ANALYSIS OF MUTANT PARASITES

THAT HAVE A DEFECT IN ADHESION

Denise MATTEI

Mar08

Cours International « Atelier Paludisme »10 Mars au 18 Avril 1008 – Institut Pasteur de Madagascar

TRAFFICKING OF VIRULENCE FACTORS IN INFECTED

RED BLOOD CELLS

SEARCH FOR TRAFFICKINGMUTANT PARASITES

Mar08

knobs

PfEMP-2

Band 3

Knob-associated proteins

PfHRP-1

Glycophorin C

Adductin

4.9

Tropomodulin

Tropomyosin

and Actin

Spectrin

4.2

4.1

Ankyrin

PfEMP1

PfEMP-3

rifin

other

molecules?PfEMP1

Mar08

OBSERVATIONS

CLAG = cytoadherence linked asexual gene

Identified through the study of parasite lines that no longer bound to C32melanoma cells = subtelomeric deletion in chr 9 (Biggs, BA et al. PNAS,1989)

.

Genetic knock-out (Trenholme, KR et al. PNAS, 2000) or

antisense RNA inhibition Gardiner, DL et al. MBP, 2000) => loss of the CD36 binding phenotype

.CLAG9 may be involved in cytoadhesion by binding directly to CD36or indirectly, through a role in PfEMP1 transport

Mar08

WORKING HYPOTHESISIs CLAG9 another CD36 binding molecule?

EXPERIMENTAL APPROACH

• P. falciparum lines selected to bind to CD36 or to CSA. Adhesion to these receptors occurs in a mutually exclusive manner

⇒ possibility to study the role of CLAG9 in cytoadherent parasites that do not bind to CD36

• Specific antibodies

=> to analyse the location of clag9 during the asexual blood stage development

CSA CD36

CHO

Receptor «panning »

FCR3 CSA FCR3 CD36

PannedFCR3

Scherf et al., 1998, EMBO J. 17 , 5418

monomorphic parasites

Mar08

Transcription of the clag9 gene

--

-2 6 10 14 18 22 26 30 34 38 42 46

9.49

7.46

4.40

3D7

hrs

The clag9 gene is transcribed in theschizont stages of both CD36 andCSA-selected parasites

Mar08

Clag9 is part of the RhopH complex in the rhoptries of merozoites

r anti-clag9FITC

mab7H8/50TRICT

nuclei

DAPI

merged

r anti-clag9 TRICT

mAb 4E10

Oregon green

merge DAPI

Mar08

Ling et al 2004. Mol.Microbiol.52:107

is transferred into the ring-stage parasite

and

Clag9 RhopH2

Localisation of CLAG9 to the rhoptries

Ling et al.2004. Mol.Microbiol.

Mar08

CLAG9

• clag9 gene is transcribed and expressed in late asexual blood stages in CD36 and non-CD36 binders

• Clag9 is present in merozoites as part of the RhopHcomplex in the rhoptries

• Anti-CLAG9 antibodies do not detect it at theerythrocyte membrane of parasitized RBC (trophozoites/schizonts)

Mar08

Analysis of clag9 natural mutant parasites

T9-96 and D10

Mar08

C32 cellsCD36 >> ICAM-1 >>CSA

FCR3CD36 D10

T9-96 and D10 do not cytoadhere to any known adhesion receptor

Mar08

OBSERVATIONS

• T9-96 and D10 do not cytoadhere• The complex RhopH is detected in the ring stages

HYPOTHESIS

clag9 is necessary to modify the parasite’s and/or parasitophorous vacuole membrane

EXPERIMENTAL APPROACH

Analysis by immunofluorescence of clag9neg linesand FCR3CD36

Mar08

RESULTS

The images were similar independently of the genotype

‘PfEMP1’ is located in association with the Maurer’s clefts

PfEMP1Alexa

Pf332TRICT

DAPI merge

FCR3CD36

D10

T9.96

Mar08

CONCLUSIONclag9neg parasites can export polypeptides to the different cellular compartments

FCR3CD36

D10

T9.96

Mab89Alexa

DAPI merge

Mar08

OBSERVATIONS

• ‘ PfEMP1’-like molecule is exported in association with Maurer’s clefts

• ‘PfEMP1’ ?

• Is it exposed on the red blood cell surface ?

EXPERIMENTAL APPROACH

• Labelling of red blood cell surface of clag9neg strains• Trypsin resistance

Mar08

CLAG9neg lines express a PfEMP1surface protein

-200kDa-

PfEMP1

•High molecular weight

•TX100-insol/SDS soluble

•Trypsin sensitive

T9.96

0 10010 µg TrypsinB

T9.9

6

D10FC

R3

CD

36

A

PfEMP1

Mar08

OBSERVATIONS

• The isolates T9.96 and D10 express PfEMP1 on the surface but do notcytoadhere to any known receptor

HYPOTHESIS

• Adhesion negative parasites express a FUNCTIONAL var gene havingunique adhesive properties BUT can not switch expression to classical var genes

• Adhesion negative parasites display a NON FUNCTIONAL conformation* Post-translational modification to establish a functional state (protease, kinase, etc…)

EXPERIMENTAL APPROACH

• Northern blot analysis• Cloning and expression of the CIDR domain BINDING ASSAYS

Mar08

PfEMP1 is transcribed in the clag9neg

parasite lines T9.96 and D10

SR TR S

D10 FCR3CD36

D10cl9

SR TR S

D10 FCR3CD36

ATS

- 7.46 -- 4.40 -

- 9.49 -

- 2.57 -

- 1.35 -

8Kb

Kb

Mar08

Functionality studies of the PfEMP1- CIDR domain in CD36 binding of

T9-96 and D10

Mar08

A classical var gene is transcribed and expressedin mutant parasites

ATS: acidic terminal segment; CIDR (α,β,γ): cisteine-rich interdomain region; DBL (α,β,γ,δ,ε): Duffy-binding-like domain;

NTS: N-terminal segment; TM: transmembrane domain;

Tandem association

DBL1αααα CIDR1ααααNTS

CR1blood gr Aheparinhep sulfate

CD36CD31IgM

Semi-conservedhead structure

TM ATS

CD31

DBL2δδδδ CIDR2ββββ

Mar08

C L K NNKKTCGKKKCNRDCKCYE RW VKRKKEEFKKIKDHFGKQKD MQPYID PDMTLKILLNYVFLQ DMKDANG NPQHIAKIQELLE KKKVELEDNLNKNTIIDYMFEDDLEEINKC

Robinson et alRobinson et al. 2003.. 2003.MolMol..MicrobiolMicrobiol. 47:1265. 47:1265--7878

D10cl9D10cl9

M1 M2 M3C9 C12

The CIDR domain from D10 is predicted to bind to CD36

Mar08

0

2

4

6

8

10

100 50 25 12,5 6,25 3,125 1,56 0,781

µg/ml

MC Cl4

PGEx

M2 Cl6

MC Cl1

PGEX 2

CSA Cl15

M2 Cl6 2

MC Cl4 2

MC Cl1 2

CD36 Binding Assays recombinant CIDR domains

Mar08

FCR3 CD36 on differents receptors

CD36 ICAM qC1QR CSA CSC

D10 on differents receptors

CD36 ICAM qC1QR CSA CSC

Adhesion on different purified receptors

Mar08

CIDR domain (M2 minimum CD36 binding site)-pDisplay vector transfected in COS-7 cells

M2 domain on the surface

CD36 protein

Anti-CD36 labelled beads

Binding assays with Dynal beads

Mar08

D10

MC+ control

anti-tag

HA merge

Mar08

CD36 Binding Properties of D10 CIDR1

TransfectedCOS7 cells

CSA(-) control

Conclusions

• Cytoadhesion mutants express a PfEMP1 molecule atthe surface of the parasitized red blood cell

• The mutant parasites can not bind to the commonadhesion phenotypes (CD36, ICAM-1, CSA, Rosetting, etc.)

• Panning on endothelial cells does not restore adhesionphenotype

Non-functional PfEMP1 surface expression !

M

Mar08

ATOMIC FORCE MICROSCOPY

Mar08

RBC infected by Plasmodium falciparum

Mar08

Control

Mar08

3D7 Parasitised red blood cell (PRBC)

Knobs sizes ø

50-60 nm

FCR3 PRBC

Knobs sizes ø

50-60 nm

D10 PRBC

Height

Amplitude error

Knobs sizes range from ø 20 nm to 100 nm

1.5 µm

Mar08

D10 and T9.96 carry a large chromosome 9 deletion

PF

I1730w

cla

g9

PF

I1725

w

PF

I1715

w

PF

I1720w

gig

PF

I1710w

BP

OR

F

PF

I1760w

rex4

PF

I1770

w

PF

I1765

c

PF

I1775

w

PF

I1780

w

PF

I1785

w

PF

I1790

w

PF

I1800

w

PF

I1795

c

PF

I1805w

rif

in

PF

I1810w

rifi

n

PF

I1825w

rifi

n

PF

I1815c

rifin

Centromere

Telomere

PF

I1820w

PfE

MP

1

PF

I1830c

PfE

MP

1

PF

I1735c

rex

1

PF

I1755c

rex

3

PF

I1740c

rex

2

PF

I1745

c

Chromosome truncation in mutant parasites

PF

I1705

w

PF

I1750

c

Mar08

HYPOTHESIS

• Adhesion negative parasites display a NON FUNCTIONAL conformation* Post-translational modification to establish a functional state(protease, kinase, etc…)

PERSPECTIVES

* Clag 9 KO => clag9 functional role * other KO => chr9 region => genotype and phenotype analysis

* SYSTEMS BIOLOGY => RNAs transcribed/translated ptn-ptn interactions cellular location

Mar08

BIHP (Institut Pasteur, Paris)

Artur ScherfSuwanna ChaorattanakaweJosé Manuel L. NogueiraCaroline DavisChristine Scheidig-BenatarYvon SterkersEmeric Roux

Parasitologie Expérimentale(Université de la Méditerranée, Marseille)

Jürg GysinCatherine Lepolard

National Institute for Medical Research(Mill Hill, London, UK)

Tony HolderIrene T. Ling

Ehime UniversitySchool of Medicine(Ehime, Japan)

Osamu Kaneko

Cellular Microbiology and Infectious Pathogeny(Institute Pasteur Lille)

Frank LafontSebastien Janel

ANALYSIS OF MUTANT PARASITES

THAT HAVE A DEFECT IN ADHESION

Denise MATTEI

Mar08

Cours International « Atelier Paludisme »10 Mars au 18 Avril 1008 – Institut Pasteur de Madagascar

THE PfRhopH COMPLEX

RhopH Complexapprox. 480 kDa

RhopH1/ClagPFI1730w - 155kDa

RhopH2PFI1445w - 140 kDa

RhopH3PFI0265c - 110 kDa

Clag 2

Clag 3.1

Clag 3.2

Clag 8

Clag 9

Kaneko et al. 2005.MBP

Why the PfEMP1expressed by the ‘clagneg’

isolates is non functional ?

HYPOTHESIS

• wrong conformation ?• post-translational modification ?

how can it be tested?

apr07

Perspectives

• A NEW var GENE HAVING UNIQUE ASPECTS?(non CD36, non CSA binding)

AND/OR

• A NEW HOST ENDOTHELIAL ADHESION RECEPTOR ?

OR

•A KNOWN PfEMP1 WHICH DISPLAYS A NON FUNCTIONALCONFORMATION

• clag9 KNOCK-OUT

1000 100 0 1000 100 0

D10FCR3 CD36

HRP

HSP 70

ATSHRP

HSP 70

Trypsin µg

175

83

62

47.5

32.5

kDaN

R

B

C

N

R

B

C

Analysis of surface-exposed PfEMP1 on PRBCby trypsin cleavage

0 100 100 0 100 100

HRP

HSP 70

D10 FCR3 CD36

HSP 70 HRP

ATS

175

83

62

47.5

32.5

kDa N

R

B

C

N

R

B

CTrypsin

Chymotrypsin µg

Analysis of surface-exposed PfEMP1 on PRBCby trypsin and chymotrypsin cleavage

Identification of natural clag9 mutant parasite lines

T9-96

3D7

chr9

NRBCFCR3

CD36

FCR3CSA

T9-96

3D7

175

83

62

47.5

32.5

kDa

clag9

T9-96 3D7

r P

Ir

ant i-c

lag9

clag9 probe

Mar08

Localisation of CLAG9

r anti-clag9 FITC

mab7H8/50TRICT

nuclei

DAPI

merged

pep1 pep2

Clag9-GST 100 aa

Clag9 is encoded by members of the clag multigene family

Ling,I et al. 2004. Mol.Microbiol. 52:107-118

Mar08

r anti-clag9

TRICT

mAb 4E10

Oregon green

merge DAPI

Rabbit anti-CLAG9 reactswith ring-infected erythrocytes

Mar08

Perspectives

• A NEW var GENE HAVING UNIQUE ASPECTS?(non CD36, non CSA binding)

AND/OR

• A NEW HOST ENDOTHELIAL ADHESION RECEPTOR ?

OR

• A KNOWN PfEMP1 WHICH DISPLAYS A NON FUNCTIONAL CONFORMATION

• clag9 KNOCK-OUT

Mar08

Why PfEMP1 expressed by the ‘clag9neg’isolates are non functional ?

HYPOTHESIS

* wrong conformation * post-translational modification

Mar08