News

Progress in Toxoplasrnosi~ Research J.E. Smith, J.C. Boothroyd, C. Hunter and E. Petersen

Drymen, UK July 1996

This biennial conference serves as a marker of progre.% in Toxoplasmosis research. The 1994 meeting, in down- town C!eveland, highlighted advances in understanding o r the structural and molecular organizat~cn of the para- site and advocated its suitability as a modPl system for studying the cellular biology of apicomplexan paras,tes. while the advent of feline and ovine vaccines boded well for immunological control of the diseasek The 1996 con- ference, in the contrasting surround- ings of rui-al Scotland, will be remem- bered as the point at which molecular genetic approaches came to the fore, and perhaps also for a shift away from immunological control and to- wards biochemical studies under- pinning chemotherapy.

New Molecular Tools

Several new and important mol- ecular tools were described. D. Roos (University of Pennsylvania, USA) re- ported that the choice and amount of S'-untranslated region could have a hundredfold impact on the efficiency of t~nsformation using different selectable markers. He also described the ex- ploitation of powerful forward and reverse selections possible using the gene encoding hypoxanthine xanthine g.~nine phosphoribosyl transferase (HXGPRT) to introduce and to knock- out genes, respectively 2. D. Soldati (Heidelberg University, Germany) de- scribed work towards a regulated sys- tem for gene expmssio n using the tet~ cycline operator-repressor system, and <uccess in using the cre--IoxPI sys- tem for efficient excision of a gene once imerted, M Black (Stanford Uni- versity, USA) described selection of an episomal vector containing a sequence of ~S00 bp from a genomic library that caused plasmids carrying it to be repli- cated in the parasite at an average level of one episome per parasite. Promoter- trap methodology has been used to clone bradyzoite-specific loci. and char- a~erization of the resulting genes is in progress (L. Knoll, Stanford University, USA).

In addition to an increased ability to manipulate genes, the identification and

Parositology Today. vol. 13, no 7. 1997

clonin8 of ger, cs fo' future pro;cots ~s enormously enhanced by the recent large-scale production and sequenc~n~ of expressed sequence tags (ESTs). The Toxopldsma database, contmn~ng over 100C0 tachyzorce ESTs, has been set up to include references, strain *n- formation and genetic map anfom~atlon (M. Aslett and J. Aji~ka. Cambndge U1,wersi~y, UK). Effe-ts are also under way to produce bradyzoite ESTs (j.C. Boothroyd, Stanford Un~vers*ty. USA). Many of the genes idenb~ed ~n the EST have no apparent Sdccharo- myces homologues, and thus might be important for the pathogenes~s of the parasite. Physical mappm~ of the genome is also under way, and S. Kahn (Cambndge University, UK) described the construction of a physical map for chromosome lb.

The molecular genet~: analyses of several important genes were pre- sented at the meeting. J. Schwartzman (Dartmouth-Hitchcock Medical Centre, USA) described an unusuaily small myosin-encoding gene as well as a pair of larger, affematwely spliced members of this complex gene family. A new addition to the family of rhoptry- encoding genes, ROP4, was found to be a homologue of ROP2 (M Foum,aux, INSERM, Lille, France). Work on the gone :amily encoding the abundant N]Pa~P~ ha~ u~ed chimeric genes to map the nucleotide-substrate-specificity domain of the different isoenzymes (K. Joiner. )aie University. USA).

Host Cell Invasion

Knockou* mutants for the oene en- coding the ~ufface protein SAG3 were shown to have reduced capacity for host cell invasion (S. Tomavo, In~itut Pasteur, Li!ie, France). This molecule has ~tn ictllrnl nv~Han with the maior tachv- zoite surface protein SAG I (Ref. 4), which is known to be involved in at- tachment. Although the identity of the hos~ cell receptors mediating a~ach- ment remains elusive, some insight was ~'iven by the observation that atta,:h- ment peaks during S phase of the host cell cycle (j. Grimwood, Dar~mouth- Hitchcock Medical Center, USA). Per- haps the rno~ ~nteresting results in this area were the simultaneous repo~s on the genes encoding the microneme proteins. MlCl, MIC3 (J-F. Dubremetz, the ER marker calnexin.

Cop. ,~h @ I,;97, Efs{%,~., S¢t,q<e { t o 2-; ' ~h t . . . . . . . . . ~ O~'~ " : ; ~8 i97S l700 S016g'173~497~01070 ]

INSERM, Lille, France) and M1C2 (j. Ajioka, Cambridge Unwers~ty, UK). These are ~mong the few proteins that show homology between api- compTexan oarasites. MICI is a homo- Iogue of the thrombospondin-related TRAP protein of Plasmodium. MIC2 also has thrombospondin homo!o~y. but is related to the EtDi00 protein of Eimena micronemes, ,vhile HIC3 has two epidermal-grovOth-factor-like do- mains, similar to MSPI of Plosmodium. The observations of D. Sibiey (Washington University, St Louis. USA) tha~ MIC2 is released locally adjacent to the moving junction argue that thi~ protein has a role in invasior~. Fina, y. studies of bradyzoite (J.E. Smith, Lee.~s University, UK) and sporozoite 5 (M. Whrte, Montana State University, USA) invasion su~=st Lhat there might be stage-specific differences. Certainly in the case of the sporozoite there is evidence that, following con- version to tachyzoites, they escape into a second vacuole.

After entry, the question arises as to how different secretory molecu!es ~re targeted within the pa,~sitophorous vacuole (PC). Exo~osis of o~anelles is separately regulated during entry. rhoptry release be:ng induced by dg,~Z junctie~ formation, while dense granule re!ease is sensitive ~o c'l~ochalasin D, and posterior release of the intra- phagosomal membrane (IPM) network to calcium (D. Sibley, In~titut Pasteur, Lille, France). The molecular basis of differential ta.~eting within the vacuole is being approached by epitope tag- ging 6. Recombfnant GRA2 and GP~&5 am ~tably integrated into the IPM and PV memb,-anes, while deletion mutants reveal the importance of hydrophobic transmembrane domains to this pro- cess (C. Mercier, D. Sibley, L Lecordier and M-F Ce~hrnn In~tih ~t Part.lit l ill~ France). It is interesting to note here that differences in GRA5 and GRA6 nobilities indicate differential post- translational modification in virulent (type I) and avirulent (type 2) strains (M-F. Ce~bron). Finally. K. Joiner re- po,-ted that the close associatio n b~.- tween the host endoplasmic reticulum (F-R) and the PV membrane was de- pendent on active invasion - fraction- ation of membranes from infected cells revealed co-localization of GRA3 and

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N e w s iii i i

The pas[ t w o ~ears have seen a m&:~ed M-rease in research on devel- opmenta~ b=.oio~y. Ir, tl insic rates of ~'rowth an~ b~dy'zoite production ;.. t [~t'weefi type I and type 2 strains. ~ q S t r a t i n ~ an e!ement Of ~n~.llC ~.o~tro~ of statq;e conven,,on (i.E. Smith). Howe~er. in ~ddi:,on, tachyzoite- L~:adyzo~te c~-,~e.,-:,,.~n rates are £~- s;tive to env:r,;r~t~e ntA! :righters such as pH. temperature, anti-mizochondrial C~',~S, n i t~ oxide" (U. Gross. Werzburg Lhive*~ity. Gent:any) and. most ~- cent}y, tumour P~rcs,s factor ~ (T,~4F-e; ,L R~card. CNRS. Grenoble. France). L Weiss (Albert Einstein Medical Ce~::e~: New York. USA) su~ested that inducible heat-shock protein 70 (hsp700 was involved ~n the heat-shock ,nd~.-tion of d;ffe~ntiation, gsin~ in- habiters and augmenters of hsp70i #d~:tlon. he showed a ton'elation beb~een leve!s of hsp70i and the effi- ciency of cy~ generation. However, ~.-.o¢i~o~-t strains for another hsp hom- o~'Ogue (~C-I ) . which is constitutively expr~.ssed in brad yzoite.-, showed no aiteGt~on in their abiley to differentiate ~,q wire (VV, Bohne, Pennsy,~ania Uni- ve~%ity, USA).

Ewdence is accumulating that brad~2oite.specific genes are regulated at the transcriptional level. S. Parmley (Pato Alto Medical Foundation, USA) found that repot'[or constructs carrying the putatwe pro noter sequences from the genes encodinoo tne tach,vzo~e (LDH i ) or brad/zoite specific (LDH2) isoforms of lactate dehydrogenase #-¢.3wed faithful developmental regu- lat in of expression. Similarly. M. Soete (INS~RM. Liile, France) found that c~hirr~c constructs of tachyzoite (SAG I) and ioradyzoite (SAG4) surface-antigen- e~:~oding ~nes gave faithful expression according to the promoter used.

Clinical pnorities in chemotherapy were clearly biased towards develnpin~ combined drug therapies for the treat- merit of AIDS patients. Although pyri- rnethamine sulphadiazine treatment ef- f ~ n t l y reduced tachyzor, e replication. atavafior~ne hzd promising ~'stogeP.iC e6.~'rs (F. Derouin. Hopita! St Louis, Paris. France). Interest stimulated by the sequencing and localization of the 35-kb ptastid-organeile (D. Roos) has led to the discovery of a novel tar- ~et arn{notransferase, unique to the or- ganeile, and to the operation of other metabolic pathways more typically

associated with plants (F. Roberts, Columbia Michael Reese Hospital, Chicago, USA). Screening of synthetic compounds against the purified Toxoplasm~l NTPase enzyme revealed a :r=ne inhibitor~ effe~ with quercetin (T. Asa~, Keio Uni~cr:,:,ty, lap,n), v,'hile the activity of cyclosponn-A-dedved compounds su~.~ested that a P-R.!yco- protein might be a potential target (K. Joiner). Other molecules of interest ,nclude HXGPRT (D. Roos}, cysteine proteinases (S. Reed, University of California San Diego Medical Center, San Diego, USA) m~d DNA polym- erase c~ CA. Johnson, University Tech- naiad,, Sydney, Australia), while basic studies on cell-cycle enzymes (K. Kim, Albert Einstein Medical Center, New York, USA) and on the mitochondrion (C. Tourset, INSERM, Lille, France) might reveal new targets.

Protective Immune Responses

In ~he area of immunology, the role of interteukM-12-induced (IL-I?.- induced) production of interferon "y (IFN-~/) in resistance to Toxoplasma was illustrated by studies in which mice deficient in either IL-12 or IFN-~, were shown to be susceptible to infection 9 CA. Sher, National Institutes of Health, Bethesda. USA). Sher also described studies showing that a lipid component of Toxoplasma can stimulate macro- phage production of IL-12, and that neutrophils from infected mice pro- duce IL-i2. Interestingly, male mice are more resistant to infection than are females, and this correlates with in- creased production of IL-12 following infection (VV. Walker, Strathclyde University, UK), While IL- 12, IFN-~/and TNF-c~ are associated with protective immune responses, their production by human peripheral blood monocytes stimulated with Toxoplasma results in the suppression of mitogen-induced lymphocyte proliferation (J. Channon, Dartmouth, USA).

Vaccine Oevelonment and Disease

Data from field trials using the Toxo- plasma $48 strain established the ira- put[once nf vaccination in preventing congenital transmission in sheep (A. Vermeulen, Intervet. The Nethedands). Other approaches to vaccine develop- ment were outlined in studies using cytokJnes as adjuvants in mudne models. IL-12 (E. Candolfi, Inst~ute of Parasitology, Strasbour~, France) or IL-15 (L Kasper, Dartmouth, USA), in

conjunction with parasite antigen preparations, enhancea development. of protective T-cell responses to Toxo- plasma. In particular, IL-I5 induced the development of a protective CD8+ T- cell response m. In contrast to the pro- te~ive -o!e of T cells, Y. Suzuki (Pale Alto, USA) derriere!rated that ~ + , CD4* T cell'_, were responsible both for the d-:'elnpment or large areaS of necrosis in the small intestine and for the death of C57BIJ6 mice after per- orai infection. R. Saavedra (University National Autonoma, Mexico) and I. Prigione (G. Gaslini Institute, Geneva, Italy) showed that the ROP2 (Ft Sr~avedra) and GP, A2 (I. Prioione) antigens were recognized by CD4 + T- eel! dnnes from seropositive individu- als, while mice immunized with confor- mationally appropriate SAG I were partially pro~ected from virulent chal- lenge (E. Pete, sen, Statens Seruminsti- tut, Copenhagen, Denmark).

Factors. that predispose to the de- velopment of neurological toxoplas- mosis were explored in clinical studies and mudne models. As patients sere- positive for Toxoplasma developed AIDS or AIDS-associated toxoplasmic encephalitis, their ability to produce IFN-~t or IL-12 in response to parasite preparations was greatly impaired (R. Gazzinelli, UFMG, Belo Horizonte, MG, Brazil), Further studies using HIV- transgenic mice suggested that oppor- tunistic pathogens, such as Toxoplasma, can accelerate the progression of HIV infection. In infants with severe congeni- tal toxoplasmosis, the frequency of the human leukocyte antigen class II allele DQ3 was increased, although the fre- quency of this allele in the mothers did not differ from that in the nom~al population (D. Mack, Michael Reese, Chicago, USA). Parallel studies revealed "that major histocompatibility complex (MHC) class II knockout mice were more susceptible to toxoplasmic en- cephalitis, but that MHC class II genes from humans could reconstitute the resistance. The importance of IL-6 in re- s;~ance to to×op!asmic encepha!~is w~_s emphasized by results from IL-6 knock- out mice (J. Alexandeq Strathclyde University, UK), as wel! as data which showed that astrocytes pl educe IL-6 in response to infection (H-G, Fischer, Dusseldorf Universrty, Germany).

Congenital screening revealed higher matemal-foe[al transmission rates in France (M-H. Bessieres, Paul Sabatier University, Toulouse) than in Denmark (M. Lebech, Statens Seruminstitut, Copenhagen. Denmark). A new im- munoglobulin G avidity test was round

Perdsitology Today, vol. 13, no. 7, !997

News

to be a useful indicator of early human thank the Commission o~ the European (A. Hassl, Clinical Institute of Hygiene, Union BIOMED 2 Programme contract Vienna, Austria) and feline infecticn no. BMHI-CT92-1572. b~oMeneux. Inten~et (K. Simon, Tier~rzliche Flochschule. and Cambridge Bio:c~ence for 3uppo,i Hannover, Germany). However, the importance of strain variation in disease References pathology and epidemioiogy t,as /eL LO i 5chwartzman. Jr) Bc.c~h,oy~. jC and

Kasper. L.H (1994) ] Euk ~4,crobol ,H. be addressed, despite the availability of !9S 215 genetic markers (M-L. Darde, Centre Ho~pitaiien Limoges, France).

Acknow!edgements The 4th International Biennial Toxoplasma Conference was held 22 26 July 1996 in Dr/men, UK. The meeting was organized by Prof. J, Aley~nder of the University of Strathclyde, UK, and was a~tended b 7 over 80 delegates. The onganizer and delegates

2 Dor,~d. FLG.K. ~t al (1996)j 8~ol Chem 2T; 14010 ;40~9

3 VVan. KL. Bi;iri'weli. jpI and A,ok2. J,,/

179-186 4 Cesbron Delauw. M.F ct d (1994) ] 6~ol

Chem 269. 16217-16222 5 Speer, C.A. e~ e. (1995) Mol B~orhem Pdm

s~t0L 75.75 86 & Mercier C.. Ce~bm~ Dc!auw bl.~ and

S~bley. L.D. (1996) Mot B,ol Cell 7. I S ] 3

7 Gross. U et al ([996) Paras~tol. r0day 12. 30 33

8 Yang, S.M. and Parmley. S.F. (1997) Gene I84, 1~12

9 Scha~col,kersten, T.M. et ~1 (1996)]. Irnmuno~ 57, 404S~4054

I0 Khan, IA. and Kasper. UH. (19Ve~ ]. Immund. 157. 2103--2i08

Judith E. Smith is at the Depar~ment 0f ~ielogy, L~cds Un~venJ~y, UK LS2 9JT. john C Boothroyd is at the Department of Micro- bioiogv and immunology. Stanford University. CA 94305-5402, USA. Chris Hunter ts at the School of Vetennary Medicine. University of Pennsylvania, Philadelphia, PA 19104-6008, USA. Esklld Petersen is at ~re S~ate~s S-_:um ~ns~itu~, G,pcnh~;gen. Denrr a~ Te~. +44 1132 332892, Fax: +44 1132 ~32882, e-mail: pobies@lucs-mac novell.leed~ ac uk

C o m m e n t

I • Trypanosomes with Mu.tlcoloured Coats

Individual Trypanosoma brucei parasites in the mammalian host express only one variable antigen type (VAr'D~ at a time. Why should this be? VVe believe we know why tlypanosomes express variable antigens, as opposed to not expressing them at all, and we under- stand many aspects of how they ex- press them and how they switch from expression of one to that of another in the process of antigenic variation, but a full explanation as to why they express them singly on individual cells as yet eludes us.

Several other pathogens also under- go antigenic vali~tion, and appear to ensure that expression of different vari- able antigens is also mutually exclusive. Thus, although systems of antigenic vari- ation are mechanistically very different in, for instance, Neisser/o g0n0rrh0eae, Borrelia hem~sii, Plasmodium folciporum and T~,panosoma bruce/, phenomeno- logical similarities i suggest that answer- in L the question p.~ed ~* tho ~ ,+~ t should interest a wide audience.

Some background on variable anti- gen expression in trypanosomes is re- quired. Each parasite cell is entirely coated in a monolayer of a single mol- ecular species of glycoprotein, the variant surface glycoprotein (VSG), which serves to protect that cell from non-specific killing mechanisms of the host, such as activation of complement via the alternative pathway. VSGs, ho..vever, stimulate adaptive immune responses, and they appear to be the

Parasitology Today, vol. 13. no. 7, 1997

C , M , R . T u r n e r

only antigens of the cell against which effective killing responses can be gener- ated. Trypanosomes escape f~om these consequences of speofic immunity by undergoing antigenic variation. Each VSG is transcribed from a separate gene of which there are, very roughly, I000 in the genome, VSG transcription occurs in expression sites, of which there are about 20, but only one of these is active at any one ume. In a dividing population ef cells, trypano- somes switch expression using a vadet;, of mechanisms. The result is that each cell individually expresses only one VSG, but the population of parasites tha~ constr(ute an infection can express a vast anay of different VSGs. A direct experiment has shown that the num- ber of VSGs that c~n ~e ~xpresced !: > !01 , and an indirect approach has suggested the number of VSG genes available for expression is roughly 1000 (Re[ 2).

(in T. brucei infections, there is a rough, but direct, tonelation between parasffe load and degree of pathology) 4. Clearly, by utilizing only one VAT at a time, tryp- anosomes can maximize the potentia: for transmission, given a VAT reper- toire of finite size and a mechanism fo- selecting singularity of expression as a heritable trait. The best evidence that VAT repertoires are of a finite size comes from observation of self-cure of trypanosome infections in large mammals s,6.

Whether singularity, of expression is a selectable trait is a more contentious issue, An early study 7 exploited a cloned line of trypanosomes that spon- taneously arose in in vitro culture and which simultaneously expressed two VATs. While co-expression of the two 'VATs was stable in wtr0, :t was unstable in vivo: parasites inoculated into mice rapidly moved to expression of only one VAT. These data suggested se!ec-

T-~kin~ ~ cim~lo ~ro!ogir~.! view of tlon for singularity of expression, bc¢a an infection does suggest why singular- more recent and extensive study 8 indi- ity of expression is important. Trypano- somes undergo antigenic variation to promote transmission from marnmal to tsetse, by prolonging the duration of infection within density-determined boundaries. ] he lower boundary is ap- proximately, one tsetse-infective tryp- anosome per 20 I~I of blood (20 I~I is the approximate volume of blood in a tsetse bloodmeal) 3 and the upper

cares no selective advantage. In this work from George Cross' group, a. VSG expression site was genetically engineered to express two VSG genes simultaneously. For each of three cloned lines expressing a different pair c,'VATs the re_sults were essentially the same, VSGs were produced in similar amounts and evenly distributed ore, the cell surface as homodimer~ The

boundary is determined by the density- double-expressor cell lines were as in- dependent generation of pathology fective to rats and had similar in vJvo

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