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exploit such habitats. The development of new fluorescence-based reporters permits the analysis of live bacterial gene responses with single-cell resolution. Acknowledgements

We thank Evi Strauss,Nina Salamaand Tim McDanielfor their input and editingexpertise, and J. Shea and D. Holdenfor sharingstrains and unpublished results. This work was supported by PHS grant AI 26195 and by unrestrictedgifts from Lederle-PraxisBiologicals and Bristol-MyersSquibb. References

1 Garciadel Portillo,F. and Finlay,B.B.(1995) Trends MicrobioI. 3, 373-380 2 Finlay,B.B.and Cossart,P. (1997) Science 276, 718-725 3 Garciadel Portillo,F. et al. (1992) Mol. Microbiol. 6, 3289-3297 4 GarcfaVdscovi,E., Soncini,F.C. and Groisman,E.A.(1996) Cell 84, 165-174

5 Fields,P.I., Groisman,E.A.and Heffron,F. (1989) Science 243, 1059-1062 6 Meighen,E. (1993)FASEBJ. 7, 1016-1022 7 Contag,C.H. et al. (1995) Mol. MicrobioI. 18, 593-603 8 Pettersson,J. et al. (1996) Science 273, 1231-1233 9 Mahan,M.J. etal. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 669-673 10 Heitboff,D.M. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 934-939 11 Rhen,M., Riikonen,P. and Taira, S. (1993) Mol. Microbiol. 10, 45-56 12 Kwaik,Y.A.and Pederson,Li. (1996) Mol. Microbiol. 21, 543-556 13 Zhang,J.P. and Normark,S. (1996) Science 273, 1234-1236 14 Cubitt,A.B.et al. (1995) Trends Biochem. Sci. 20, 448-455 15 Valdivia,R.H. and Falkow,S. (1996) Mol. Microbiol. 22, 367-389 16 Shea,J.E. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2593-2597

Pore-forming proteins in pathogenic protozoan parasites M. Fbtima Horta inside the cell generates osmotic Pore-forming proteins (PFPs) may play lthough pathogenic propressure, causing a water influx. important roles in pathogenesis by tozoa differ in many protozoan parasites by either directly The cell disrupts through a proways, many of them process known as colloid-osmotic damaging the plasma membrane of the duce cytolytic proteins that disrupt target cell membranes by host cells or ensuring intraceUular survival lysis3,4. Death of host cells mediated of the parasites by promoting their exit forming discrete channels in from lysosomal vacuoles. The L e i s h m a n i a by PFPs from bacterial pathothe lipid bilayer. These poregens has frequently been deamazonensis pore-forming cytolysin, forming proteins (PFPs) are scribed (for reviews, see Refs leishporin, may play a crucial role in the thought to play a significant 4-6). Protozoan pathogens can pathogenesis of leishmaniasis. role in the pathogenesis of now be added to the list of PFPmany protozoan infections 1,2. M.F. Horta is at the producing organisms, and the Channel formation by PFPs is Dept de Bioquimica e Imunologia, notion of a cause-and-effect rea well-defined mechanism of Instituto de CiSncias Biol6gicas, lationship between cytolysins membrane damage used in bioUniversidade Federal de Minas Gerais, and host tissue damage can logical systems ranging from 31270-010 Belo Horizonte, MG, Brazil. tel: +55 31 441 5 7 7 7 , fax: +55 31 441 5963, now be extended to parasitic bacteria to vertebrates: the C9 e-mail: [email protected] diseases. component of the complement system and perforin from cytoThe outsider's attack lytic lymphocytes in vertebrates are good examples. E n t a m o e b a histolytica, which is responsible for human PFPs bind to the plasma membrane of a target cell amoebiasis, was the first protozoan in which PFPs were and insert into the lipid bilayer by changing their condiscovered 7,s. Amoebiasis is an enteric illness that may formation and exposing hydrophobic domains. A spread to multiple organs when the parasite invades chain reaction follows in which the altered molecules the intestinal mucosa. The main manifestation of the bind others, oligomerizing around a central axis to form disease is the destruction of host tissues, which results transmembrane pores that grow in diameter by the from parasite contact-mediated cytolysis. The cytolytic successive annexation of new monomers. Some PFPs effect has been attributed to a family of pore-forming act as monomers that adopt circular or semicircular peptides, termed amoebapores, that are localized within structures without mutual association. Ions and small cytoplasmic granular vesicles of the trophozoites 9. molecules can then pass freely across the lipid bilayer, Amoebapores form transmembrane channels with an and the high concentration of macromolecules trapped

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internal diameter averaging 2 nm (Ref. 7) and are most active at acid pH (Refs 10,11). The cDNA-deduced amino acid sequence of amoebapores reveals a peptide of 77 amino acids 12. Both pathogenic and nonpathogenic isolates of E. histolytica possess cytolytic and pore-forming activities. However, the nonpathogenic isolates are threefold less cytolytic, with 60-80% less pore-forming activity than the pathogenic forms 1°,11. Naegleria fowleri, a free-living amoeboflagellate that can cause invasive meningoencephalitis in humans, also expresses a PFP. As the disease is a fulminating infection with massive tissue destruction 13, it has been suggested that a parasite-induced cytolytic process takes place. Indeed, N. fowleri has been reported to be cytotoxic in vitro ~4-16, and multiple cytolytic activities have been described in the amoebae lysates T M . The major cytolytic component is a heat-resistant protein, called N-PFP (Ref. 19), that is associated with the parasite surface membrane 2°. It is a 66-kDa or 50-54-kDa molecule under reducing or nonreducing conditions, respectively, that produces channels of 3.6-5.2 nm diameter. N-PFP accounts for 70-90% of the total cytolytic activity of N. fowleri lysates 2° and, therefore, may account for the tissue injury seen in vivo. How E. histolytica and N. fowleri cytolysins kill host cells in vivo is still unclear. It has been hypothesized that amoebapores are secreted by trophozoites in vitro in response to stimuli that mimic parasite-target contact s. However, recent work 21 indicates otherwise: amoebapore release appears to be associated with disintegration of the parasites, and intact amoebae fail to secrete the peptides, even after stimulation with concanavalin A, bacterial lipopolysaccharide (LPS) or the calcium ionophore, A23187. Likewise, N-PFP is not secreted by N. fowleri in vitro 17, and contact between the target cell and the parasite seems to be a requisite for cytolysis~4,15. In E. histolytica, PFP secretion may occur by exocytosis of the cytolysin-containing granular vesicles. Contact-dependent cytotoxicity is also thought to be mediated by a protozoan PFP in Trichomonas vaginalis infection. However, it has been shown that cytolysis by T. vaginalis can also occur in a contact-independent fashion, during which a change in pH triggers the release of the lytic molecules =. The enemy from within

Insertion of parasite PFPs into plasma membranes does not always result in the immediate death of the host cells. For example, the facultative intracellular bacterium Listeria monocytogenes, initially trapped inside phagocytic vacuoles, is unable to survive in this compartment. To escape into the cytoplasm, the bacterium uses its pore-forming cytolysin, listeriolysin O (LLO; Ref. 4), to disrupt the vacuolar membrane 23 without affecting the plasma membrane and killing host cells. Trypanosoma cruzi, the causative agent of Chagas' disease, may rely on an analogous mechanism to gain a safe environment inside its host cell: a C9-related PFP, called Tc-TOX, which is secreted by the intracellular stage amastigote 24. Appropriately, Tc-TOX, a molecule of 60-66 kDa (nonreduced) or 70-75 kDa (reduced),

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Fig. 1. Balb/c peritoneal macrophages infected with Leishmania amazonensis. Amastigotes live inside parasitophorous vacuoles until the macrophages are disrupted. Parasites are attached to the inner membrane of the vacuoles, which are often extended to the borders of the cell in contact with the inner plasma membrane (arrow heads). Scale bar = 5 pm.

seems to be responsible for vacuolar disruption by the parasite. It forms large pores of -10 nm, which can lyse erythrocytes and nucleated cells. Analogous to LLO, Tc-TOX is optimally active at pH 5.5, but is inactive at neutral pH, suggesting that it acts in the acidic intracellular vacuoles formed shortly after parasite invasion. In fact, it can be localized to the lumen of the parasitecontaining phagosomes 24. Significantly, treatment of infected cells with drugs that raise the pH of intracellular compartments inhibits parasite escape into the cytosol2s. 'Porin' out

The most recently discovered member of the PFPproducing protozoa is another trypanosomatid. Recently, we have shown that extracts of Leishmania amazonensis contain a cytolytic activity that damages erythrocytes and nucleated cells, including macrophages (its vertebrate host cells) 26,27. This activity is mediated by a heat-labile protein that is found in promastigotes and amastigotes and is optimally active at pH 5.0-5.5. In contrast to Tc-TOX and LLO, it is still active at neutral pH. L. amazonensis-mediated haemolysis is inhibited by macromolecules, such as polyethyleneglycol 6000, indicating its colloid-osmotic nature, which is typical of pore formation 2v. Indeed, whole-cell patchclamp experiments have demonstrated that L. amazonensis extracts induce voltage-dependent nonselective channels in the macrophage membrane, indicating that L. amazonensis cytolysin is a PFP (F.S.M. Noronha et al., unpublished), which we have called leishporin. Preliminary results indicate that the size of leishporin is -55-65 kDa. The mechanism of pore formation by leishporin resembles that of classical PFPs. Patch-clamp experiments and osmotic protection by molecules of various diameters have shown that the diameters of the pores increase in size (from -1.1 nm to >6.1 nm) with time or with the concentration of the cytolytic extract. This suggests that pores are formed by aggregation or polymerization of single protein units.

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But what is the functional role Removed of the L. amazonensis PFP in vivo ? peptide Once inoculated into the vertebrate Pro-PFP host, the promastigotes (the extracellular form in the sandfly vector) are ingested by macrophages and transform into amastigotes. Limited / In contrast to T. cruzi, the phagoproteolysis lysosome environment does not of precursor intimidate L. amazonensis, and the amastigotes not only survive but also replicate inside these acidic parasitophorous vacuoles without escaping into the cytoLipid plasm (see Fig. 1). However, at ~ ~ bilayer later stages of the parasite life cycle, both the vacuolar and plasma membranes are disrupted 28 to release the amastigotes, which then infect adjacent cells. All descrip)teolysis tions of the disruption of infected inhibitor cells are vague: the cells are said Functional to 'burst', an event that is suppore posed to be purely mechanical. Considering that the parasitoPFP + inhibitor ~ ' l l k ~ V ~ Degraded ~ A • inhibitor phorous vacuole can harbour numerous parasites, thus swelling and/or fusing with other phagoFig. 2. Possible mechanisms of activation of Leishmania amazonensis pore-forming protein (PFP). A cytolytically inactive pro-PFP may be cleaved by limited proteolysis, removing a peptide and yieldcytic vesicles as the parasites multiing the active PFP. Alternatively, the PFP may initially be bound to an inhibitor, which is subsequently ply28,29 (see Fig. 1), purely medegraded by proteolysis, releasingthe active PFP. In both cases, the active PFP can polymerize into chanical rupture seems unlikely. the lipid bilayer, forming a transmembrane functional pore. Our recent findings of an acidactive PFP in L. amazonensis suggest that amastigotes may use this molecule to disrupt neutral pH. Another possibility could be that the conhost cells26,27. nection between the phagolysosome outer membrane and the plasma inner membrane of L. amazonensisLaunching the missile: aiming at two targets infected macrophages (see Fig. 1) favours the interaction of the PFP with the inner membrane. It is also Leishporin does not exist freely in the cytosol of L. amazonensis: it co-sediments with the membrane fracpossible that these cytolysins are tightly regulated and tion but appears to be trapped inside membranous act only at specific stages of the parasite life cycle, in vesicles in a soluble form 27. However, promastigotes do which case it would be reasonable to assume that the not secrete the active molecule, even upon stimulation plasma membrane of T. cruzi-infected macrophages with calcium ionophores (A23187 or ionomicin) z7 or could also be disrupted by Tc-TOX at later stages. Our most recent data on leishporin concerns its mode concanavalin A (F.S.M. Noronha and M.F. Horta, of activation. We have found that the cytolytic activity unpublished). We are currently investigating whether of parasite extracts increases approximately fivefold amastigotes are able to secrete leishporin. Amastigotes lodge in the macrophages by firmly adhering to the in- when kept at 4°C for 7d or at 37°C for 24h. This increase is totally blocked by protease inhibitors, and ner surface of the phagolysosomes, which, in turn, can both parasite-derived and exogenous soluble proteases become distended as the parasites multiply to the boundcan generate cytolytic activity (F.R. Almeida-Campos aries of the cell (Fig. 1). In vivo, it is possible that this and M.F. Horta, unpublished). These and other results intimate membrane contact triggers a secretion mechanism. As the lysin seems to be a luminal protein, a ves- have led us to believe that the activity of leishporin icle exocytosis mechanism for delivering the cytolysin arises from proteolysis. We are investigating two hypotheses: (1) that the active PFP is produced by limited to its target can be envisaged. proteolysis of an inactive precursor and (2) that an inIn contrast to T. cruzi, L. amazonensis amastigotes hibitory molecule bound to the cytolysin is proteolytiare faced with the task of disrupting two membranes: cally degraded to release the active cytolysin (Fig. 2). first the phagolysosomal membrane and then the plasma membrane. A puzzling question is why T. cruzi PFP only lyses the phagosomal membrane, leaving the host The war is just beginning plasma membrane intact. The pH dependence of the Much investigation is needed to substantiate our assumptions and to determine whether the production two PFPs may offer one explanation: whereas Tc-TOX of PFPs is a general feature of pathogenic protozoans. is active only at acid pH, leishporin is also lytic at

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Questions for future research

• Is the expression of pore-forming proteins (PFPs) a common feature of all pathogenic protozoa? • What signals trigger protozoan PFP-mediated disruption of plasma and/or vacuolar membranes in vivo? • Do all protozoan cytolysins need to be previously activated to create pores? • How do PFP-producing parasites avoid self-lysis? Do they secrete a lyrically inactive PFP that becomes activated outside the parasite? Does the parasite express membrane molecules that abort channel formation? ° How would PFP-knockout parasites behave? Would they still be virulent? ° Are all protozoan PFPs structurally related?

Nevertheless, recent data make the hypothesis of protozoan PFPs as virulence factors quite attractive. Two other protozoans should be considered as potential PFP-producers: P l a s m o d i u m f a l c i p a r u m and T o x o p l a s m a gondii. These species reside in parasitophorous vacuoles within erythrocytes and nucleated cells, respectively, where the presence of channels 3° or putative channels 31 has been reported. Although it is not certain whether these pores are of parasitic origin, recent data are consistent with this hypothesis 32,33. Identifying the leishporin gene, disclosing its mechanism of action and defining its function(s) are primary goals. Although it does not preclude other roles for leishporin (see Ref. 27), the assumption that this molecule is involved in macrophage rupture leads to a shift in the current thinking that macrophage bursting is a direct result of excessive parasite burden. This PFP may be a crucial molecule for the pathogenesis of leishmaniasis, acting as both a tissue-damaging and an infectionspreading factor. As leishmaniasis encompasses selfhealing skin ulcers and mucocutaneous lesions to fatal visceral forms 28, it would be interesting to search for a correlation between the pathologies caused by the different species and the presence of PFPs. Extending this correlation to other protozoan parasites would open a whole new field of investigation. The scenario looks promising. Acknowledgements

I thank F. Juarez Ramalho-Pinto, F~itimaS.M. Noronha, Flfivia R.A. Campos, Jane Lima dos Santos and Santuza M. Teixeira for their invaluable assistance. The research in my laboratory is supported by Financiadora de Estudos e Proletos (FINEP), Fundaq~o de Amparo 5 Pesquisa de Minas Gerais (FAPEMIG)and Conselho Nacional de Desenvolvimento Cientffico e Tecnoldgico (CNPq).

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In the other Trends journals A selection of recently published articles of interest to TIM readers. • HIV and chemokines: ligands sharing cell-surface receptors, by P.R. Clapham - Trends in Cell Biology 7 , 2 6 4 - 2 6 8 • Salicylic acid and disease resistance in plants, by J. Durner, J. Shah and D.F. Klessig- Trends in Plant Science 2 , 2 6 6 - 2 7 4 • Bacterial surface display: trends and progress, by S. Stfihl and M. U h l 6 n - Trends in Biotechnology 15, 1 8 5 - 1 9 2

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