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Reviews 36 Franco, A. et al. (1988) Expression of class I and class II major histocompatibility complex antigens on human hepatocytes. Hepatology 8, 449–454 37 Thursz, M.R. et al. (1995) Association of hepatitis B surface antigen carriage with severe malaria in Gambian children. Nat. Med. 1, 374–375 38 Ikeda, T. et al. (1986) Evidence for a deficiency of interferon production in patients with chronic hepatitis B virus infection acquired in adult life. Hepatology 6, 962–965 39 Foster, G.R. et al. (1993) Expression of the terminal protein of hepatitis B virus is associated with failure to respond to interferon therapy. Hepatology 17, 757–762 40 Kullberg, M. C. et al. (1992) Infection with Schistosoma mansoni alters Th1/Th2 cytokine responses to a non-parasite antigen. J. Immunol. 148, 3264–3270 41 Actor, J.K. et al. (1993) Helminth infection results in decreased virus-specific CD81 cytotoxic T-cell and Th1 cytokine responses as well as delayed virus clearance. Proc. Natl. Acad. Sci. U. S. A. 90, 948–952 42 Lowin, B. et al. (1994) Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathways. Nature 370, 650–652 43 Esser, M.T. et al. (1996) Distinct T cell receptor signaling requirements for perforin- or FasL-mediated cytotoxicity. J. Exp. Med. 183, 1697–1706 44 Denkers, E.Y. et al. (1997) Perforin-mediated cytolysis plays a limited role in host resistance to Toxoplasma godii. J. Immunol. 159, 1903–1908 45 Lalvani, A. et al. (1997) Rapid effector function in CD81 memory T cells. J. Exp. Med. 186, 859–865 46 Nussler, A.K. et al. (1993) In vivo induction of the nitric oxide pathway in hepatocytes after injection with irradiated malaria sporozoites, malaria blood parasites or adjuvants. Eur. J. Immunol. 23, 882–887 47 Tsuji, M. et al. (1995) Development of antimalaria immunity in mice lacking IFN-gamma receptor. J. Immunol. 154, 5338–5344

48 Doolan, D.L. et al. (1996) Circumventing genetic restriction of protection against malaria with multigene DNA immunization: CD81 T cell-, interferon g-, and nitric oxide-dependent immunity. J. Exp. Med. 183, 1739–1746 49 Renggli, J. et al. (1997) Elimination of P. bergei liver stages is independent of Fas (CD95/Apo-1) or perforin mediated cytotoxicity. Parasite Immunol. 19, 145–148 50 Nardin, E.H. and Nussenzweig, R.S. (1993) T cell responses to pre-erythrocytic stages of malaria: role in protection and vaccine development against pre-erythrocytic stages. Annu. Rev. Immunol. 11, 687–727 51 Nakamoto, Y. et al. (1997) Differential target cell sensitivity to CTL-activated death pathways in hepatitis B virus transgenic mice. J. Immunol. 158, 5692–5697 52 Altman, J.D. et al. (1996) Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94–96 53 Lalvani, A. et al. (1994) An HLA-based approach to the design of a CTL-inducing vaccine against Plasmodium falciparum. Res. Immunol. 145, 461–468 54 Gilbert, S.C. et al. (1997) A protein particle vaccine containing multiple malaria epitopes. Nat. Biotechnol. 15, 1280–1294 55 Ockenhouse, C.F. et al. (1998) Phase I/II safety, immunogenicity, and efficacy trial of NYVAC-Pf7, a pox-vectored, multiantigen, multistage vaccine candidate for Plasmodium falciparum malaria. J. Infect. Dis. 177, 1664–1673 56 Shi, Y. et al. (1999) Immunogenicity and in vitro protective efficacy of a recombinant multistage Plasmodium falciparum candidate vaccine. Proc. Natl. Acad. Sci. U. S. A. 96, 1167–1169 57 Blum-Tirouvanziam, U. et al. (1995) Localization of HLA-A2.1restricted T cell epitopes in the circumsporozoite protein of Plasmodium falciparum. J. Immunol. 154, 3922–3931 58 Wizel, B. et al. (1995) HLA-A2-restricted cytotoxic T lymphocyte responses to multiple Plasmodium falciparum sporozoite surface protein 2 epitopes in sporozoite-immunized volunteers. J. Immunol. 155, 766–775

Pathways Involved in Environmental Sensing in Trypanosomatids M. Parsons and L. Ruben Digenetic parasites, such as those of the order Kinetoplastida, must respond to extracellular and intracellular signals as they adapt to new environments within their different hosts. Evidence for signal transduction has been obtained for Trypanosoma brucei, T. cruzi and Leishmania, as reviewed here by Marilyn Parsons and Larry Ruben. Although the broad picture suggests similarities with the mammalian host, there are large gaps in our understanding of these processes; this probably contributes to a perception of differences. Nonetheless, current evidence suggests that the trypanosomatids might lack certain classes of signalling molecules found in other organisms. Responses to environmental changes are mediated by signaling pathways that coordinate processes involved in cell growth, development and function. Generally, in eukaryotes, this coordination is Marilyn Parsons is at the Seattle Biomedical Research Institute, 4 Nickerson St, Seattle, WA 98109, USA and the Department of Pathobiology, School of Public Health and Community Medicine, University of Washington, Seattle, WA 98195, USA. Larry Ruben is at the Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275, USA. Tel: +1 206 284 8846 ext 315, Fax: +1 206 284 0313, e-mail: [email protected] 56

achieved by regulatory circuits that involve protein kinases and phosphatases, G proteins and second messengers. Changes in their activities can be initiated by external signals, environmental changes and internal homeostatic or cycling mechanisms, and in turn result in the modulation of the activities and interactions of other proteins. Modular domains that evolved to function specifically in protein–protein interactions are key to the transmission of many of these signals. The signals are integrated to yield a physiological response – in higher eukaryotes, these might include changes in the cytoskeleton, altered gene transcription or secretion. Initially, the study of signal transduction in model eukaryotes focused on specific molecules and their biochemical activities. Subsequently, great progress has been made towards connecting the steps of many pathways. These advances have relied on several key factors, many of which are not available in parasite systems. Among them are: the availability of a large battery of specific antibodies and cloned genes; the detection of intermolecular interactions; the definition of mechanisms of activation; the ability to modulate gene function through transfection; and the ability to modulate specific pathways using drugs and physiological inducers.

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Reviews Unique characteristics of molecules that generate signals, their receptors, the downstream proteins or even the interactions of proteins in a pathway might be targeted in the design of new therapies. Much of the current research in parasites is predicated on the assumption that disruption of signal transduction, or induction of an inappropriate signal, would compromise the ability of parasites to survive in the mammalian host. This review will provide an overview of the potential mediators of responses to environmental changes that have been identified in trypanosomatids, and then discuss in more detail several of the second messenger systems that function in these parasites. Owing to space limitations, literature citations are representative rather than comprehensive.

factors has not yet been validated by many other laboratories. Trimeric G proteins function as molecular switches that can modulate downstream events during transmembrane signalling. Association of receptor with ligand initiates exchange of GDP for GTP by the a subunit and subsequent dissociation from the bg dimer6, leading to changes in interaction with other proteins and a signalling cascade. Leishmania, T. cruzi and T. brucei proteins have been identified that possess some or all of the following hallmarks of trimeric G proteins: (1) the ability to bind GTP; (2) an appropriate molecular weight; (3) crossreactivity with specific peptide antibodies; and (4) ADP-ribosylation in response to bacterial toxins that target G proteins in higher eukaryotes7–9 (reviewed in Ref. 10). It is unclear whether bacterial toxins can be used to manipulate G protein activity within intact trypanosomatids because these cells may lack surface receptors for the toxins. Perhaps the closest to a functional assay has been the demonstration that partially purified membrane proteins from T. cruzi inhibit glucagon stimulation of mammalian adenylate cyclase in a manner that can be reversed with pertussis toxin10. These data are suggestive of Gi, although other inhibitory factors could have been involved. Overall, the ability of trypanosomatids to utilize trimeric G proteins in response to extracellular signals is not firmly established. Except for receptor adenylate cyclases, genes encoding molecules that transduce signals following interactions with specific extracellular ligands have not yet been found. The ‘missing’ genes include those specifying receptor protein kinases and phosphatases, serpentine receptors and heterotrimeric G proteins. Although there are sequences in the database with some homology to the missing proteins, the extent and level of homology is not convincing. Proof of relevance will require not only the cloning of full-length genes, but also functional analyses using genetic and biochemical approaches. The life cycle and cell cycle of kinetoplastid parasites are intertwined; there is often an alternation of dividing and cell cycle-arrested forms through the life cycle. Parasites have evolved mechanisms to coordinate

Pathways and signalling molecules in trypanosomatids Through gene discovery, molecules related to many of the key players in signal transduction in higher eukaryotes have been found in trypanosomatids (Table 1). However, the emerging data suggest that the parasites might have a streamlined set of signalling molecules. Indeed, as discussed below and depicted in Fig. 1, the spectrum of molecules implicated in the receipt of extracellular signals in trypanosomatids is very limited, and the types of responses observed are also limited. Certain types of modular protein interaction domains are conspicuous in their absence from the gene databases [eg. SRC homology regions 2 and 3 (SH2 and SH3 domains, respectively)]. Many signalling pathways in other organisms culminate in the activation of transcription factors; such factors have not been identified in trypanosomatids. Because the major mechanisms for gene regulation in these parasites appear to be posttranscriptional, we speculate that signalling pathways regulating gene expression might target molecules regulating RNA processing or turnover. Many organisms induce signalling pathways in response to metabolites; whether trypanosomes do so is not yet known. To date, studies of signal transduction in parasites have focused on differentiation, infectivity and cell growth. Unlike mammals, the Kinetoplastida have not been shown to display rapid, triggered changes in exocytosis, cell shape, transcription or metabolism. Little is known about the types of extracellular macromol- Table I. Trypanosomatid components potentially involved in environmental sensing ecules that will alter kinetoplastid behaviour, or the type of response Signal/sensing mechanism Role in Kinetoplastids Refs that is expected. The idea that spe- cAMP regulators and effectors Cell differentiation 3, 10, 11, 16, 27–30, 40 Host cell invasion 10, 31–47 cific molecules secreted by the host Ca21 regulators and effectors Cell death or parasite modulate parasite proUnknown liferation and development is very Absent: stimulus– attractive. Among the molecules reresponse coupling? ported to have physiological effects Inositol phosphates and Unknown pathways 37, 48–50, 52 on trypanosomatids are growth facphosphatidylinositol phosphates tors, cytokines and adrenergic lig- PH domains Regulated membrane ands1–3. One of the more interesting targeting (presumed) 56 cases is that of interferon g (IFN-g), Mitotic kinases and regulators Cell cycle control 11 which is reported to exert a growth Stage-regulated protein kinases Unknown pathways 11, 17, 20 Absent: stimulus– regulatory effect on Trypanosoma response coupling? brucei4. Intriguingly, it also appears Phosphoproteins (kinase targets) Unknown pathways 15, 18, 19, 24 to modulate the activity of Kfr1, a Cell cycle control 21–23, 25 protein kinase structurally related to Protein phosphatases Unknown pathways mitogen-activated protein (MAP) Heat-shock proteins Cell differentiation 59 5 kinase . Despite these provocative Protein chaperones findings, most work on extracellular Parasitology Today, vol. 16, no. 2, 2000

57

Reviews Ca2+ channels

Adenylate Serpentine cyclase receptors

Catalytic receptors

Ligand receptors Plasma membrane

PIK

PtdIns PH PIPLC domains

PDE Ca2+

Trimeric G proteins

SH2, SH3 interaction domains

cAMP

RAS InsP CaBPs

Receptor-associated kinases

PKA

Intracellular Ca2+ stores

Soluble kinases

Transcription factors

Phosphoproteins Nucleus

Phosphatases Parasitology Today

Fig. 1. A simplified diagram of cell signaling. Many of the components and connections present in mammalian cells have not yet been found in trypanosomatids. PDE, cAMP phosphodiesterase; PIK, phosphatidylinositol kinases; PIPLC, phosphoinositide phospholipase C; PKA, cAMP-dependent protein kinase; PtdIns, phophatidylinositol; SH2, SRC homology region 2. Dashed arrows and dark grey shading indicate missing components and connections; solid arrows and pale grey shading indicate known components and connections.

the complexities of the cell cycle, as shown by the presence of genes encoding similar signalling proteins to those involved in the yeast and human cell cycle. Proteins that coordinate mitosis are present, including those with homology to cyclin-dependent and other cell cycle-regulated kinases, cyclins and other regulatory proteins11. Genes functioning in responses to environmental stresses, such as heat-shock proteins, are found in all organisms, including trypanosomatids. In higher eukaryotes, MAP kinase pathways regulate cell growth, apoptosis and stress responses. Genes encoding molecules with homology to protein kinases in the MAP kinase pathways have been identified in the trypanosomatids5,12 (accession numbers AF034925 and AF050218). Interestingly, certain environmental stresses, such as changes in temperature, pH or nutrients, act as triggers for the induction of developmental programmes. This convergence suggests that many of the same molecules might function in cell responses to stress, cell cycle control and differentiation. For example, in T. brucei growth arrest (and subsequent death) induced by treatment with concanavalin A and cell cycle arrest induced by development to stumpy forms are both marked by high expression of a transcript encoding an intracellular modulator of protein kinase C localization13,14. Although the parasite molecules controlling differentiation to insect stages are not known, 58

it is interesting that, in Leishmania, changes in protein phosphorylation have been observed within minutes of the environmental shift15 (D. Zilberstein, pers. commun.). Recent data indicate that, within 10 min after the shift to low temperature in the presence of cis-aconitate, trypanosomes commit to specific aspects of differentiation to procyclic forms14. In T. brucei, changes in adenylate cyclase activity are observed several hours after the shift16. In contrast to these kinetic analyses, most studies of signalling molecules in development have focused on steady-state differences between developmental stages. There are numerous differences in the phosphoprotein profiles and protein kinase activities in the different developmental stages of trypanosomes17,18 and Leishmania19. Genes encoding highly developmentally regulated protein kinases have been identified11,20, but neither the factors inducing their expression nor their downstream targets are known. Genes encoding several major classes of protein serine/threonine phosphatases have been cloned21–23. Although no genes encoding tyrosine kinases or phosphatases have been cloned, evidence for their activity has been obtained24–26. Also specified in trypanosomatid genomes are proteins participating in second messenger systems. These molecules include enzymes that regulate or are regulated by second messengers (see below). Parasitology Today, vol. 16, no. 2, 2000

Reviews Cyclic AMP In one case, a conceptual link has been made between a ligand and a specific receptor-mediated signalling response. Bloodstream forms of T. brucei secrete a factor that stimulates the parasites to transform into nondividing stumpy forms27. Although the chemical nature of stumpy-induction factor is not known, it appears to act through induction of cAMP formation. The action of this molecule can be mimicked by the addition of cAMP analogues. This dovetails nicely with the fact that the genes encoding receptor adenylate cyclases are the only known trypanosomatid genes encoding a receptor with a recognizable cytoplasmic signalling domain28,29. These molecules are similar in structure to the receptor guanylate cyclases found in mammalian cells and contain a putative extracellular ligand-binding domain and a cytoplasmic adenylate cyclase domain. In both Leishmania and T. brucei, the genes exist as a multigene family, in which members differ significantly in the extracellular portion. These differences suggest that the proteins might interact with different ligands to modulate adenylate cyclase activity. However, like receptor guanylate cyclases, there is no evidence that activation of the trypanosomatid adenylate cyclases involves trimeric G proteins. Work in a variety of laboratories suggests that cAMP might be important in regulating parasite development. As noted above, bursts of adenylate cyclase activity occur as T. brucei differentiates from bloodstream to procyclic form16. The addition of non-hydrolysable cAMP analogues or stimulation of cAMP formation through pharmacological agents can modulate development and proliferation of T. cruzi3. Most cAMP-induced effects are mediated through protein kinase A. Genes encoding protein kinase A-related molecules have been identified in trypanosomatids11. The targets of protein kinase A have not yet been identified in the parasites. Phosphodiesterases act to return cAMP levels to baseline. Although the enzymes have not yet been cloned in these organisms, the activity has been characterized10,30 Calcium Ca21 is universally recognized by all eukaryotic cells as a signalling molecule. Not surprisingly, kinetoplastids contain the biochemical machinery necessary to produce and terminate Ca21 signals. In T. brucei, the nucleus acquires Ca21 passively31. In addition, all kinetoplastids examined to date contain three separate membrane compartments capable of unambiguously transporting Ca21 in an energy-dependent manner, namely: the mitochondrion, plasma membrane and acidocalcisomes32. In addition, endoplasmic reticulum (ER) Ca21 transport has been deduced from the cloning of genes encoding ER P-type Ca21-ATPases33. Vanadate-sensitive Ca21 transport has also been detected, but has not been linked directly to the ER pool. Collectively, the redundant transporting organelles safeguard against uncontrolled changes in intracellular Ca21 ([Ca2+]i)31. When multiple Ca21-transporting organelles are disrupted with reactive oxygen species, the result for T. brucei is Ca21-dependent fragmentation of nuclear DNA, loss of motility and cell death34. The acidocalcisome is perhaps the most distinctive component of the homeostatic organelle system. It has properties similar to plant and fungal Ca21 reservoirs and might play a dual role, regulating intracellular pH as well Parasitology Today, vol. 16, no. 2, 2000

as Ca21 (Ref. 35). Whereas the ER Ca21 pool is of central importance to the regulation of cell responses in mammalian cells, the role of the ER Ca21 pool in trypanosomatids remains enigmatic. This problem arises because physiological activators capable of releasing unambiguously stored Ca21 from the ER have not been found. The ER pool appears to be refractory to inositol (1,4,5)trisphosphate [Ins(1,4,5)P3] and perhaps thapsigargin36,37. Organellar storage compartments are predicted to be of special significance for the propagation of Ca21 signals in intracellular trypanosomatids, because the host cytoplasm contains very low levels of Ca21. In T. cruzi, more Ca21 is contained in amastigote acidocalcisomes than in promastigotes, perhaps to accommodate the intracellular life style35. By contrast, the extracellular stages have access to a large reservoir of extracellular Ca21, which might be effectively utilized in signalling. Although the stimulatory molecules that might initiate a Ca21 influx are not known, some progress has been made in elucidating the mechanism of influx in T. brucei by bypassing receptor function with amphiphilic peptides and amines38. These molecules produce a specific Ca21 influx in bloodstream and procyclic forms that appears to be mediated by activation of a cell-associated phospholipase A2 and concommitant release of arachidonic acid from membranes38. The presence of this plasma membrane-associated pathway strongly suggests that extracellular triggers might modulate Ca21 influx in these parasites. This mechanism of regulation clearly differs from most non-electrically coupled cells of the mammalian host, where phospholipase C and Ins(1,4,5)P3 are of central importance. The response of the cell to Ca21 is determined by the cellular complement of Ca21-binding proteins (CaBPs). Trypanosomatids contain a variety of CaBPs, including the universal regulatory protein calmodulin (CaM)10. To date, CaM dependency has been shown only for a limited number of target enzymes in trypanosomatids. These include cyclic nucleotide phosphodiesterase, CaM kinase II, nitric oxide synthase (reviewed in Ref. 10) and plasma membrane Ca21-ATPase (reviewed in Ref. 32). In addition, the multifunctional elongation factor 1a binds CaM39; a fact that has been confirmed for mammals and plants. Other CaBPs include isoforms of adenylate cyclase40, a protein kinase10, endonuclease41 and EH-5/CUB42. A variety of putative CaBPs have also been cloned from expressed sequence tags (ESTs). Along with CaM, the flagellum of kinetoplastids contains one or more EF-hand CaBPs43. The intriguing localization of these proteins to the flagellum suggests a potential role in motility or environmental sensing. Specific processes regulated by Ca21 flux and CaBPs have not been well characterized in trypanosomatids. In essence, the kinetoplastid Ca21 pathway is the answer to a question that has yet to be posed and discovering the timing and the purpose of Ca21 flux will be the next major challenge. A major unresolved aspect of Ca21 research centres upon extracellular signals that might initiate Ca21 flux and trigger the Ca21 pathways. No soluble molecules, such as hormones and growth factors, have yet been found that affect [Ca2+]i in T. brucei38. A role for Ca21 has been surmised for the invasion process in T. cruzi, where changes in Ca21 concentrations within the parasite and host have been reported44. Contact is associated with Ca21 flux in the 59

Reviews parasite cytoplasm. The source of mobilized Ca21 has not been determined. However, the raised [Ca2+]i appears to be important for invasion because stabilization of [Ca2+]i with BAPTA or Quin-2 inhibits this process. Developmental changes and cell growth rate might also have Ca21-dependent components. Partial disruption of the CaM gene array in T. brucei produces a slow growth phenotype45, and developmentally regulated CaM-binding proteins have been detected in T. cruzi46 and T. brucei39. Changes in stored Ca21 also accompany the development process35,47. Overall, the kinetoplastid Ca21 system shares broad similarities with the mammalian host. However, differences in cell surface receptors, mechanism of channel regulation, homeostatic organelles and CaBPs indicate the presence of unique signalling components. How these components are coordinated to alter parasite behaviour is a key unresolved area for future research. Phosphoinositidides Several inositol phosphates (InsPs) and phosphatidylinositol phosphates (PtdInsPs) play major roles as second messengers in signalling pathways. Analogous to the situation with Ca21, a complete InsP–PtdInsP signalling pathway would include triggered changes in InsP and PtdInsP levels, as well as proteins that respond to changes in the spectrum of these molecules. PtdInsPs are the lipid conjugate forms, which reside in membranes. Therefore, they are in an ideal location to respond rapidly to changes initiated at the plasma membrane. Phosphorylation of PtdIns is mediated by position-specific enzymes, including phosphatidylinositol 3-kinase (PI3K), which generates PtdIns(3,4)P2 and PtdIns(3,4,5)P3. InsPs are cytoplasmic molecules generated by cleavage of PtdInsP by phospholipase C; a gene encoding this enzyme has been identified in T. cruzi (accession numbers AB022677 and AF093565). This enzyme is distinct from glycosylphosphatidylinositol phospholipase C. In mammalian cells, PI3K and phospholipase C can be activated in response to ligand binding to cell surface receptors. Foetal calf serum and the cholinergic agent carbachol stimulate the production of inositol phosphates in T. cruzi, implying the presence of a regulated phospholipase C48,49. PtdInsPs and InsPs have not been well studied in trypanosomatids. Their presence has been documented in bloodstream and procyclic forms of T. brucei, where the ratios of the specific forms differ37. Ins(4)P, Ins(4,5)P2 and Ins(1,4,5)P3 have been identified in the parasites, with the concentrations of the last two being much higher in procyclic forms than in slender bloodforms. PtdInsPs have been studied in the epimastigote stage of T. cruzi50. The parasites appeared to lack significant levels of PtdIns(3,4)P2, although it should be noted that modifications at the 3 position (by PI3K) are usually induced by interactions with specific ligands51. With respect to the enzymes of PtdIns metabolism, a gene encoding the C-terminal half of a putative PI3K has been cloned from T. brucei52, suggesting that PtdIns phosphorylated at the 3 position could be present under certain conditions. InsPs and PtdInsPs exert their effect by binding to and modulating protein function. As noted above, Ins(1,4,5)P3 is a key effector of [Ca21]i release in higher eukaryotes, but not in trypanosomes37. A type of 60

modular structure, termed the PH domain, interacts with PtdInsPs and InsPs53. Different PH domains have different specificities for the various phosphorylated forms (see, for example, Ref. 54). The presence of specific InsPs or PtdInsPs can regulate the localization of PH-domain proteins to the membrane or cytoplasm. These second messengers can also regulate the activity of PH-domain proteins55. PH domains have been identified on several protein kinases of trypanosomatids (in the databases), including one that is developmentally regulated in T. brucei56. These findings raise the possibility that InsPs and phosphoinositides are important second messengers in these parasites. Perspective The examination of responses of parasites to environmental changes is still in its infancy. There are several considerations worth raising with respect to this early assessment of signalling pathways. Highthroughput genomic and EST tag sequencing have provided many data that have not been integrated yet to form a cohesive picture – and this sequence information will grow dramatically even before this article is published. Thus, there are certainly partial gene sequences relevant to signalling already in these databases that have not yet been analysed by investigators in the field. It is also possible that classes of genes cited as missing are actually present, but with only a few representatives in the genome. The comparison of Saccharomyces cerevisae and Caenorhabditis elegans genomes57 is instructive here. Saccharomyces cerevisae completely lacks some classes of signalling molecules found in C. elegans (eg. nuclear hormone receptors and epidermal growth factor repeat domains), whereas others are well represented (protein kinases and cyclic nucleotide binding domains). Other classes have numerous representatives in the worm but only a few in yeast (pheromones and SH2). The close association of trypanosomatids with metazoan hosts might require a signalling complexity somewhat greater than yeast. In any case, completion of the parasite genome projects will be required for an accurate assessment of the constellation of signalling molecules present. The ordered approach of the Leishmania Genome Project does allow a preliminary view of the relative importance of signalling to the parasites, as measured by the proportion of genes devoted to these processes. As catalogued by the authors, ~6% of the genes on chromosome 1 and 3 in L. major are predicted to be involved in signal transduction58, encoding such molecules as cell cycle regulators, protein kinases or phosphatases. These data suggest that 400–700 different signalling proteins are present in the parasites. Of these, only a handful has been studied to any degree, and virtually no linkages between signalling molecules have been made. Gene discovery, coupled with genomic approaches to functional analyses, might provide a mechanism for rapidly building our knowledge of parasite molecules involved in responses to environmental cues. Acknowledgements Owing to space limitations, the authors regret being unable to include all of the important contributions of our colleagues. The generation of this manuscript was supported in part by NIH AI31077 and by NIH AI24627. Parasitology Today, vol. 16, no. 2, 2000

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Lymphocyte Polyclonal Activation: A Pitfall for Vaccine Design against Infectious Agents B. Reina-San-Martín, A. Cosson and P. Minoprio In this article, Bernardo Reina-San-Martín, Alain Cosson and Paola Minoprio summarize the marked alterations in the immune system functions after infection that might account for the poor success of effective parasite vaccine development. Many of the studies on oligoclonal B- and T-cell responses to parasite antigens aiming at vaccination strategies would seem to ignore more general, and perhaps fundamental, aspects of parasite–immune system interactions. In essence, because of its consequences on immunopathology and parasite escape, the authors ascribe a central importance in the pathogenesis of parasitic diseases to the ‘nonspecific’ polyclonal lymphocyte activation that occurs during infection. Hence, novel targets and strategies for immune intervention should be considered. It is a common belief that parasites, viruses, fungi and bacteria often evade the immune system through adaptive mechanisms, that render the immune response powerless. Classic approaches to vaccine development have focused on the study of specific, parasite-directed mechanisms such as ‘immunodominant’, ‘immunopathological’ and ‘protective’ epitopes, in search of molecules able to trigger protective immunity while avoiding evasion. However, there is an important gap in this thinking: the immunologically relevant interactions between the infectious agents and the host are not limited to specific immune responses. Parasites, viruses, fungi and bacteria can actually obliterate specific immune responses by simply triggering the machinery of polyclonal lymphocyte responses, thus resulting in a general lack of specificity of antibodies or T-cell responses to the microbial antigens during infection and in the immunosuppressive state that follows. This apparently reduced availability of lymphocyte clones able to respond to the infectious agent (and to heterologous antigens as well) can actually be explained either by conventional immunosuppression or by the extensive engagement of most lymphocyte populations in effector functions that are not clonally specific (hyperstimulation). The onset of autoimmunity, another unwanted and frequent consequence of infectious processes, can Bernardo Reina-San-Martín, Alain Cosson and Paola Minoprio are at the Department of Immunology, Institut Pasteur, 25 rue du Dr. Roux, 75724, Paris, France. Tel: +33 1 45688615, Fax: +33 1 40613185, e-mail: [email protected] 62

also be explained by the establishment of a long-lasting polyclonal activation, with the bulk of lymphocyte populations activated by the infection embodying hostdirected cell clones involved in the evolution of selfaggressive mechanisms. Vaccine strategies aimed at neutralization of mitogens/superantigens and the control of nonspecific responses are considered here; for additional references, see http://www.pasteur.fr/recherche/unites/tcruzi/ minoprio/PTrefs.html). Immune system-driven approach to infection There are obvious difficulties faced by the immune system in order to eliminate a parasite. It is striking that a normal immune system is able promptly to reject tissues or organs that differ from the host by just a few amino acids in a single major histocompatibility complex (MHC) molecule. In contrast, the immune system is unable to eliminate parasites bearing a very complex and extremely different antigenic composition to that of the host. Nevertheless, the immune responses induced by parasite infections are sufficiently ‘strong’ to lead to progressive autoimmune pathologies that frequently can kill the host. Paradoxically, immune mechanisms that are inappropriate to eliminate the parasite are capable of destroying the host itself. Infectious agents share the ability to activate a high fraction of total lymphoid cells, many of which differentiate to exhibit effector functions. The consequences of this quasi-panclonal activation of the immune system are: (1) the development of nonspecific B- and T-cell responses; (2) the immunosuppression of humoral and cellular responses to homologous and heterologous antigens; and (3) the onset of autoimmune processes that might arise from the expansion of selfreactive clones. The magnitude of these responses and the profound perturbation of immune homeostasis they bring about are major hindrances to the development of effective vaccine strategies. Thus, lymphocyte activation in infection is essentially the result of mitogenic and/or superantigenic microbial components that elicit relatively poor specific responses. However, classic vaccination approaches have attempted to ‘neutralize’ ‘immunogenic molecules’ that do not prevent panclonal activation and are, therefore, ineffective in controlling mitogen-dependent immune disorders and parasite evasion.

0169-4758/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0169-4758(99)01591-4

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