Protist, Vol. 152, 169–173, September 2001 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/protist

A Provocative Foreword (L.S.)

For historical reasons the study of the major parasitictrypanosomatids such as the Old World and NewWorld Leishmania and the African and South Ameri-can trypanosomes has always been somewhat insu-lar. This insularity was caused by the peculiarities ofthe diseases caused by these parasites, the specificvector biologies and epidemiology involved, and thegeographical localization of the diseases (which ofcourse determines consumer interest!). With the ad-vent of molecular analysis it has become clear thatthere are no major biological or molecular differ-ences between trypanosomatids and that cross fer-tilization between these fields is beneficial and evenimperative. I would argue for functional or problem-oriented meetings rather than genus-specific meet-ings. However when a field reaches a certain densityof researchers, a realistic compromise must befound between inclusiveness and exclusiveness,and it is beneficial to clinicians and field biologists tobe able to talk to biochemical and molecular typesand vice-versa. And of course there is that warm,fuzzy feeling of being surrounded by your peers whoare working on the same organism.

The Meeting (L.S.)

The first international symposium of “leishmaniacs”(copyright, I believe, is jointly held by Chuck Green-blatt and K.P. Chang!) was held in Istanbul in 1997and it was extremely successful, so that a secondmeeting was held in Crete, May 20–24, 2001. TheCrete Symposium meeting (had over 400 partici-pants, showing that there are a lot of people work-ing on various Leishmania parasites and diseasesthroughout the world. It was especially nice to seeso many scientists from countries where leishmani-asis is endemic or epidemic. Part of the problem ofLeishmanial insularity was solved by inviting peo-

ple to talk on results using other trypanosomatidssuch as Trypanosoma and Crithidia.

There were 8 general lectures on topics rangingfrom genomics, vaccine development, chemother-apy and evolution. Following the general lectures,there were three simultaneous sessions with shorttalks on cell and molecular biology, immunologyand epidemiology, chemotherapy and drug devel-opment, diagnosis, reservoirs, and sand fly vec-tors. There were also a large number of posters inafternoon sessions. From the initial “LeishmaniacManifesto” of K.P. Chang to the breaking of dishesduring the Greek dancing in the hotel Taverna, thiswas a extremely well organized and interestingmeeting in a fantastic locale. The meeting andaudio-visual facilities were also excellent, includingcomputer projectors for Powerpoint presentations.Thanks and appreciation should be given Ketty So-teriadou, Ziya Alkan and K.P. Chang for a great job!

I (L.S.) have created a web site (with permission ofthe Organizing Committee) with pdf files of the ab-stracts from this meeting.

The URL is: http://www.rna.ucla.edu/crete/index.htmlI have also taken the liberty of entering all the atten-

dees into the database of the Molecular ParasitologyNetwork at http://www.arc.ucla.edu/mpn/faculty.cfm.Each person can log on and enter and revise theirown information.

A Modest Proposal (L.S.)

An interesting poster (I. Bauer, James Cook Univer-sity, Australia) showed that few tourists to endemiccountries actually knew anything about Leishmania-sis, and this probably extends to most researchersother than true leishmaniacs, including members ofcompetitive grant review committees. This reviewersuggests that perhaps this lack of recognition is re-lated to the non-descriptive name of the disease,which most people can not spell or even pronounce.

Meeting Report: WorldLeish 2, Crete, Greece,May 20–24, 2001

Protist

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PROTIST NEWS

I don’t want to cast any aspersions on the good Dr.Leishman, but look for example at Sleeping Sick-ness, the common name for African trypanosomia-sis, or River Blindness, the common name for onco-cerciasis. A major problem of course is that leishma-niasis has multiple syndromes, depending on theparasite species and the host response. But I feelthat we desperately need a new name (or names) forour disease so we can better communicate withpeople outside our field! Trends in Parasitology maybe the venue for a competition to seek an appropri-ate and useful new name.

The Science

There was a lot of really good science at this meet-ing. One problem was that we were torn betweenthe interesting sessions that we would have at-tended, but could not because of the three concur-rent streams. This took away somewhat from thepurpose of the meeting which was to cross fertilizeall these fields and start a dialogue between the cel-lular, the molecular and vector biology folk. Howeverthere was plenty of time during the poster sessionsand meals to meet colleagues and discuss science.We cannot begin to cover all of the 257 different pre-sentations, but L.S. in addition to provocative pro-posals will attempt to present the highlights from hispoint of view, and E.H. will provide a summary of in-teresting vaccine-related developments and prob-lematic issues.

Genomics and Proteomics (L.S.)

The various genome sequencing projects havebrought a revolution to biology and this is also oc-curring in parasitology. The Leishmania genome se-quencing is progressing, (although not as fast asthe T. brucei and the Plasmodium sequencing pro-jects) and a T. cruzi project is just initiating. Theanalysis of several small chromosomes and randomgenomic fragments from Leishmania (Peter Mylerand Ken Stuart, Seattle Biomedical Research Insti-tute; Al Ivens, Sanger Center, UK) has already led tonew ideas on gene organization and regulation. Ap-proximately 24 Mb of the 33.5 Mb genome of L.major has already been sequenced. Fifty percent ofthis sequence is coding, with one gene approxi-mately every 3.7 kb. There is extensive synteny be-tween trypanosomatid species, which augurs wellfor cross species comparisons. Apparent largetranscriptional units, both divergent and convergentorganized were observed in several chromosomes.UV inactivation kinetics is being used to zero in on

the elusive Pol II promoter (P. Myler, SBRI) and theequally elusive centromere sequences (C. Blaineau,Lab Genome des Parasites, Monpellier). Preliminaryproteomic analyses were performed using two di-mensional gels and even mass spectrometry (K.Stuart, SBRI; P. Nugent and D. Smith, Imperial Col-lege, U.K.; and M. Ouellette, CNTR, Quebec). Un-fortunately the technical problems associated withperforming mass spectrometry identification of pro-teins from spots on 2D gels have not yet beensolved (M. Ouellette, CNTR), but some promisingresults have been obtained for differentiation of L.donovani (M. Thiel, Bernard Nocht Inst., Hamburg).And microarray technology is being applied toproblems of stage specific gene expression inLeishmania even before completion of the genomesequence (J. Blackwell, Welcome Trust Lab., Cam-bridge).

The Kinetoplast-Mitochondrion (L.S.)

An novel localization of a protein that binds to theorigin of replication of the kinetoplast DNA minicirclein Crithidia fasciculata (a model organism for Leish-mania!) was reported (J. Shlomai, Hebrew Univ.).This protein localized in a cell cycle-specific mannerto two regions on the flagellar side of the kDNA nu-cleoid body where minicircles are removed from thenetwork for replication. The kDNA replication storycontinues to amaze and perplex the viewer! Anothernovel finding in a Crithidia system was the demon-stration that a single nuclear gene for RNase H(RNH1) produces proteins targeted either to the nu-cleus or the mitochondrion, depending on whichAUG codon is used for translation initiation (D. Ray,UCLA).

The long standing problem of the existence of amitochondrial translation system and mitochondrialribosomes was solved (D. Maslov, UC Riverside).The problem appeared to be that the mitochondrialtranslation products are extremely hydrophobic andmigrate abnormally in gels. Protein synthesis wasalso demonstrated in isolated mitochondria.

A mitochondrial 3′ terminal uridylyl transferasewas isolated from Leishmania tarentolae and shownto be a member of the nucleotidyl transferase su-perfamily, and RNAi was used to show that this en-zyme is involved in U-insertion RNA editing (L.Simpson, UCLA). Several proteins were identifiedby mass spectrometry analysis from a purifiedmacromolecular complex from T. brucei competentfor in vitro editing and the genes cloned (K. Stuart,SBRI). Two of these were the RNA ligases involvedin editing and others were of as yet unidentifiedfunction.

170 L. Simpson and E. Handman

Regulation of gene expression during theparasite life cycle (E.H.)

An important observation in Leishmania researchhas been the differential expression of many func-tionally important proteins and glycolipids duringthe parasite differentiation from the promastigote tothe intracellular amastigote. The question ad-dressed at this meeting by several investigators hasbeen the regulation of this developmental gene ex-pression. Is there a single mechanism which deter-mines expression in amastigotes? B. Papadopoulou(Centre de Recherches, Canada) has identified ashort 150 base pairs sequence present in 3′ UTRs ofseveral open reading frames which were specificallyexpressed in amastigotes. Moreover, this short se-quence could direct stage-specific expression of aluciferase reporter. The 150 bp sequences varied intheir degree of conservation among the variousgenes and some seem to be located at the 5′ ratherthan the 3′ end of the genes they regulate. This se-quence most likely works by regulating the stabilityof the RNAs. However, this is not the only mecha-nism regulating amastigote-specific expression be-cause not all amastigote-specific genes contain thissequence. D. Smith (Imperial College, UK) hasmapped the 3′ UTRs of several genes which are dif-ferentially expressed during the promastigote differ-entiation from the less infective procyclic form to theinfective metacyclic form and found that the meta-cyclic-specific genes have a conserved 286 bp 3′UTR and precise polyadenylation sites. As with theamastigote-specific mRNAs, it is at the level ofmRNA stability that the regulation of gene expres-sion occurs. It would be of interest to know if the150 bp sequence described by Papadopoulou’sgroup is contained within the 286 nucleotides ofSmith’s group. Similar messages about the impor-tance of the 3′ UTRs for regulation of gene expres-sion come from studies on HSP83 and HSP100 ex-pression (M. Shapira (University of Beer Sheva, Is-rael) and J. Clos (Bernard Nocht Institute, Germany),as well as cysteine proteases (J. Mottram and G.Coombs, University of Glasgow, U.K).

On a more general and practical note, studies byMensa-Wilmot’s group (University of Georgia, USA)have examined the role of sequences in the 5′ UTRsof genes in the regulation of the level of protein ex-pression. Using an interesting approach of syntheticUTRs containing motifs from the Shine-Dalganobox, the E. coli lacZ spacer, and nucleotides preced-ing the translation initiation site, they have gener-ated a series of sequences, which confer dramati-cally different degrees of expression of a reportergene. The productive sequences termed transla-

tional enhancers have been engineered into ex-pression vectors. Moreover, such translational en-hancers have been identified in some highly ex-pressed Leishmania genes.

Evolution of Leishmania and otherTrypanosomatids (L.S.)

Someone once told me that evolution is somethingyou do when you get old. But Fred Opperdoes (Re-search Unit for Tropical Diseases, Brussels), who iscertainly still in his prime, presented a very interest-ing hypothesis on the evolution of kinetoplastid pro-tists and actually backed it up with some new factsand some old facts that had not fit into the paradigmpreviously. He found that several enzymes for car-bohydrate metabolism tree with chloroplast andcyanobacterial enzymes. As was recently suggestedindependently by Krepinsky et al. (Eur. J. Biochem.268, 2678, 2001) and also P. Beech (Protist 151,299–305, 2000), the ancestor of Euglenozoa protistsmay have had a plastid organelle which became achloroplast in the Euglenoids and was lost in thekinetoplastids after the genes from the plastidmoved to the nucleus. One problem in this hypothe-sis is that the plastid proteins would have to havesomehow acquired a signal sequence for the perox-isome-like organelle, the glycosome. Also theknown occurrence of rampant lateral gene transferin evolution raises a flag of caution. This hypothesishowever makes many seemingly aberrant facts un-derstandable, such as the plant-like alternative oxi-dase (G. Hill, Meharry Medical School) present inblood stream African trypanosomes.

Chemotherapy (E.H.)There were two informative lectures on treatment ofvisceral and cutaneous Leishmaniasis (H.W. Murray,N.Y. Presbyterian Hospital; J.D. Berman, WalterReed Army Institute of Medicine). Multiple agentsare now available for the treatment of visceral dis-ease other than the old standard, pentavalent anti-mony. These include different formulations of am-photericin B, aminosidine and oral miltefosine. Im-munochemotherapeutic approaches are also in clin-ical trial phases. However, we were somewhat crest-fallen by the review of the current state of treatmentfor cutaneous leishmaniasis and the prospects forthe future. It was stressed that the cure rate must begreater than a placebo cure rate in controlled stud-ies for a new drug to be considered effective. Andtherein lies the problem in a spectrum of diseases,some of which cure naturally and some of which

Meeting Report: WorldLeish 2 171

may not cure. So, it is important to first determine theparasite type, emphasizing the importance of classi-fication of the Leishmania species by isoenzyme ormolecular methods. Old World disease caused byL. major and L. tropica and New World diseasecaused by L. mexicana usually show natural cure.Disease caused by L. brasiliensis complex cells(causing disease which may reactivate even yearsafter initial cure) species is still treated by pentava-lent antimony. There was a comprehensive review ofthe current drugs including those developed on a ra-tional basis such as allopurinol, and these mostly donot work. However, there is a suggestion that oralmiltefosine may be effective. We did not go awayfeeling extremely confident about the future prospectsof drug development for cutaneous disease.

Issues related to the development of aLeishmania vaccine (E.H.)

Several presentations dealt with the results fromclinical vaccine trials and the consensus seems tobe that the killed vaccine has not been as successfulas hoped when tested rigorously in the field. Themajor question then is where should we go fromhere ? Having invested in this model, should we justsimply ditch it and move on to new approaches? Ifso, maybe it is time to address the fundamentalquestions below, which have been flagged in sev-eral presentations.

How to select vaccine candidates?Some vaccine candidates selected with human ormouse immune serum have been successful in themouse model, but others have not worked out. Thisapproach seems to have been useful for identifica-tion of protective secreted antigens. Reed (Corixa)indicated that selection of antigens based on reac-tivity of T cell lines turned out not to be useful in theLeishmania system.

One example of a failed antigen was presented byChenik et al. (Inst. Pasteur of Tunis). This was a20 kDa antigen from L. infantum (conserved inL. major) which induced excellent Th1 responses invitro in human monocytes, yet did not protectagainst infection with L. major in mice. Vaccinationwas done in BALB/c mice using a recombinant BCGexpressing the antigen and it seemed to induce anexclusive Th1 response. Is the problem with theantigen, the adjuvant or the delivery or the use of aninappropriate animal model? Is it the large parasitedose used for infection?

On a more positive note, data presented by sev-eral members of the Reed group (Corixa) indicatedthat a multisubunit vaccine including the stress-

inducible LmSTI1 protein, LeIF and the thiol specificantioxidant peroxidoxin induced protection in themouse model when administered as a polyprotein(Leish-111f) and in mice and monkeys, when admin-istered as a polygenic plasmid DNA. Interestingly, amajor contribution of LeIF to the vaccine seemed tobe an increased production of IFN-γ.

Sacks (Lab. of Parasitic Diseases, NIH) also pre-sented a success story demonstrating that DNAvaccination with three antigens LACK, MAPS andM15 was far more potent than vaccination with theprotein antigens with adjuvant, both in terms of re-ducing pathology and in terms of eliminating theparasites from the skin and thus preventing furthertransmission. The success of the DNA vaccinationmay depend on the induction of CD8+ T cells.

Is the mouse a good model for testing humanvaccines? Which mouse model?Sacks advocated a mouse model which combinesseveral features of natural transmission: a very lowdose of metacyclic parasites injected intradermallyin the ear. The BALB/c mouse is probably bestsuited to assess the pathology of leishmaniasiswhich is induced by a combination of host immuneresponses. For example, Sacks demonstrated thatCD8+ T cells are required for the formation of the le-sion and b-microglobulin null mice which lack CD8 Tcells display no pathology despite the presence oflarge numbers of parasites in the skin. Moreover, thepersistence of parasites in the clinically cured im-mune individual appeared to be supported by thepresence of IL-10; mice lacking IL-10 or micetreated with neutralizing antibodies to the IL-10 re-ceptor eliminated all parasites. Studies presentedby Farrell and colleagues (Univ. of Pennsylvania) in-dicated a similar role for IL-10 in susceptibility to vis-ceral leishmanias.

What parasite to use for challenge infection?Discussions in the corridors indicated that muchwork has focused on the host, but not enoughthought has been given to the parasite. Isolation ofparasites and in vitro culture selects for organismswhich grow well under these conditions. Selection ofmetacyclic parasites using lectins in the case of L.major, and antibodies for L. mexicana introduces an-other conundrum. A most interesting presentation in-dicating the importance of the quality of the chal-lenge infection was given by Rogers and Bates (Liv-erpool School of Tropical Medicine) who showedthat parasite-derived glycoconjugates in the sandflysaliva introduced with the parasites increases theirvirulence and promotes lesion development. How-ever, saliva itself had only a marginal role in virulence.

172 L. Simpson and E. Handman

What about long-term memory induced by vaccination and parasite persistence in the immune individual?Unfortunately, as demonstrated by Sacks and col-leagues, once the persistent parasites were elimi-nated, immunity waned. Although often postulated,this is the first demonstration, to my knowledge, forthe need of constant, “trickle” antigen presence forthe maintenance of long-term T cell memory toLeishmania. A major question for future vaccine de-velopment is how should long-term T cell memorybe induced? How should it be maintained? Wouldfrequent infectious bites in the field be sufficient forboosting immunity? Should the vaccine aim to pre-vent infection, to eliminate the parasites or to pre-vent host pathology?

Summary

This meeting showed once again that the study ofLeishmania parasites and parasite-host/vector inter-actions at the molecular, biochemical, cellular, im-munological, epidemiological and clinical levels isalive and well. There will be another Worldleish Sym-posium in 2005 but the location has not yet been de-cided.

What are we to expect from the next WorldleishIII? Research on microbial pathogenesis and im-munology is changing rapidly and fundamentally,from the characterization of individual host or para-site factors to a comprehensive analysis of host-pathogen interactions. In our field, this will becomepossible with the completion of the Leishmaniagenome sequence and the availability of the human

and mouse genomes. We will be able to obtain aglobal picture of gene content, expression and regu-lation thus allowing us to focus detailed studies onrelevant aspects. We will be able to elucidate the or-ganization and dynamics of the metabolic, signallingand regulatory networks through which parasitesestablish infection and subvert the function of thehost immune system. The power of this approachderives from a combination of genomics and pro-teomics and we have already seen the beginnings ofthese approaches this year from Blackwell, Nugentand Smith, Ouellette, Stuart and Forget.

A missing area has been structural biology. How-ever, a combination of rapid advances in X-ray crys-tallographic techniques in the last few years and theavailability of large amounts of sequence informa-tion has resulted in new thinking in the field, namely“structural genomics”. No doubt we will see more ofthis next time around.

So, the message for leishmaniacs is that there is alot to do and much fun to be had doing it!

Larry Simpsona,1 and Emanuela Handmanb

aHHMI-UCLA, 6780 Mac Donald Research Laboratories, 675 Charles E. Young Dr. S.,

Los Angeles, CA 90095-1662, USAbDivision of Infection and Immunity,

The Walter and Eliza Hall Instituteof Medical Research,

Parkville, Victoria 3050, Australia

1Corresponding author;fax 1 310 206 8967

e-mail simpson@hhmi.ucla.edu

Meeting Report: WorldLeish 2 173