is believed that mutations in the HPD alter the ability of an HSP40 to functionally interact with an HSP70 and therefore it is not clear which functions type IV HSP40s fulfil. We have begun to analyse the J-like domain of PF14_0013 (PF3D7_1401100), a PEXEL containing type IV HSP40 which displays a YPK tripeptide in place of the HPD. We have constructed a chimeric protein (PF14’-J) containing the J-like domain of PF14_0013 followed by the canonical domains of a bacterial HSP40. The chimeric protein was used in complementation assays using a thermosensitive Escherichia coli strain (OD259) which lacks HSP40s. PF14-J was not able to functionally replace the endogenous HSP40s. We therefore mutated the YPK motif to the canonical HPD. Also in this case the mutated PF14-J was not able to reverse the thermosensitivity of the E. coli OD259 strain. These results suggest that the J-like domain of this type IV HSP40 may not be a functional J-domain in the typical sense of a co-chaperone. PF14_0013 as well as other type IV HSP40s may perform specialised tasks which do not necessarily include the stimulation of the ATPase activity of HSP70s.    P30 Severe malaria infection impairs germinal centre responses and suppresses the generation of B cell memory Victoria Ryg-Cornejo1, Lisa Ioannidis 1, Stephen Nutt 1, Axel Kallies 1 and Diana Hansen1 1The Walter & Eliza Hall Institute of Medical Research, Parkville, Australia Naturally acquired immunity to the parasite Plasmodium plays an important role in protection against malaria infections, which remain a widespread cause of morbidity and mortality worldwide. No evidence exists of sterile immunity to the disease and the development of sustained clinically protective antibody responses has been shown to require repeated infections. While many studies have focused on the complex nature of these responses against the antigenically diverse parasite, few have addressed the effect of malaria infection on the generation of memory B cell (MBC) responses. A study of children in areas of high seasonal transmission revealed a delay in malaria-specific MBC generation despite continual exposure to the parasite. In contrast, in a low transmission setting, lasting MBC responses were detected in adults following a single exposure to the parasite. These data

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indicate clinical malaria infe ctions may hinder the generation and maintenance of malaria-specific MBC populations. To further investigate this notion, C57BL/5 mice were infected with P. berghei ANKA or immunised with an equivalent antigenic load of attenuated parasites, and mice were drug-cured prior to the onset of severe malaria symptoms. Long-lived populations of B cells are generated during the germinal centre (GC) reaction in secondary lymphoid organs, such as the spleen, and histological studies of this organ revealed that clinical malaria infections profoundly impede the correct generation of GC structures. Further, compared to immunised control animals, infected animals had reduced numbers of GC B cells and T follicular helper cells, both critical to an effective GC reaction. Importantly, the frequency and relative affinity maturation of MBC populations appeared to be compromised by malaria infections relative to immunised controls. Taken together these data indicate that clinical malaria negatively impacts the development of long-term humoral immunity by disrupting critical early stages in the development of the B cell response. P31 Functional characterisation of the cyclin proteins in Plasmodium falciparum Siti Nurfatimah MS, Teresa G. Carvalho, Christian Doerig Department of Microbiology, Monash University, Wellington Road, Clayton VIC 3800, Australia Plasmodium falciparum causes the most severe form of human malaria and has the highest rate of mortality. Due to the increase of drug resistance and to the lack of an effective vaccine, new therapeutic targets are urgently needed. Recently, protein kinases (PKs) have been proposed as an attractive drug target as they have been successfully targeted in cancer and other human diseases. The P. falciparum kinome comprises 65 PKs. About half of them are likely to be essential for the proliferation of the parasite inside red blood cells, and the function of most remains to be elucidated. The CMGC group of kinases, to which Cyclin-dependent kinases (CDKs) belong to constitute a family of cell cycle regulators. The activation of CDKs during cell cycle progression is tightly controlled by the interaction with a particular cyclin subunit. Several CDKs have been characterised in P. falciparum and are essential for the erythrocyte asexual cycle. Equally, four cyclins have been identified in P. falciparum and two of these have shown the ability to activate PfPK5 (a putative CDK1 functional homologue) in vitro. We aim at characterising the function of each cyclin during the asexual stages of P. falciparum. We are currently producing transfected parasite lines expressing a mcherry-tag of each cyclin. These lines will be used to determine the subcellular localisation of the proteins in vivo, and to identify the binding partners of each cyclin by performing pull-downs and mass spectrometry. Further, we are also expressing recombinant fusion proteins of the four cyclins and will test the ability of these to activate different CDKs using in vitro kinase assays. A complete picture on the function of the cyclins and cyclins/CDKs interactions will provide an in-depth understanding of the biological role of P. falciparum and may lead to novel targets for antimalarial in the future. P32 Production, characterisat ion and immunogenicity of plant-made Plasmodium falciparum Merozoite Surface Protein 4, a malaria vaccine candidate, in Nicotiana benthamiana Kart ika M. Setyabudi1 , D iane E. Webster2 , Hans J . Net ter1 , and Ross. L . Coppel1 . 1Department o f Microbio logy, Monash Univers i ty , V ic tor ia 3800, Austra l ia ; 2School o f B io log ica l Sc iences, Monash Univers i ty , V ic tor ia 3800, Austra l ia Malaria remains a major public health problem causing approximately one million deaths and up to 500 million cases annually. It is caused by infection with protozoa of the genus Plasmodium, with Plasmodium falciparum being responsible for the most severe form of malaria. Vaccination against malaria is considered as the most efficient tool for prevention of malaria and reduction of malaria transmission. A number of proteins have shown promise as components of a subunit malaria vaccine including P. falciparum Merozoite Surface Protein (PfMSP) 4. It is synthesized at the late ring stage and transported to the parasite surface. Besides identifying the right antigen target, the high cost of vaccination is another challenge in the development of a malaria vaccine. Plants have gained wide acceptance for the cost-effective production of recombinant pharmaceutical proteins. Advantages that plants offer include the potential of industrial scale, lower risk of contamination from potential human pathogens, fewer constraints in distribution and potential for oral delivery. In our study, we optimised the production of PfMSP4 in plants using codon optimisation for expression in E. coli, yeast or mammalian cells and subcellular targeting. Characterisation of the recombinant

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protein indicates that it is the correct size and antigenically similar to native protein. We also found that subcutaneous and oral immunisations of mice using the plant-made PfMSP4 elicited the antigen-specific antibodies. By conducting this study, we aim to add to research supporting plants as a biofactory for the production of a safe, affordable and effective malaria vaccine. P33 Investigating phosphatidic acid synthesis in the Plasmodium apicoplast Melanie J Shears1,3, Vanessa Mollard1, James I MacRae2, Malcolm J McConville2, Cyrille Y Botté1,3, Geoffrey I McFadden1. 1 The School of Botany, The University of Melbourne, Australia; 2 The Department of Biochemistry and Molecular Biology, The Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne, Australia; 3 ApicoLipid group, Laboratoire Adaptation et Pathogénie des Microorganismes UMR5163, CNRS, Université Joseph Fourier Grenoble I, La Tronche, France. Replication of Plasmodium in the human host is dependent on the parasite obtaining sufficient lipid for membrane biogenesis. As the majority of membrane lipid species are synthesized from a phosphatidic acid precursor, the pathways for the generation of this key lipid species are considered promising targets for drug intervention. Analysis of the Plasmodium falciparum genome indicates that the parasite likely possesses two pathways for phosphatidic acid synthesis; one in the endoplasmic reticulum and a second in the apicoplast. We characterized the first enzyme of the apicoplast phosphatidic acid synthesis pathway in P. falciparum, a glycerol-3-phosphate acyltransferase named PfATS1. We confirm PfATS1 is localized to the apicoplast, and show it can functionally replace the equivalent enzyme in the phosphatidic acid synthesis pathway of Escherichia coli. We demonstrate by gene knockout that PfATS1 is dispensable in blood stage parasites, with preliminary data indicating loss of the enzyme does not significantly alter either the growth rate or the lipid composition of the parasite. Together, these results provide the first experimental evidence for the presence of a phosphatidic acid synthesis pathway in the apicoplast, and raise intriguing questions about the role of the pathway in parasite lipid metabolism.

P34 The genetic diversity of Plasmodium falciparum var genes in Papua New Guinea

Sofonias Tessema1, 2, Livingstone Tavul4, John C. Reeder3, 5, Peter Siba4, Ivo Mueller1, 2,6, Alyssa E Barry1, 2

1The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3052, Australia; 2Department of Medical Biology, University of Melbourne, Melbourne, Victoria 3052, Australia; 3Department of Epidemiology and Preventative Medicine, Monash University, Melbourne, Australia; 4Papua New Guinea Institute of Medical Research, Goroka, Papua New Guinea; 5Centre for Population Health, Burnet Institute, Melbourne, Australia; 6Centre de Recerca en Salut Internacional de Barcelona (CRESIB), Barcelona, Spain The diversity of var genes has been shown to be immense in different geographical regions. However, it was reported to be limited in Amele1, Madang Province of Papua New Guinea (PNG) and in Portvelho, Brazil2. The aim of this study was to estimate the extent of var gene diversity in two geographically distinct parasite populations from PNG. The DBLα domains of the var genes of 70 P. falciparum isolates from PNG (Mugil = 35, Wosera = 35) were PCR amplified, gel-purified, cloned and sequenced. High quality sequences were analyzed; the number of unique DBLα types were determined and compared with previous reports. The sequence analyses showed that the estimated diversity of DBLα types is 1094 (CI: 923-1326) in Wosera and 906 (CI: 766-1100) in Mugil, PNG. The diversity of DBLα in Wosera and Mugil is two times higher than Amele, PNG1 and eight times lower than Africa2. The Sorensen Index (pairwise nucleotide sequence sharing among isolates) of Mugil and Wosera was found to be lower as compared to areas of limited var gene diversity (Amele, PNG1 and Portvelho, Brazil3). We confirmed the comparably low diversity of var genes in PNG with geographical variation. References:

1Barry et al. (2007), PLOS Pathogen 3(3): 0349-0357; 2Chen & Barry et al. (2011), PLoS

ONE 6(2): 1-12; 3Albrecht et al. (2010) Gene 453(1-2): 37–44. P35 Malaria immunity in pregnancy in times of changing malaria epidemiology

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Andrew Teo1, Wina Hasang1, Gaoqian Feng2 Louise M. Randall1, James G. Beeson2, Graham V. Brown3 and Stephen J. Rogerson1 1Department of Medicine (Royal Melbourne Hospital), University of Melbourne, Melbourne, Australia; 2Macfarlane Burnet Institute of Medical Research, Melbourne, Australia.; 3The University of Melbourne, The Nossal Institute for Global Health, Parkville, Australia

Background: Pregnancy-specific malaria antibodies can protect women from pregnancy-associated malaria. As malaria control is intensified, pregnant women may be less exposed to malaria, thus affecting the acquisition of protective antibody. Methods: Plasma samples were collected at delivery from Malawian and Melanesian pregnant women. IgG levels to schizont extract, merozoite antigens and VAR2CSA-DBL5 were measured by ELISA, IgG levels to variant surface antigens (VSA) of infected erythrocytes (IEs) were measured by flow cytometry, and functional opsonizing antibody levels to merozoites and IEs were determined using flow cytometry based phagocytosis assays. Results: Women were enrolled during different time periods that reflect high (enrolled earlier) and low (enrolled later) prevalence of parasitemia at delivery. In Malawi, pregnant women enrolled later had significantly lower IgG to merozoite antigens; MSP2 p=0.02, MSP3 p=0.05 and Rh2A9 p=0.004, and to VAR2CSA-DBL5 p=0.02. Interestingly, total IgG or opsonizing antibody levels to both non-pregnancy-associated parasite line, E8B-ICAM, and pregnancy-malaria associated CS2 IE did not vary between the early enrolled and later enrolled women. In Papua New Guinea, the decline in antibody responses measured by ELISA was more apparent (schizont extract, MSP2, MSP3, Rh2A9 and VAR2CSA-DBL5 all p=<0.0001) whereas levels of opsonic IgG to E8B-ICAM and CS2 IE, and levels of merozoite-opsonizing antibodies did not vary over time. Conclusion: Antibody levels to schizont extract, merozoite antigens and VAR2CSA-DBL5, measured by ELISA, declined over time, possibly as a result of decreasing malaria exposure. Anti-merozoite IgG may have shorter half-lives than IgG to VSA. The latter antibody responses may be carried forward to the women’s subsequent pregnancy, thus offering longer-lasting protection against malaria during pregnancy. The differences in total levels of IgG to merozoite antigens, but not in levels of opsonic IgG to merozoites suggest that the quality but not quantity of IgG may be important in offering protection against malaria infection. P36 A Role For A Female Gametocyte Specific ABC Transporter in Sexual Commitment and Neutral Lipid Biosynthesis Phuong N. Tran1,2, Matthew W. A. Dixon3, Simon H. J. Brown4, Todd W. Mitchell4, Kiaran Kirk1 and Alexander G. Maier1 1Research School of Biology, The Australian National University, Canberra, ACT, 0200; 2Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086 Australia; 3Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC 3010, Australia; 4School of Health Sciences, University of Wollongong, Wollongong, NSW 2522, Australia Transmission of malaria from human to mosquito is mediated by the sexual precursor cells of the malaria parasite (gametocytes), which circulate as intraerythrocytic forms in the blood of the host. Many transmission-blocking agents have been investigated and the focus of research has shifted recently towards the sexual stage. Many open questions remain about the fundamental biology of the sexual stages of the parasite, in particular what trigger the switch from the asexual pathway to the production of the sexual stages and the physiological changes involved in the differentiation. In this study, we examined the role of an ABC transporter of P. falciparum that is predominantly expressed in gametocytes. To investigate the location and function of the protein of interest, GFP-tagged, HA tagged, clonal knockout, and complement transfected cell lines were generated. Co-localization studies with different organelle markers and lipid labelling using confocal and super-resolution microscopies suggest that the molecule resides on a lipid body containing neutral lipids. Interestingly we found that the ABC transporter is a female gametocyte-specific molecule and may play a role in the sexual commitment. More importantly, lipidomic analysis reveals that the transporter might be a key player in the biosynthesis of neutral lipids in gametocytes. Identifying the function of this female gametocyte-specific ABC transporter will not only increase our understanding of lipid metabolism of the sexual stage parasites but also provide new insights into a fundamental biological mechanism of the sexual differentiation in malaria parasites.

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P37 Naturally acquired antibodies to P. falciparum blood stage antigens associated with protection from malaria in Papa New Guinea

Rupert I Weaver1 2*, Linda I Reiling 1 and Gaoqian I Feng1, Ivo I Mueller3, Peter I Siba4, Tsuboi I Takafumi 5, Jack I Richards1, Freya I Fowkes1, James I Beeson1 1Burnet Institute,2The University of Melbourne, 3 Walter and Eliza Hall Institute of Medical Research, 4 Papua New Guinea Institute of Medical Research, Madang, 5Cell-Free Science and Technology Center Ehime University, Matsuyama, Ehime, Japan

Background: Malaria is a leading cause of mortality around the globe; children bear the greatest burden of disease and a vaccine is urgently needed. The development of a vaccine is supported by the fact individuals naturally develop immunity to malaria after repeated exposure to infection. Antibodies to blood stage antigens of the parasite, including the merozoite antigens that are important for invasion of human red blood cells, are thought to be important for protective immunity. However, different subclasses of antibodies mediate different effector functions. Knowledge of which mechanisms and antibody subclasses provide protection and what are the important targets of this response remains incomplete. A greater understanding of the immune responses will aid vaccine development by providing biomarkers of immunity and identifying targets of the protective immune response. Aims/Methods: The aim of this research is to investigate the IgG subclass responses to the blood stage antigens, including the essential invasion ligand PfRh5, and determine the association between IgG subclass responses with protection from malaria. This will be investigated using serum samples taken at baseline from a treatment re-infection cohort study of 206 Papa New Guinean children between the ages of 4 to 14 years. The children were prospectively followed every 2 weeks for a period of 6 months allowing antibody responses to be associated with their clinical outcome. Anticipated results: Previous studies have shown antibodies to PfRh5 and other invasion ligands have been associated with protection from clinical malaria. Our initial results demonstrate that IgG1 and IgG3 are the dominant antibodies produced against PfRh5, and studies to determine the association between these responses and protective immunity are ongoing. Prior research has shown the principal subclass response to blood stage antigens associated with protection have been predominantly IgG1 and 3. Conclusions: This research will help prioritize antigens as potential vaccine candidates and define the nature of responses required for protective immunity to guide vaccine development. P38 A hierarchy of receptor-ligand interactions in malaria parasite invasion and their association with deformation, calcium influx and dehydration of host erythrocytes Paul R. Gilson1,2, Tana Taechalertpaisarn1,3, Greta E. Weiss1, Wai-hong Tham4, Nienke de Jong1, Katherine L. Harvey1,5, Freya Fowkes1, Paul N. Barlow6, Julian C. Rayner7, Gavin J. Wright8, Alan F. Cowman3,4 and Brendan S. Crabb1 1Burnet Institute, 85 Commercial Road, Melbourne, Victoria, Australia 3004; 2Department of Immunology, Monash University, Victoria, Australia; 3Department of Medical Biology, The University of Melbourne, Victoria, Australia; 4The Walter & Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia 3052; 5The Department of Microbiology & Immunology, The University of Melbourne, Victoria, Australia; 6Schools of Chemistry and Biological Sciences, University of Edinburgh, UK; 7Malaria Programme, Wellcome Trust Sanger Institute, Cambridge CB10 1SA, UK; 8Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, Cambridge CB10 1HH, UK The invasion of Plasmodium falciparum merozoites into host erythrocytes requires multiple receptor-ligand interactions during an 11s pre-entry stage. Here, we use real-time imaging to observe the effects of ablating four known interactions; an early ‘heparin-inhibited’ interaction, the EBA/Rh-mediated ‘alternate pathways’, Rh5 to basigin binding and the AMA1 to RON2 interaction. We show that these interactions are distinct and occur in the aforementioned order. We demonstrate that vigorous erythrocyte deformation at an early pre-invasion stage strongly favours subsequent successful invasion. This deformation is mediated by the 'alternate-ligands' and requires engagement of the actomyosin motor. Downstream of this, Rh5 binding to basigin is necessary for the erythrocyte dehydration (echinocytosis) that is observed 30-60s after invasion. We show that echinocytosis is triggered by a calcium flux in the erythrocyte. Surprisingly, the source of calcium appears to be the secretory organelles of the invading merozoite implicating an open connection between the merozoite

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and erythrocyte exists prior to tight junction formation. This open connection requires Rh5 binding to the erythrocyte basigin receptor. It would provide a passage for translocation of the parasite-derived RON complex that is known to occur at this time. The AMA1 to RON2 interaction occurs downstream of these events, promoting the formation of the tight-junction that is necessary for invasion. This study provides a rational basis to combine sequentially acting key merozoite vaccine candidates for potentially synergistic control of P. falciparum malaria blood-stages. P39 Development and optimization of a glucose-6-phosphate-dehyrdogense (G6PD) deficiency assay Andrew Guy1,2, Ismael Barreto3, Jessamine Welsh1, Joe Mannita4, Chuong Tran5, Céline Barnadas6, James Beeson1,7, Paul Ramsland1,2, Jack Richards1,7,8 1Centre for Biomedical Research, Burnet Institute, Melbourne; 2Department of Immunology, Monash University; 3National Health Laboratory, Dili, Timor-Leste; 4Victorian Infectious Diseases Reference Laboratory, Melbourne; 5Biochemistry Department, Royal Children’s Hospital, Melbourne; 6Walter and Eliza Hall Institute of Medical Research, Melbourne; 7Department of Immunology, Monash University; 8Victorian Infectious Diseases Service, Royal Melbourne Hospital, Melbourne. Glucose-6-Phosphate-dehydrogenase (G6PD) deficiency is a heritable condition that can cause hemolytic anemia in individuals when exposed to oxidative stresses. A particularly important cause of oxidative stress is the anti-malarial drug primaquine which is used for the treatment of P. vivax hypnozoites and as a gametocidal agent. It can cause severe hemolytic anemia and even death in G6PD deficient individuals. The prevalence of severe G6PD deficiency is generally higher in malaria endemic regions and may reach up to 20% in some areas. It is generally accepted that testing for G6PD deficiency must occur before commencing treatment with primaquine. Unfortunately, current methods for testing for G6PD deficiency are time consuming, expensive and sometimes unreliable. The aim of this work was to create a rapid, cost-effective and accurate test to quantitate G6PD activity. This will be used to diagnose individuals for clinical management but also for population-based screening. A published WST-8/1-methoxy PMS method was adapted, and the results were compared to existing established assays including the gold standard UV spectrophotometry assay. The new WST-8/1-methoxy PMS assay has created a faster (15 minutes) quantitative assay which can be used on whole blood samples and in a high throughput format. This work seeks to provide a rapid and affordable G6PD assay that will make primaquine treatment easier and safer to administer.

P40 Two Myosin Essential Light Chains are critical for motility and host cell invasion in Toxoplasma gondii Melanie Williams 1,2, Hernan Alonso 1, Brian Smith 3, Christopher Tonkin 1,2 1The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia, 2The Department of Medical Biology, University of Melbourne, Melbourne, Australia, 3La Trobe Institute of Molecular Sciences, La Trobe University, Melbourne, Australia. Virulence of apicomplexan parasites relies on actomyosin-based motility to traverse through host tissue, invade and ultimately egress host cells. Motility is initiated in response to intracellular calcium signalling events and accomplished through the generation of mechanical force by a unique molecular motor, termed the glideosome. The glideosome consists of a Myosin heavy chain (MyoA), together with its Myosin Light Chain (MLC1) and several scaffold proteins, the Gliding Associated Proteins (GAPs). While the role of MyoA is to convert chemical energy in the form of ATP to mechanical energy in the form of movement, the mechanisms that regulate MyoA activity and thus force production are unknown. We have recently identified two Myosin Essential Light Chains (ELC1 and ELC2) in Toxoplasma gondii and show that both proteins interact specifically with the tail region of MyoA. Using a conditional gene regulation system, we investigate their roles in vivo and observe significant impairment of motility, invasion and egress when ELC1 and ELC2 are simultaneously suppressed. Utilising a structural model of the MyoAtail-Light Chain complex to inform mutational analyses of the ELCs, we identify the key residues required for ELC function in vivo. These results demonstrate the importance to parasite motility, of calcium binding to ELC and intermolecular interactions between the glideosome components. To understand the mechanism by which these interactions promote force production, we have tagged MyoA for affinity purification of the native glideosome. Using this, we will

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reveal the effect on glideosome function by kinetic analyses and have begun to reveal the macromolecular structure of the glideosome using single-particle cryo-electron microscopy. P41 New pulse assays that mimic in-vivo exposure reveal differences in sensitivity of P. falciparum to artemisinin Stanley C. Xie1, Nectarios Klonis1, Con Dogovski1, James McCaw2, Maria P. Crespo-Ortiz1, Sophie Zaloumis3, Julie A. Simpson3 and Leann Tilley1

1Department of Biochemistry and Molecular Biology and Australian Research Council Centre of Excellence for Coherent X-Ray Science, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia; 2Vaccine and Immunisation Research Group, Murdoch Children’s Research Institute and Melbourne School of Population Health, University of Melbourne, Parkville, Victoria 3010, Australia; and 3Centre for Molecular, Environmental, Genetic, and Analytic Epidemiology, Melbourne School of Population Health, University of Melbourne, Parkville, Victoria 3010, Australia

Artemisinins (ARTs) are the most effective class of antimalarials against Plasmodium falciparum. However, ART resistance has emerged in regions along the Cambodia-Thailand border and many patients in that area experience delayed clearance of blood parasites. Frustratingly, the parasites isolated from those patients do not always show reduced ART sensitivity in standard 3-day in vitro assays. Since ART antimalarials have short in vivo half-lives of 1 to 2 hours, we have applied short drug pulses to parasites in an effort to better mimic in vivo conditions. We hypothesised that short drug pulses may reveal stage and stain-dependent differences in drug sensitivity that are not apparent in standard 3-day assays. We have examined and compared the two laboratory strains D10 and 7G8. In standard 3-day assays, D10 has 2-3 fold higher ART IC50 values than 7G8 parasites. In pulse assays, tightly synchronised parasites were subjected to 4 h drug pulses at different stages throughout the intraerythrocytic asexual lifecycle. Parasite viability following drug exposure was monitored in the cycle following the drug pulse by flow cytometry using the nucleotide-binding dye, Syto61. Under these conditions, the 7G8 strain exhibited up to 100 fold higher ART sensitivity than D10 parasites. Furthermore, parasites treated with 4 h drug pulses showed significant stage-dependent differences in drug sensitivity. Very early rings (less than 6 h post invasion) were very sensitive to ART. Apart from this very early stage, most of ring stage parasites (mid-ring to late-ring) were relatively insensitive to ART compared to trophozoites and schizonts1. We have applied pulse assays to field isolates from the Pailin region in Cambodia that exhibit similar ART IC50 values in standard 3-day assays. Our results show that pulse assays can reveal large differences in sensitivity of field strains to ARTs that are not evident in standard assays, which may be clinically relevant. P42 Conditional knockdown of essential invasion gene in Plasmodium falciparum Alan Yap1, Paul Gilson2, Danny Wilson1, Matthew O’Neill1, Mauro Azevedo2, Brendan Crabb2, Alan Cowman1 1Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia; 2 The Burnet Institute for Medical Research and Public Health, Melbourne, Australia

Plasmodium falciparum merozoites actively invade host erythrocytes during blood stage malaria, the stage that causes clinical symptoms. Invasion of erythrocytes is a complex process that involves a cascade of protein-protein interactions. To date, multiple merozoite proteins have been described as essential based on the inability to knock-out the proteins in vitro. In order to elucidate the functions of these essential proteins an inducible gene regulation system is required. Currently, there is a dearth of inducible systems that are robust and efficient in Plasmodium falciparum. Here we present evidence for the efficient knockdown of apical membrane antigen-1 (AMA-1) using a dimerizable Cre recombinase (DiCre) system. AMA-1 is a leading malaria vaccine candidate and essential for replication of parasites in the blood stage. Parasites expressing DiCre were generated and upon addition of rapamycin there was a knockdown of AMA-1 gene and protein expression of up to 90% within 48 hours. This led to a growth reduction following knockdown of AMA-1, which is related to decreased invasion efficiency as demonstrated by flow cytometry and live/fixed cell imaging. This project reveals the potential utility of DiCre in deleting essential parasite genes that cannot be knocked out using conventional methods, hence greatly facilitating further functional analysis.

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P43 Maintenance of immunity during pregnancy to surface antigens of P. falciparum-parasitized red blood cells Xi Zen Yap1, Mirja Hommel1, Rose McGready2,3, Francois Nosten3, Freya Fowkes1, James Beeson1 1Burnet Institute, Victoria, Australia; 2 Centre for Vaccinology and Tropical Medicine, Churchill Hospital, Oxford, UK; 3 Shoklo Malaria Research Unit, Mae Sot, Tak Province, Thailand The immune response to malaria develops with repeated exposure. Susceptibility to symptomatic malaria decreases with age and is associated with the acquisition of antimalarial antibodies, and adults in endemic areas generally possess immunity to symptomatic malaria. Pregnant women are the exception and experience higher rates of malaria infection, which impacts upon maternal and newborn health. This increased susceptibility to malaria is thought to be partially due to the expression of chondroitin sulphate A (CSA) and other pregnancy-specific ligands on the placenta, enabling interactions with P. falciparum-infected erythrocytes expressing the adhesion protein VAR2CSA, a variant of the P. falciparum erythrocyte membrane protein 1 (PfEMP1). These interactions facilitate sequestration of parasites in the placenta. Antibodies to VAR2CSA are thought to protect pregnant women from malaria, but the generation and maintenance of immunity to malaria in pregnant women is poorly understood. Previous studies have suggested that the antibody response to recombinant VAR2CSA in pregnant women may be extremely long-lived compared to the antibody response against merozoite antigens. It is unclear whether the longevity of this response is specific to VAR2CSA, or whether antibodies to antigens on the surface of parasitized red blood cells (pRBCs) are better maintained than antibodies to merozoite antigens. This study therefore aims to measure antibody levels against and determine the longevity of the antibody response to different variants of PfEMP1 during pregnancy. This study will examine a cohort of women from the Thai-Burma border. In order to assess the longevity of antibody responses, serum samples have been taken from each individual at repeated timepoints from enrolment to delivery. Serum samples will be tested for antibodies to pRBC surface antigens using placental- and CD36-binding parasite isolates. The findings of this study may help to generate a better understanding of the generation and maintenance of antimalarial immunity in pregnant women. P44 Remodelling of the Erythrocyte Cytoskeleton During P. falciparum Merozoite Invasion Elizabeth S. Zuccala1,2, Timothy J Satchwell3, Fiona Angrisano,1,2 , Danushka S. Marapana1,2, Yan-Hong Tan1, Ashley M Toye3 & Jake Baum1,2 1Division of Infection & Immunity, Walter & Eliza Hall Institute of Medical Research, Parkville, Australia; 2Department of Medical Biology, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Australia; 3School of Biochemistry, University of Bristol, Bristol, United Kingdom Red blood cells are remarkably resilient, strong and dynamic structures. These properties are required for their passage through small capillaries and are imparted by the cytoskeleton, a network of proteins that underlies and links to the cell membrane. To successfully invade, the blood stage malaria parasite must induce drastic changes to the structure of the target erythrocyte, including the formation of a tight junction and a new cellular compartment, the parasitophorous vacuole. These key modifications involve the infolding of the red blood cell membrane, membrane fusion and fission events and the secretion of parasite proteins into the host cell. While detailed cellular descriptions of merozoite invasion have been achieved over the past few decades, however, comparatively little is known about the molecular basis of how the host cell responds to parasite entry. We hypothesise that to invade, merozoites interface with endogenous erythrocyte pathways that regulate membrane and cytoskeletal remodelling, and here present cellular and molecular data on the host factors involved in facilitating merozoite invasion. Using high definition fluorescence imaging we have constructed a map of how different erythrocyte cytoskeletal components reorganise around invading merozoites to identify key targets of remodelling. To investigate the role of the host cell in parasite uptake we have explored the effect of inhibitors that target various host cell pathways on merozoite invasion. Finally, we resent preliminary quantitative phospho-proteomic data on the host cell response to invading merozoites. By shedding light on the host-cell contribution to invasion we hope to uncover novel chemotherapeutic targets to stop invasion and hence prevent or treat malaria disease.

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P45 Red blood cell deformability throughout pregnancy Kerryn Moore1, 2, Marcus J Rijken3, 4, 5, Germana Bancone3, 4, Julie A Simpson1, Freya J I Fowkes1, 2, Rose McGready3, 4, 6, Arjen Dondorp4, 6, Francois Nosten3, 4, 6

1. Centre for Molecular, Environmental, Genetic and Analytic Epidemiology, University of Melbourne, Parkville, Victoria, Australia; 2. The Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Victoria, Australia; 3. Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Mae Sot, Thailand; 4. Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 5. Department of Obstetrics, University Medical Centre Utrecht, Utrecht, The Netherlands; 6. Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Oxford, UK. For adequate blood flow, red blood cells (RBCs) deform to permit flow through the microvasculature. Reduced deformability of infected and uninfected RBCs has been observed in severe malaria patients, which reduces blood flow and perfusion, and increases splenic removal of RBCs. A single episode of treated malaria during pregnancy may reduce RBC deformability beyond the period of actual parasitaemia, which may contribute to the negative impact of malaria on birthweight despite prompt treatment. We sought to describe RBC deformability throughout pregnancy in women living in an area of low malaria transmission. 279 pregnant women attending antenatal care on the Thai-Burma border were recruited (median gestation 12 weeks) and followed until delivery. RBC deformability (elongation index, EI) was measured every 5 weeks using a laser assisted optical rotational analyser (LORCA). All cases of malaria, detected by peripheral blood smear, were immediately treated. Additional deformability measurements were taken at each malaria episode, and two weeks later. RBC deformability was related to gestational age and malaria using linear mixed-effects models. Estimated mean EI (shear stress 1.69 Pa) was 0.226 (95% CI: 0.222, 0.231) at 12 weeks gestation. EI was highly variable within each woman throughout pregnancy, but there was no association between EI and gestational age at a population level (mean change: 0.00017/week; 95%CI: -0.00003, 0.00038; p = 0.097). 77 (28%) women became infected with Plasmodium spp. during follow-up, with a total of 110 episodes (1-8 episodes/woman). EI decreases between malaria infection (all species) and 14 days post-diagnosis (nadir ~0.206), before gradually returning to baseline over several weeks (mean change: 0.0008/day; 95% CI: 0.0002, 0.0014; p = 0.006). Malaria reduces deformability, and is gradually restored following treatment and parasite clearance. Further research is needed to determine clinical significance by exploring the association between reduced RBC deformability and birthweight for gestational age. P46 Elucidation of invasion molecular mechanisms in malaria infection by atomic force microscopy Xavier Sisquella1, Joseph O’Neill1, Michelle Gee2, Waihong Tham1, Alan Cowman1

1Walter+Eliza Hall Institute of Medical Research, Parkville, Australia; 2Department of Chemistry, University of Melbourne, Parkville, Australia During the blood stage of malaria parasite infection, P. falciparum invades human erythrocytes (RBCs) in order to survive within the human host. Prior to the intraerythrocytic phase, merozoites (the invasive form of P. falciparum) go through a number of stages (initial attachment, reorientation and final invasion) that involve several merozoite adhesins that interact and adhere to erythrocyte receptors. The aim of the presented work is to understand the role of P. falciparum adhesins in invasion. Atomic force microscopy (AFM) allowed the analysis of RBCs’ stiffness under the action of proteins shed by the parasite. RBCs in the presence of invasion parasite proteins (EBA-175, PfRh4 and MSPDBL2) become more deformable suggesting that those specific ligand – receptor interactions activate a signaling pathway aiming to weaken the RBC cytoskeleton and enable the parasite to invade. Western-blot analysis of RBCs membrane ghosts after being assayed for each parasite adhesin binding enabled identification of cytoskeletal proteins intervening in this signaling process. The combination of a biophysical characterization technique such as AFM with standard protein biochemistry methods represents a challenging, feasible and interdisciplinary approach, which enhances improved interpretation of the molecular mechanisms of invasion and a better understanding of the basic mechanisms that govern this complex process which will allow us to identify attractive candidates for a vaccine against blood stage infection.

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P47 Insight into the pathogenesis of foetal growth restriction in placental malaria: decreased placental glucose transporter-1 expression Upeksha Chandrasiri1, Caroline L.L. Chua1, Alexandra J. Umbers1, Ebbie Chaluluka2, Jocelyn D. Glazier3, Stephen J Rogerson1,4, Philippe Boeuf1,4

1 Department of Medicine, University of Melbourne, Parkville, VIC Australia; 2 College of Medicine, University of Malawi, Blantyre, Malawi; 3 Maternal and Foetal Health Research Centre, Institute of Human Development, Manchester Academic Health Sciences Centre, University of Manchester, St. Mary's Hospital, Manchester, UK; 4 Victorian Infectious Disease Service, Royal Melbourne Hospital, Parkville, VIC Australia

Malaria in pregnancy can lead to placental malaria defined as the accumulation of Plasmodium falciparum-infected erythrocytes in the placental intervillous blood spaces where they get in direct contact with the syncytiotrophoblast, the nutrient transporting epithelium of the placenta. Placental malaria can trigger a local inflammatory response (intervillositis). Women with placental malaria and intervillositis are at increased risk of delivering low birth weight infants compared to women with placental malaria without inflammation. This suggests a pathogenetic role for monocytes in foetal growth restriction associated with placental malaria. However, the supporting mechanisms are unknown. Transplacental glucose transport is a strong regulator of foetal growth. Glucose transporter 1 (GLUT-1) is the main transporter of glucose across the placenta. It is expressed in the mother-facing microvillous plasma membrane (or MVM) and foetus-facing basal membrane (or BM) of the syncytiotrophoblast. BM GLUT-1 expression represents the rate-limiting step for transplacental glucose transport. We hypothesized that placental malaria, especially when complicated by intervillositis, was associated with decreased BM GLUT-1 expression and, therefore, impaired glucose supply to the foetus and foetal growth restriction. In a cohort of Malawian pregnant women, we used Western-blotting to quantify GLUT-1 expression in MVM and BM from uninfected placentas (n=18) and infected placentas with (n=8) and without (n=20) intervillositis. We found comparable MVM GLUT-1 expression between uninfected placentas or placental malaria cases with or without intervillositis, whereas BM GLUT-1 expression was lowest in placental malaria with intervillositis compared to the other groups (P≤.0016) and correlated negatively with monocyte infiltrate density (r=-0.43; P=.003) and positively with birth weight (r=0.28; P=.06). Our results suggest that intervillositis, more than placental malaria per se, might cause foetal growth restriction through impaired transplacental glucose transport. The identification of the molecular mechanisms at play could help the design of intervention strategies aimed at improving pregnancy outcomes for malaria-infected women.

P48 The role of ring exported protein 1 in virulence protein trafficking in Plasmodium falciparum

Emma McHugh 1 2 3 Don Gardiner 4 Katharine Trenholme 4 Leann Tilley 1 2 3 and Matthew Dixon 1 2 3

1Biochemistry and Molecular Biology, The University of Melbourne, Parkville, VIC, Australia; 2ARC Centre of Excellence for Coherent X-ray Science, The University of Melbourne, Parkville, VIC, Australia; 3Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC, Australia; 4Malaria Biology Laboratory, Queensland Institute of Medical Research, Herston, QLD, Australia

The most severe forms of human malaria are caused by Plasmodium falciparum, an apicomplexan parasite that undergoes asexual replication within red blood cells. Infected erythrocytes gain the ability to adhere to endothelial cells in the host microvasculature through a virulence complex on the cell surface. This process, termed cytoadherence, requires P. falciparum to produce and export a range of proteins to the surface of the host erythrocyte which possesses no endogenous protein trafficking apparatus. Membranous parasite-derived structures called Maurer’s clefts located in the erythrocyte cytoplasm play a key role in the sorting of proteins involved in cytoadherence. Ring exported protein 1 (REX1) is a Maurer’s cleft protein required for expression of the major virulence protein on the red blood cell surface. The C terminus of REX1 contains a repeat region which varies between parasite strains and diversity of these repeats is thought to be linked to parasite virulence. In order to investigate the role of the C terminal repeat region of REX1 in virulence protein trafficking, we have

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performed binding assays to characterise the cytoadherence phenotype of several REX1 truncation, replacement and wild type parasites. We have observed altered Maurer’s cleft structure in parasites with deletion of the C terminal repeat region. Trypsin cleavage assays, where the protease trypsin is used to cleave the extracellular portion of the major virulence protein, have been performed to assess surface expression of virulence protein.

P49 Computational analysis of Plasmodium ookinete motility suggests a critical role for cell shape in malaria parasite colonisation of the mosquito midgut Andrey Kan1*, Yan-Hong Tan2*, Fiona Angrisano2,4, Eric Hanssen5, Kelly L. Rogers3, Lachlan Whitehead3, Vanessa P. Mollard6, Anton Cozijnsen6, Michael J. Delves7, Simon Crawford6, Robert E. Sinden7, Geoffrey I. McFadden6, Christopher Leckie1, James Bailey1 and Jake Baum2,3,7

1 Victoria Research Laboratory, National ICT Australia (NICTA), Department of Computing and Information Systems, University of Melbourne, Victoria 3052, Australia; Infection and Immunity Division2 and Centre for Dynamic Imaging3, The Walter and Eliza Hall of Institute of Medical Research, Parkville, Victoria 3052, Australia; 4 Department of Medical Biology, University of Melbourne, Victoria 3052, Australia; 5 Electron Microscopy Unit Bio21 Molecular Science and Biotechnology Institute and Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia. 6 School of Botany, The University of Melbourne, Parkville 3010, Victoria, Australia. 7 Department of Life Sciences, Imperial College of Science, Technology and Medicine, London, SW7 2AZ, United Kingdom. *These authors contributed equally The parasites that cause malaria, protozoan pathogens from the genus Plasmodium, require cell movement to complete their two-host lifecycle. The motile lifecycle forms achieve motility, called gliding, via the activity of an internal actomyosin motor. However, whilst based on such widely conserved proteins as actin and myosin, the biomechanics of gliding are not completely understood. Current models envisage a precisely organised motor that produces rearward directional force, which, linked to surface-bound adhesins, is passaged to the posterior propelling the parasite forwards. This model is believed to apply to all three motile Plasmodium lifecycle-stages: the sporozoite, merozoite and ookinete. However, only the insect-stage ookinete demonstrates continuous gliding in the absence of an intracellular developmental phase requiring host-cell entry and, as such, uniquely represents an opportunity to test fundamental questions about the biomechanics of cell movement in the absence of invasion. Here we develop a quantitative imaging platform to study ookinete movement in three-dimensions (3D) to understand the key factors underlying ookinete motion and address the critical question of how parasites colonize the mosquito midgut. Using single-cell tracking and numerical analysis of fluorescent parasite trajectories, our computational analysis of ookinete gliding demonstrates their conformity to a right-handed helical mode of movement. By exploring wild-type and cytoskeletal knockout ookinetes using varied structural electron microscopy techniques and comparison of their 3D movement we demonstrate that shape of the Plasmodium cell may be more important than direction of motor force in driving parasite cells forward. In relaxing organizational requirements on the motor we can highlight the potentially overlooked importance of cell shape as an adaptive strategy for Plasmodium cell dissemination and, as such, parasite transmission.

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Delegate list Surname First Name Laboratory Email Achtman Ariel Schofield (WEHI) achtman@wehi.EDU.AU, Andrew Dean Richards (Burnet) dean.andrew@burnet.edu.au Angrisano Fiona Baum (WEHI) angrisano@wehi.EDU.AU Armistead Jennifer Boddey (WEHI) armistead.j@wehi.edu.au Arnott Alicia Reeder (Burnet) alicia@burnet.edu.au Barber Bridget Anstey (MSHR, Darwin) bridgetbarber@hotmail.com Barnadas Celine Mueller (WEHI) barnadas@wehi.edu.au Barry Alyssa Barry (WEHI) barry@wehi.edu.au Batinovic Steven Tilley (BIO21, Melb Uni) s.batinovic@student.unimelb.edu.au Beeson James Beeson (Burnet) beeson@burnet.edu.au

Blume Martin McConville (BIO21, Melb Uni) martin.blume@unimelb.edu.au Boddey Justin Boddey (WEHI) boddey@wehi.EDU.AU

Boeuf Philippe Rogerson (Dept Med, Melb Uni) pboeuf@unimelb.edu.au

Brewster Jessica Barry (WEHI) brewster.j@wehi.edu.au Brown Graham Brown (Nossal) gvb@unimelb.edu.au Buffet Christelle Boeuf (Dept Med, Melb Uni) christelle.buffet@unimelb.edu.au Bursac Dejan de Koning Ward (Deakin) d.bursac@deakin.edu.au Carmagnac Amandine Schofield (WEHI) carmagnac@wehi.edu.au Carvalho Teresa Doerig (Monash) teresa.carvalho@monash.edu Chan Jo-Anne Beeson (Burnet) jchan@burnet.edu.au

Chandrasiri Upeksha Rogerson (Dept Med, Melb Uni) upekshac@student.unimelb.edu.au

Charman Susan Charman (Monash) Susan.Charman@monash.edu Charnaud Sarah Crabb (Burnet) charnaud@burnet.edu.au Chisolm Scott de Koning-Ward (Deakin) schishol@deakin.edu.au Chiu Christopher Hansen (WEHI) chiu@wehi.edu.au Chua Charlie McConville (BIO21, Melb Uni) h.chua@student.unimelb.edu.au Coffey Michael Tonkin (WEHI) coffey.m@wehi.edu.au Conway Caitlin Doerig (Monash) cgcon2@student.monash.edu Cooke Brian Cooke (Monash) Brian.Cooke@monash.edu Counihan Natalie de Koning-Ward (Deakin) n.counihan@deakin.edu.au Cozijnsen Anton McFadden (Botany, Uni Melb) a.cozijnsen@unimelb.edu.au Davenport Miles Davenport (Uni NSW) m.davenport@unsw.edu.au Dawson Karen Ralph (BIO21, Melb Uni) k.dawson@student.unimelb.edu.au de Koning-Ward Tania de Koning-Ward (Deakin) tania.dekoning-ward@deakin.edu.au

Dimasuay Kris Rogerson (Dept Med, Melb Uni) kdimasuay@student.unimelb.edu.au

Dixon Matt Tilley (BIO 21, Melb Uni) matthew.dixon@unimleb.edu.au

Doerig Christian Doerig (Monash) christian.doerig@monash.edu Duffy Sandra Avery (Eskitis, Griffith) sandra.duffy@griffith.edu.au Duffy Michael Duffy (Dept Med, Melb Uni) mduffy@unimelb.edu.au Elliott Salenna Beeson (Burnet) selliott@burnet.edu.au Elsworth Brendan Crabb (Burnet) elsworth@burnet.edu.au Erickson Sara Cowman (WEHI) erickson@wehi.edu.au Evans Krystal Schofield (WEHI) evans@wehi.edu.au Feng Gaoqian Beeson (Burnet) G.feng2@pgrad.unimelb.edu.au

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Forsyth Wasan Schofield (WEHI) forsyth@wehi.EDU.AU Fowkes Freya Fowkes (Burnet) fowkes@burnet.edu.au Franca Camila Mueller (WEHI) franca.c@wehi.edu.au Garcia-Bustos Jose Garcia-Bustos (Monash) jose.garcia-bustos@monash.edu Gazdik Michelle Boddey (WEHI) gazdik@wehi.edu.au Ge Xiaopeng Foley (Latrobe) xpge@students.latrobe.edu.au Ghosh Sreejoyee de Koning-Ward (Deakin) sghosh@deakin.edu.au Gilson Paul Gilson (Burnet) gilson@burnet.edu.au Gohil Sejal Cooke (Monash) Sejal.Gohil@monash.edu Gruszczyk Jakub Tham (WEHI) jakub.gruszczyk@gmail.com Guy Andrew Beeson (Burnet) andrewguy@burnet.edu.au Haase Silvia Baum (WEHI) haase@wehi.edu.au

Hansa Annjaleen Rogerson (Dept Med, Melb Uni) aphan1@student.monash.edu

Hansen Diana Hansen (WEHI) hansen@wehi.EDU.AU Hanson Josh Anstey (MSHR, Darwin) drjoshhanson@gmail.com Harrison Abby Barry (WEHI) aharrison@wehi.edu.au Harvey Katherine Gilson/Crabb (Burnet) katherine@burnet.edu.au

Hasang Wina Rogerson (Dept Med, Melb Uni) whasang@unimelb.edu.au

Hazirin Mita Barry (WEHI) hazairin.m@wehi.edu.au Healer Julie Cowman (WEHI) healer@wehi.edu.au Herrmann Susann Ralph (BIO21, Melb Uni) susann.herrmann@unimelb.edu.au Herten-Crabb Asha Fowkes (Burnet) asha@burnet.edu.au Hill Danika Schofield (WEHI) hill@wehi.edu.au Hliscs Marion Tilley (BIO21, Melb Uni) marion.hliscs@unimelb.edu.au Holleran John Avery (Eskitis, Griffith) john.holleran@griffith.edu.au Hooker David Doerig (Monash) david.hooker@monash.edu Horne-Debets Joshua Wykes (QIMR Beghofer MRI) Joshua.Horne-Debets@qimrberghofer.edu.au Huang Ziwei Wei Shen (Monash) windy.ziwei.huang@monash.edu Ioannidis Lisa Hansen (WEHI) ioannidis@wehi.edu.au Irani Vashti Richards/Beeson (Burnet) vashti.irani@burnet.edu.au Jabbarzare Marzieh Rogerson (Dept Med, Melb Uni) jabbarzare_marzieh@yahoo.com Jackson Katherine Ralph (BIO21, Melb Uni) kathputnam@gmail.com Javati Sarah Barandas/Mueller (WEHI) sarah.javati@pngimr.org.pg Jennison Charlie Barry (WEHI) jennison@wehi.edu.au John von Freyend Simona Doerig (Monash) simona.johnvonfreyend@monash.edu Josling Gabrielle Duffy (Dept Med, Melb Uni) g.josling@student.unimelb.edu.au Kalanon Ming de Koning Ward (Deakin) mkalanon@gmail.com Katris Nicholas Waller (Botany) n.katris@ugrad.unimelb.edu.au Kennedy Alexander Tham (WEHI) a.kennedy2@student.unimelb.edu.au Kenny Shannon Tilley (BIO21, Melb Uni) shannon.kenny@unimelb.edu.au Koepfli Cristian Mueller (WEHI) koepfli@wehi.edu.au Lakkavaram Asha de Koning-Ward (Deakin) alakkava@deakin.edu.au Li Wai Suen Connie Mueller (WEHI) liwaisuen@wehi.edu.au Lim Erin Ralph (BIO21, Melb Uni) eel@unimelb.edu.au Lim Nicholas Tham (WEHI) nlim@wehi.edu.au

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Lin Clara Cowman (WEHI) lin@wehi.edu.au Loganathan Sasdekumar Avery (Eskitis, Griffith) s.loganathan@griffith.edu.au Lucantoni Leonardo Avery (Eskitis, Griffith) l.lucantoni@griffith.edu.au Lucet Isabelle Lucet (Monash) isabelle.lucet@monash.edu Ma Charles Cooke (Monash) Charles.Ma@monash.edu Maier Alexander Maier (ANU, Canberra) alex.maier@anu.edu.au Maiwald Michael Ralph (BIO21, Melb Uni) michael.maiwald@unimelb.edu.au Malau Elisheba Mueller (WEHI) malau@wehi.edu.au Marapana Danushka Cowman (WEHI) marapana@wehi.edu.au Matthews Kathryn de Koning-Ward (Deakin) k.mifsud@deakin.edu.au Mazhari Ramin Schofield (WEHI) mazhari@wehi.edu.au McConville Malcolm McConville (BIO21, Melb Uni) malcolmm@unimelb.edu.au McFadden Geoff McFadden (Botany, Uni Melb) gim@unimelb.edu.au McHugh Emma Tilley (BIO21, Melb Uni) e.mchugh@student.unimelb.edu.au McLean Alistair Fowkes (Burnet) alistair@burnet.edu.au Mollard Vanessa McFadden (Botany, Uni Melb) vpas@unimelb.edu.au Moore Kerryn Fowkes (Burnet) kerrynmoore.kam@burnet.edu.au Morahan Belinda Garcia-Bustos (Monash) belinda.morahan@monash.edu Morrissette Naomi Cowman (WEHI) morrissette.n@wehi.edu.au Morton Sarah McFadden (Botany, Uni Melb) s.morton@student.unimelb.edu.au Mueller Ivo Mueller (WEHI) mueller@wehi.edu.au Nebl Tom O'Connor (WEHI) nebl@wehi.edu.au Nie Catherine Gilson/Crabb (Burnet) nie@burnet.edu.au O'Neill Matt Boddey (WEHI) moneill@wehi.edu.au Olshina Maya Baum (WEHI) olshina@wehi.edu.au Papageorgiou Marco Duffy (Dept Med, Melb Uni) m.papageorgiou@student.unimelb.edu.au Pasaje Charisse Ralph (BIO21, Melb Uni) cpasaje@student.unimelb.edu.au

Pesce Eva-Rachele Blatch (College of Health & Biomed Eva-Rachele.Pesce@vu.edu.au

Petter Michaela Duffy (Dept Med, Melb Uni) mpetter@unimelb.edu.au Phuong Thuan Schofield (WEHI) phuong@wehi.edu.au Powell Rosanna Fowkes (Burnet) rpowell@burnet.edu.au Proellocks Nicholas Cooke (Monash) nicholas.proellocks@monash.edu Rajasekaran Pravin Boddey (WEHI) rajasekaran@wehi.edu.au Ralph Stuart Ralph (BIO21, Melb Uni) saralph@unimelb.edu.au,

Randall Louise Rogerson (Dept Med, Melb Uni) louise.randall@unimelb.edu.au

Reiling Linda Beeson (Burnet) reiling@burnet.edu.au Richards Jack Richards (Burnet) richards@burnet.edu.au Robinson Leanne Mueller (WEHI) robinson@wehi.edu.au

Rogerson Stephen Rogerson (Dept Med, Melb Uni) sroger@unimelb.edu.au

Ryg-Cornejo Victoria Hansen (WEHI) rygcornejo@wehi.edu.au Samad Nadira Waller (Botany) nsamad@student.unimelb.edu.au Sanders Paul Gilson/Crabb (Burnet) sanders@burnet.edu.au Sayers Claire McFadden (Botany, Uni Melb) csayers@student.unimelb.edu.au Schofield Louis Schofield (WEHI) schofield@wehi.edu.au Selvarajah Shamista Duffy (Dept Med, Melb Uni) s.selvarajah@ugrad.unimelb.edu.au Setyabudi Kartika Coppel (Monash) Kartika.Setyabudi@monash.edu

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Sexton Anna Cooke (Monash) anna.sexton@monash.edu Shahpudin Siti Doerig (Monash) siti.mohdshahpudin@monash.edu Shears Melanie McFadden (Botany, Uni Melb) shearsm@unimelb.edu.au Siddiqui Ghizal Cooke (Monash) Ghizal.Siddiqui@monash.edu Sisquella Xavier Cowman (WEHI) sisquella@wehi.EDU.AU Sleebs Brad Boddey (WEHI) sleebs@wehi.edu.au Speed Terry Speed (WEHI) speed@wehi.edu.au

Stewart Rebecca Tonkin (WEHI) rstewart@wehi.edu.au

Sturm Angelika McFadden (Botany, Uni Melb) asturm@unimelb.edu.au Tan Yan Hong Baum (WEHI) ytan@wehi.edu.au Tan Stephanie Schofield (WEHI) stan@wehi.edu.au Tang Jingyi Duffy (Dept Med, Melb Uni) tangj@student.unimelb.edu.au Teguh Silvia Tilley (BIO21, Melb Uni) steguh@student.unimelb.edu.au

Teo Andrew Rogerson (Dept Med, Melb Uni) andrew.teo@uqconnect.edu.au

Tessema Sofonias Barry (WEHI) tessema@wehi.edu.au Tessier Natacha Barry (WEHI) tessier.n@wehi.edu.au Tham Wai-Hong Tham (WEHI) tham@wehi.edu.au Tilley Leann Tilley (BIO21, Melb Uni) ltilley@unimelb.edu.au Tonkin Chris Tonkin (WEHI) tonkin@wehi.edu.au Tran Phuong Maier (ANU, Canberra) np3tran@students.latrobe.edu.au Turner Kelsey Ralph (BIO21, Melb Uni) turnerke@student.unimelb.edu.au Uddin Taher McFadden (Botany, Uni Melb) tuddin@student.unimelb.edu.au Umbers Alexander Beeson (Burnet) umbers@burnet.edu.au Volz Jennifer Cowman (WEHI) volz@wehi.EDU.AU Waller Ross Waller (Botany) rossfw@unimelb.edu.au

Waltmann Andreea Mueller (WEHI) waltmann@wehi.edu.au Weaver Rupert Beeson (Burnet) rupert@burnet.edu.au Weir Christopher Cowman (WEHI) weir.c@wehi.edu.au Weiss Gretchen Gilson/Crabb (Burnet) weiss@burnet.edu.au Welsh Jessamine Richards (Burnet) jessamine.welsh@burnet.edu.au Williams Melanie Tonkin (WEHI) mwilliams@wehi.edu.au

Wilson Danny Cowman (WEHI) dwilson@wehi.edu.au Wykes Michelle Wykes (QIMR Beghofer MRI) MichellW@qimr.edu.au Xie Stanley Tilley (BIO21, Melb Uni) c.xie@student.unimelb.edu.au Yang Annie Boddey (WEHI) yang.a@wehi.edu.au Yap Xi Zen Beeson (Burnet) xyap@burnet.edu.au Yap Alan Cowman (WEHI) yap@wehi.edu.au

Yeoh Lee Ralph (BIO21, Melb Uni) l.yeoh@pgrad.unimelb.edu.au Zaloumis Sophie Simpson (Melb Uni) sophiez@unimelb.edu.au Zuccala Liz Baum (WEHI) zuccala@wehi.edu.au

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Additional Information

Parents Room We have a private parent’s room with refrigeration facilities available for attendees to use in the Burnet Institute - next door at 85 Commercial Road. Please see the receptionist for details. Twitter #MiMelb2013 Conference Dinner – College Lawn Sponsored by Beckman Coulter

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Malaria in Melbourne 2013 would like to acknowledge the generous support of our sponsors