Meeting Report: Molecular Parasitology, Woods Hole, USA 24–28 September, 1997

Protist, Vol. 149, 7-10, February 1998 © Gustav Fischer Verlag

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Meeting Report: Molecular Parasitology, Woods Hole, USA 24-28 September, 1997 For those interested in molecular parasitology the "Molecular Parasitology Meeting" in Woods Hole is one of the highlights of the year. In recent meetings the first successful transfection studies in Plasmodium and Toxoplasma were presented to eager audiences, as was the possible role of var genes in antigenic variation in Plasmodium fa/ciparum. This year's meeting was not marked by a 'great' new discovery but was more along the lines of consolidation and detailed analysis. The trend towards more studies on apicomplexans at the expense of the kinetoplastids continued, resulting in a well balanced meeting. This report does not attempt to give a detailed summary of all the results presented, but offers a glimpse of things we felt would be of wide interest. Some progress has been made in understanding the way the var gene family of malaria is transcriptionally controlled. Work reported by Kirk Deitsch (Bethesda, USA) clearly indicated that upstream promoter sequences are not sufficient to control proper var gene expression, but have to be in the right chromosomal context. The cloning of a number of different PfEMP1 genes that could be linked to different adherence properties was reported. Quijun Chen (Stockholm, Sweden) from Mats Wahlgren's laboratory reported the cloning of an unique var gene associated with rosetting, while Rosoura Hernandez-Rivas (Cinvestav, Mexico) in collaboration with Artur Scherfs group (Paris, France) cloned a var gene associated with the binding of chondroitin sulphate A (CSA). John Reeder from Alan Cowman's laboratory (Victoria, Australia) also presented some elegant work leading to the identification of a var gene associated with CSA binding. Interestingly, this gene is different from the one identified by Scherf's group. There has been considerable interest in understanding how the malaria parasite binds to its host cell. Proteins previously identified as being involved are characterised by a unique cysteine rich domain,

known as the duffy binding-like (dbl) domain. David Peterson (Georgia, USA) reported the existence of a superfamily of genes containing the dbl domain, one member of which (Ebl-1) was linked to a rapid proliferation phenotype. Following a similar approach, Stefan Kappe (Notre Dame, USA) used sequence similarities to the carboxy-terminal cysteine rich region of erythrocyte binding proteins, to identify a whole new family of such proteins in rodent malaria. More light has been shed into the mechanisms by which Variant Surface Glycoprotein (VSG) gene transcription is controlled in trypanosomes. Miguel Navarro (New York, USA) showed that a T7 promoter integrated into a VSG expression site (ES) is only subjected to a VSG-type transcription control in the presence of a VSG core promoter element. This suggests that the core promoter element may playa role in chromosome structure, controlling accessibility to trans-acting factors. Along the same line, work presented by Luc Vanhamme (Brussels, Belgium) showed that a protein complex that binds to a region of the VSG core promoter also specifically interacts with single-stranded telomeric and subtelomeric repeats. He postulates that this protein complex may playa role in the regulation of VSG expression and acts from its subtelomeric location. Progress was also made in addressing the question why only one VSG ES is active at any given time. Gloria Rudenko (Amsterdam, The Netherlands) clearly demonstrated that activation of more than one ES at the same time inhibits parasite growth. Under these circumstances neither ES exhibits maximal activity, moreover, there seems to be a preferential deletion of the last active ES during switching. Since VSG genes are thought to be transcribed by RNA polymerase I, the question of whether VSG genes are transcribed in the nucleolus like rRNA genes, was addressed by Ines Chaves (Amsterdam, The Netherlands). Dual fluorescence, in situ hybridization (FISH) showed this was not the case.

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M. J. Blackman, P. K. Patnaik, and P. R. Preiser

Apicomplexan parasites like Plasmodium and Toxoplasma have two DNA containing organelles, a mitochondrion and a plastid. Both organelles are potential targets for drugs. The antibiotic thiostrepton inhibits eubacterial protein synthesis and has been shown to have antimalarial activity. Using temperature-dependent hyperchromicity profiles, John Rogers (Bethesda, USA) demonstrated that thiostrepton directly interacts with the GTPase center of the large ribosomal RNA encoded by the plastid, but not those encoded by the nucleus, suggesting that the plastid is the primary target for the drug. Maria Fischera (Philadelphia, USA) reported that in Toxoplasma, clindamycin, chloramphenicol and ciprofloxacin cause the parasite to exhibit a "delayed death" phenotype, where the effect of the drug is observed after one replication cycle. This effect associates with the selective inhibition of plastid DNA replication. Don Williamson (London, UK) presented data on the mechanism of plastid DNA replication in P. falciparum obtained by 2D neutral/neutral gel electrophoresis. The replication intermediates observed by this technique are consistent with the interpretation that two different mechanisms are used. Initially, replication initiates via a D-Ioop that then converts to a rolling circle. The basis by which malaria parasites acquire resistance to chloroquine generated a lively set of presentations. A recent report from Michael Lanzer's laboratory (Sanchez et al. J. BioI. Chem. 272: pp 2652-2658, 1997) suggests that in parasitized erythrocytes, chloroquine uptake is saturable, temperature-dependent and inhibitable by amiloride derivatives, indicative of carrier-mediated transport which has features of a Na+/W exchanger. Resistant parasites showed diminished uptake. Stefan Wunsch from the same laboratory (Wurzburg, Germany) used a pH and Na+ sensitive dye to measure intracellular pH in chloroquine-sensitive and resistant strains of P. falciparum. Resistant strains consistently showed a higher intracellular pH, and this was offered as supporting evidence for the diminished activity of the proposed Na+/W exchanger. Andrew Slater (New York, USA), on the other hand, reported data that showed chloroquine import to be neither saturable nor inhibitable by amiloride drugs in the presence of bicarbonate. This group suggested that under physiological conditions (presence of bicarbonate) chloroquine (a weak base) enters parasitized cells down an electrochemical gradient using the CI-/HC03- antiport. DIDS, a known inhibitor of this antiport, and Nigericin, which abolishes the electrochemical gradient, were both shown to block chloroquine import. It was proposed that chloroquine resistance is a result of mutation(s) that alter

ion conductances that indirectly control drug entry into the parasites's cytoplasm. Finally, Xin-zhuan Su from Tom Wellems's laboratory (Bethesda, USA) reported on efforts to directly sequence the locus responsible for conferring chloroquine resistance. This locus has been narrowed to a 36 kb region on chromosome 7 as a result of a long-standing effort to analyse the progeny of a genetic cross between a chloroquine-sensitive and resistant strain of the parasite. The sequence of this region includes a large open reading frame (8.5 kb) that is expected to code for a protein of 320 kD. The candidate gene (CG2) shows differences between sensitive and resistant strains and is predicted to have 6 membrane spanning domains. Antibodies to synthetic peptides corresponding to parts of this protein localize it to the food vacuole. Replacement of the presumptive sensitive allele with the resistant version should provide confirmatory evidence if this is indeed the gene responsible for chloroquine resistance. However, such a test has so far been precluded by the large size of this gene and consequent problems faced in obtaining an unrearranged recombinant construct - a problem all too common when dealing with the A-Trich DNA of Plasmodium. None of these problems plague work with trypanosmatids, and a successful general approach pursued by Steve Beverley (St. Louis, USA), Scott Landfear (Portland, USA), Salvatore Turco (Lexington, USA), and Buddy Ullman (Portland, USA) laboratories (often in collaboration), has been to use a large cosmid library of genomic DNA from Leishmania, to functionally complement defined mutant phenotypes. Leishmania cannot synthesize purines de novo and salvage these from their hosts using cell surface transporters. Chemical mutagenesis followed by selection with toxic purine analogs, were used to obtain parasites deficient in nucleoside transport. Functional complementation of these mutants has led to the identification of the L. donovani adenosine-pyrimidine and the inosine-guanosine transporters. Further studies will be required to evaluate if these molecules can serve as potential targets for chemotherapy. A similar approach has led to the identification of a gene-family involved in the transmission of L. major by sandflies. Differentiation of procyclic promastigotes to infectious metacyclics is accompanied by a remodelling of the surface lipophosphoglycan. During this remodelling, terminal galactose residues are capped by arabinose and this permits detachment and release of parasites from the sandfly midgut. By transfecting a wild-type L. major cosmid library into a variant that cannot do this remodelling, Deborah Dobson (St. Louis, USA) has ob-

Meeting Report: Molecular Parasitology, Woods Hole, USA 24-28 September, 1997

tained a glycosyl transferase which plays a crucial role in differentiation. Chi-Wu Chiang (Alabama, USA) in collaboration with Buddy Ullman and David Roos (Philadelphia, USA) laboratories has used insertional mutagenesis and toxic purine analogs to obtain an adenosine transport mutant in Toxoplasma gondii. The position of the insertion is thought to mark the candidate transporter gene, considerably simplifying its identification and analysis. This approach is feasible in Toxoplasma because under certain conditions DNA transfected into this organism inserts into the genome via non-homologous recombination - i.e. at random locations. DNA transfected into trypanosomatids on the other hand always integrates via homologous recombination, so an entirely different approach has to be tried to obtain random insertional mutagenesis in these organisms. Steve Beverley's laboratory has recently reported (Gueiros-Filho and Beverley, Science 276: pp 1716-1719, 1997) the successful use of a mariner transposon for this purpose in Leishmania. A similar approach is being tried in the laboratory of George Cross (New York, USA) for Trypanosoma brucei, but results reported at this meeting show the work is still undergoing teething problems. The "New Tools" session introduced some exciting new developments in the area of control of heterologous gene expression in protozoa. Workers from two laboratories described parallel approaches to developing transfection systems for the binucleate protozoan pathogen Giardia lamblia. Steven Singer (Bethesda, USA) reported that stable genomic integration of a puromycin resistance gene could be achieved by homologous recombination, but a minimal vector was maintained episomally under drug pressure. Both Singer and Chin-Hung Sun (Taipei, Taiwan) described expression of luciferase in Giardia transfectants from an episomallymaintained plasmid. In a system which is much more advanced, Elizabeth Wirtz from George Cross's laboratory reported on the co-ordinated use of a constitutively-expressed selectable marker under T7 promoter control, in concert with test gene expression under the control of a tetracycline-responsive PARP promoter. She was able to obtain tightly-regulated, inducible expression in T. brucei bloodstream forms. This will allow studies on the effects of expression of even extremely toxic gene products in trypanosomes. Transfection in Plasmodium is nowhere near as advanced as in kinetoplastids, or even in the related apicomplexan T. gondii, and since the first successes reported in 1995, progress has been difficult. Brendan Crabb (Victoria,

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Australia) described a number of new, compact vectors which allow stable, constitutive expression in P. falciparum of both a selectable marker and a transgene of interest. An important characteristic of these vectors is the head to head orientation of the respective promoters, resulting in enhanced expression of both transgene and drug resistance gene. In the case of vector pHC1, some chromosomal integration can occur and this vector has been used to obtain stable expression of the P. chabaudi AMA-1 gene in P. falciparum. These are exciting developments; for malariologists to feel on an equal footing with other parasitologists, all we need now is an inducible expression system! One potential use of inducible transgene expression in parasites is controlled expression of antisense RNAs. This technology could be of immense value in the exploration of endogenous gene function, particularly in investigations of multigene families where classical gene knockout approaches may be impractical or inappropriate. We-Wei Zhang (Montreal, Canada) described the use of antisense RNA-mediated blockade of expression of the amastigote-specific A2 gene family in L. donovani. Remarkably, greater than a 90% downregulation of A2 gene expression was obtained in stable transfectants, and although no striking phenotypic effect of A2 blockade was detectable in vitro, amastigote growth in vivo was severely retarded, suggesting that A2 gene products playa role in virulence. Egress (release) of invasive forms of apicomplexan parasites from the host cell is a fascinating but neglected area of research; for example, virtually nothing is known about the process in Plasmodium. However, in T. gondii egress can be induced using the calcium ionophore A23187, and Michael Black from John Boothroyd's laboratory (Palo Alto, USA) described attempts to identify parasite genes involved in this process by genetic complementation of ionophore-resistant mutants using a vector which is maintained stably in an episomal form in the parasite. This promising approach has yet to yield clear results. In the area of elucidating the molecular triggers involved in the cascade of events required for the opposite process i.e. host cell invasion, Vern Carruthers from David Sibley's laboratory (St. Louis, USA) presented compelling evidence that attachment of T. gondii tachyzoites to host cells - the primary step in committed invasion - requires the release of micronemal contents. Microneme discharge was found to be temperature dependent, was refractory to repeated stimulation, and could be blocked by chelation of intracellular calcium. Much remains to be determined- ego which micronemal

10M. J. Blackman, P. K. Patnaik, and P. R. Preiser proteins act as the primary adhesins in attachment?; how do these molecules relocalise to the parasite surface, since they presumably function in a membrane-bound form?; which host cell components are the receptors for these adhesive interactions? In a different but related vein, Mike Blackman (London, UK) presented data on the identification and posttranslational processing of a P. falciparum merozoite serine proteinase. This enzyme, which belongs to the subtilisin-like proteinase superfamily, localises to a subset of dense granules in the apical domain of merozoites, and appears to be released in a soluble form at host cell invasion. The enzyme's function is unknown, but evidence was presented that it could mediate the proteolytic processing of mero-

zoite surface protein-1 at invasion. Our current understanding of how an apicomplexan parasite responds at the molecular level to its initial interaction with a target host cell is undoubtedly poor, but can only get better! It was a fascinating meeting!

M. J. Blackman, P. K. Patnaik\ and P. R. Preiser Division of Parasitology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW71AA, UK Corresponding author: fax 44-181 913 8593; e-mail ppatnainimr.mrc.ac.ak 1