Proton-transporting and calcium ion-transporting ATPases of Entamoeba histolytica

Reviews

Proton-transporting and Calcium Ion-transporting ATPases of Entumoebu histolyticu J.C. Samuelson, N. Azikiwe and P-S. Shen Entamoeba histolytica is unique among human protozoan parasites in its ability to phagocytose bacteria and red blood cells and to destroy host epithelial cells via a contactmediated cytolysis. Antagonists of vacuolar acidif?cation and calcium ion-transport inhibit amebic lysis of epithelial cells in vitro. In this review, John Samuelson, Nnecka Azikiwe and Pei-Shen Shen describe the primary structures of E. histolytica V-type proton-transporting ATPase W-ATPase) and P-type calcium-transporting ATPase, which probably mediate amebic vacuolar acidification and calcium ion sequestration, respectively. The function of the amebic V-ATPase is discussed with regard to pinocytosis, bacterial killing and host cell lysis. Phylogenetic trees incorporating the sequences of the proteolipid and catalytic peptides of the amebic V-ATPase are described. The amebic P-type calcium-transporting ATPase is compared to those of the red blood cell plasma membrane and yeast vacuolar membrane. Finally, the potential of the V-ATPase proteolipid and P-type calcium ion-transporting ATPase as targets for anti-amebic antibodies or for bacteria loaded with recombinant toxins is explored. Entamoeba histolytica is the protozoan parasite that causes approximately 50 million cases of amebic dysentery and liver abscess each year in developing countries, where it is spread by the fecal-oral route’. Amebic trophozoites live as commensal organisms in the colonic lumen, where they are surrounded by bacteria, which the parasites consume and use as a food source2 (Fig. 1). These trophozoites cause dysentery when they lyse colonic epithelial cells and invade host tissues, where amebae phagocytose red blood cells (RBCs). Amebic phagocytosis of bacteria and host cells appears to be associated because a phagocytosis-defective mutant, which shows decreased bacterial killing and decreased erythrophagocytosis, is less virulent in experimental models3. Amebic phagocytosis probably involves: (I) binding of amebic lectins to sugars on bacteria and epithelial cells42; (2) vacuolar acidificatior+; (3) calcium ion release7; and (4) release of phospholipases7, proteases8 and pore-forming peptide@. This review focuses on the vacuolar membrane proton-transporting ATPase (V-ATEase), which acidifies amebic vacuolesPi2, and the P-type calcium ion-transporting ATPase, which is important in regulating intracellular calciumi3. Amebae rapidly pinocytose fluid that surrounds them into tens of small pinocytotic vacuoles, which may be identified with fluorescein-conjugated dextrani” (Fig. 2). Acidification of these pinocytotic vacuoles is John Samuelron, Nnecka A.&we

and Pel-Shen Shen are at the Department of Troplcal Public Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02 I IS, USA. Tel: +I 617 432 4671, Fax: +I 617 738 4914, e-mail:

[email protected]

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inhibited by bafilomycin A, which is a specific inhibitor of V-ATPases10J4. Amebae phagocytose starch particles, bacteria or RBCs into phagolysosomes, which are fewer and larger than the pinosomes (Fig. 2). Dependence of epithelial cell lysis on low vacuolar pH and calcium ions Amebic lysis of epithelial cells has been extensively studied in vitro by Leippe, Martinez-Palomo, Orozco, Ravdin, Petri, Stanley and co-workers. Epithelial cell lysis begins with binding of amebic lectins to carbohydrates on the surface of epithelial celW6Js. Amebae then either secrete or release vacuolar contents onto the surface of epithelial cells, which are lysed and phagocytosed by the parasite+. Epithelial cell lysis is inhibited by ammonium chloride, which raises the internal pH of the acidic vacuole+, and by calcium ion chelatorsr. This inhibition of killing may be caused by blocking secretion of the amebic vacuolar contents, because antagonists of proton- or calcium ion-transporters block secretion in other eukaryotesi6. Alternatively, the activities of amebic phospholipasesr, proteases and pore-forming peptides may be pH dependent and be less at neutral rather than at acidic pH. Amebic lysis of bacteria, epithelial cells and RBCs (Fig. 1) appear to be associated because: (1) amebic uptake and killing of bacteria, detected with DNAbinding, fluorescent dyes (which are membrane-permeant and stain live bacteria green or are membraneimpermeant and stain leaky bacteria red), was inhibited by ammonium chloride (J. Samuelson and N. Azikiwe, unpublished); (2) parasites incubated with bacteria are more virulent when injected into livers of experimental animalsz; (3) the amebic poreforming peptide, which is associated with epithelial cell lysis, has potent acid-dependent antibacterial activity9; and (4) phagocytosis-defective mutants, selected by incubation of amebae with bacteria loaded with BUdR, show decreased phagocytosis of RBCs and decreased virulence in gerbilss. The nature of the mutation(s) in the phagocytosis-deficient amebae remains to be determined. An argument against the linkage between bacterial phagocytosis and amebic virulence, however, is the fact that non-pathogenic E. histolytica phagocytose bacteria in the colonic lumen but do not invade the host epithelium and cause dysentery’. Non-pathogenic E. histolytica, now called E. dispar (by most, but not all, investigators), resemble pathogenic E. histolytica in their appearance under the light microscope, but their isoenzymes, antigens and sequences of their coding and non-coding genes differiT. Invasion of host tissues by E. histolytica apparently provides no selective advantage: non-invasive E. dispar is widely prevalenti7,‘8 and E. histolytica infection is spread by acid-resistant cysts, which are decreased in number in 0 64 4?58!9Y%29 50

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Colonic lumen Colonic wall

Fig. 2. Confocal micrographs of amebae that have pinocytosed fluorescein-conjugated dextran (a) and phagocytosed fluorescein-conjugated starch particles (b). Scale bar = IO km.

the stools of dysenteric patients’. Recent axenization (growth in the absence of bacteria) of E. dispur by Clark should facilitate studies of the interaction of these parasites with intestinal epithelial cells.

Comparison of amebae to macrophages Like amebae, macrophages are -50 pm in diameter, have a vacuole-filled cytoplasm, and phagocytose and lyse bacteria and RBCslg. Unlike amebae, macrophages primarily phagocytose opsinized bacteria and old RBCs (in the spleen), and lyze target cells via oxygen metabohtes (including peroxide, hydroxyl radicals and nitric oxide)6Jg. Vacuolar acidification is crucial for killing of bacteria by macrophages, as shown by inhibition with ammonium chloride and bafilomycin A, a specific inhibitor of the V-type ATPaser4. V-type ATPases located in the plasma membrane of macrophages are necessary for maintaining cytosolic pH in the acidic abscesses in which macrophages frequently function. Specialized macrophages called osteoclasts use plasma membrane V-type ATPases to secrete acids that degrade hydroxyapatite crystals, when osteoclasts remodel bonelg. Whether amebic V-type ATPases are located on the plasma membrane, contribute to maintenance of cytosolic pH, and are used to secrete acid remains to be determined. Acid secretion by amebae might explain why inflammatory infiltrates are typically scant within amebic abscesses. 418

Fig. I. Diagrammatic representation of amebic phagocytosis. Ameba I in the colonic lumen phagocytoses bacteria; ameba 2 attaches to, lyses and consumes epithelial cells in the colonic wall; and ameba 3 phagocytoses RBCs within tissue abscesses.

Eukaryotic V-ATEases (EC 3.6.1.35) are 400-500 kDa proton-transporting pumps, which acidify intracellular compartments, including endosomes, lysosomes, secretory granules, and the lumen of the endoplasmic reticulumlQ0. V-ATPases, which are related in structure to proton transporters of bacteria, mitochondria and chloroplasts (F-type ATPases or FoFl ATPases), comprise at least ten different peptides. The major component of the transmembrane segment of the VATPase is a 17 kDa peptide, encoded by the vma3 gene in yeast. This 17 kDa peptide is often referred to as the proteolipid because it partitions with the organic phase of a chloroform-methanol extraction21 (Fig. 3). The 17 kDa proteolipid paradoxically contains no lipid: it is composed of four hydrophobic domains, which form the proton channel. A major component of the cytosolic segment of eukaryotic V-ATPases is an -67kDa catalytic peptide, encoded by the vmal gene in yeast, which contains the ATP-binding site22. Amebic V-ATPase proteolipid and catalytic peptide To begin to explore the importance of vacuolar acidification in amebic phagocytosis, we recently cloned an E. histolyticu gene (Ehvmul) encoding the catalytic peptide of a putative amebic V-ATPase, as well as genes encoding putative V-ATPase proteolipids of E. histolyticu (Ehvmu3) and E. dispur (Edvmu3)‘1J2. The Ehvma3 open reading frame (ORF) shared a 92% positional identity with that of Edvmu3, while the Ehvmu3 and Ehvmul ORFs showed 58% and 62% positional identities, respectively, with the Succhuromyces cerevisiue vmu3 and vmul peptideG22. Conserved features in the Ehvmu3 and Edvmu3 ORFs (V-ATPase proteolipids) included four hydrophobic cw-helices (the putative transmembrane elements) and a glutamate residue, which covalently binds the V-ATPase inhibitor dicyclohexylcarbodiimide (DCCDP. Conserved features in the ORF of Ehvmul (the V-ATPase catalytic peptide) included ATP-binding site A (P-loop) and an ATEase-a-B signature sequenceu. None of the amebic V-ATPase genes contained introns, and Southern blots suggested that both Ehvmul and Ehvmu3 were ‘single copy’ genes. The function of the amebic V-ATPases might be demonstrated by complementation yeast mutants with the Ehzmal and Ehvmu3 geneszO. Alternatively, the effect of knocking out the amebic V-ATPase genes on bacterial Parasitology Today, vol.

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Reviews killing and epithelial lysis may be determined, if the required genetic techniques become available. Because V-ATPases are present in all eukaryotes while related F-ATPases are and archaebacteria, present in all eubacteria and mitochondria, V- and F-ATPase peptides have been used for evolutionary comparisonG22. In a phylogenetic tree of 20 V- and F-ATPase proteolipids, 15 eukaryotes formed one large group or clade, at the edge of which were E. histolytica and E. &spar”. A second mixed clade contained an archaebacterium and four eubacteria, the proteolipids of which have two transmembrane segments rather than four transmembrane segments present in eukaryotic V-ATPase proteolipids21. In contrast, in a phylogenetic tree of 27 V- and F-ATPase eukaryotic and archaebacteria catalytic peptides, formed a mixed clade with Plasmodium falciparum rather than E. histolytica at the edge of the eukaryotic group, while eubacteria formed a second cladel2,22. Ironically, then, eubacteria and archaebacteria are linked in the proteolipid tree (with eukaryotes as the outlier), while eukaryotes and archaebacteria are linked in the catalytic peptide tree (with eubacteria as the outlier). So far it has not been possible to decide from phylogenetic trees whether E. histolyticu had a bacterial endosymbiont and lost it or never had a bacterial endosymbiont’l,l2. Amebic P-type calcium-transporting ATPase P-type cation-transporting ATPases, which are -1OOkDa proteins that contain an ATP-binding site and ten transmembrane segments13. In an effort to determine whether amebae might have a P-type proton-transporting ATPase or a P-type calcium ion-transporting ATPase, we designed PCR primers to conserved sequences found in eukaryotic P-type transporters13. An amebic P-type proton-transporting ATPase was not found (which does not prove its absence). An E. histolyfica gene (Ehcfal) was identified that encoded a P-type calcium ion-transporting ATPase with 45% and 44% positional identities, respectively, with a human plasma membrane calcium ion-transporter and S. cerevisiae vacuolar calciumtransporting ATPase’“r2” (J. Samuelson and P-S. Shen, unpublished). The Ehctul ORF contained ten predicted hydrophobic a-helices (putative transmembrane segments) and so was distinct from a recently described ectoplasmic calcium-dependent ATPase that is likely involved in purine uptake24. The Ehctal gene product, by homology to other eukaryotic calcium ion-transporting ATPases, iS likely important in regulating cytosolic calcium, which has been shown to be critical for parasitic killing of epithelial cells in vitro7. This is the first gene encoding a plasma membrane-type calcium ion transporting-ATPase identified from a protozoan parasite, although a gene encoding an endoplasmic reticulum-type calcium ion-transporting ATPase has been identified from Plasmodium falciparum25. Proton-transporting and calcium ion-transporting ATPases as targets for anti-amebic vaccines or toxins Although the amebic V-type proton-transporting ATPase and P-type calcium-transporting ATPase have not yet been localized, it is likely that these pumps are Pormtoiogy

To&y

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V-type protorHr~a;cvting

P-type calciufnft~a~eporting

Fig. 3. Cartoon to demonstrate the possible location of targets for anti-amebic antibodies or toxins. Both the calcium ion-transporting ATPase and the V-ATPase (of which two peptides are marked) are assumed to be present on an invagination of the plasma membrane, which will later develop into a pinocytotic or phagocytotic vacuole. Circles indicate unique ectoplasmic loops on the calcium ion-transporting ATPase and V-ATPase proteolipid.

located in the parasite vacuolar and/or plasma membranes, as has been described for similar pumps in macrophages and yeasts 13,16,19,23. A potential target for anti-amebic antibodies is a distinctive 24 amino acid ectoplasmic loop between transmembrane segments 1 and 2 of the V-type ATPase proteolipid*‘,21 (Fig. 3). Similarly, the predicted calcium-transporting ATPase contains a unique 53 amino acid ectoplasmic loop between transmembrane segments 3 and 4 and a second unique 43 amino acid ectoplasmic loop between transmembrane segments 7 and 8 (J.C. Samuelson and P-S. Shen, unpublished). Presently, multi-antigenie peptides corresponding to the ectoplasmic loops are being used to raise antibodies to localize the V-ATPase and P-type ATPase and to immunize hamsters against subsequent challenge with amebae. Antibodies to the ectoplasmic loops of the amebic V-ATPase proteolipid and calcium ion-transporting ATPases might be joined to bacterial ADP-ribosylating toxins to make recombinant chimeras (‘magic bullets’Yk-‘H. These ‘magic bullets’ might be released from toxin-loaded bacteria (‘magic bacteria’) when they are phagocytosed by Entamoebae (Fig. 4). The need for ‘magic bacteria’ comes from: (1) the slow improvement in sanitary conditions in developing countries, that might prevent the spread of amebic dysentery’; (2) the difficulty in treating amebae in the

Blebs on the surface of a dying parasite {

-n

Fig. 4. Cartoon of a toxin-loaded ameba that has phagocytosed it.

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‘magic bacterium’ killing the

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Reviews colonic lumen with metronidazole, because the drug is so well absorbed29; and (3) the probable failure of anti-amebic antibodies to clear parasites from the colonic lumen. ‘Magic bacteria’, the development of which is a long-term, ‘pie-in-the-sky’ goal of our research group, might capitalize on: (1) amebic use of bacteria as a food sourcez; (2) the presence within amebae of elongation factor 2, which is the target for Pseudomonas exotoxin A and diphtheria toxirW7,sQ, of rho proteins, which are the targets for clostridial exoenzyme C3 (Ref. 311, and of YUS family proteins, which are the targets for exoenzyme S32.33;and (3) the possibility of re-engineering bacterial toxins to bind to one type of cell and not to others2Q7. The possible advantage of targeting the proton-transporting and calcium ion-transporting ATPases versus the bettercharacterized amebic vaccine candidates (Gal/ GalNac lectin or serine-rich E. hisfolyficu protein9 is that these transporters are more likely to be present in endocytic vacuoles, from which ADP-ribosylating proteins usually enter the cytosoP-28. Conclusions Vacuolar acidification and calcium ions have been shown to be important for amebic killing of bacteria and epithelial cells, so E. hisfolyticu genes encoding the proteolipid and catalytic peptide of the V-ATPase and encoding the P-type calcium ion-transporting ATPase were cloned. Although the predicted ORFs of these genes closely resemble those of other eukaryotes, the V-ATPase proteolipid and P-type ATPase each have unique ectoplasmic loops, which may be targets for anti-amebic antibodies or recombinant toxins. Acknowledgements This work was supported In part by an NIH grant and by the John D. and Cathkine T. MacArthur Foundation. Our thank go to Cesano Bianchi for help with the confocal mlcroscopy.

References 1 Martinez-Palomo, A. and Martinez-Baez, M. (1985) Rev. Infect. Dis. 5,1093-1102 2 Mirelman, D. (1987) Microbial. Rev. 51,272-284 3 Rodriguez, M.A. and Orozco, E. (1986) J. Infect. Dis. 154,27-32 4 Ravdin, J.I. (1986) Rev. Infect. Dis. 8,247-259 5 McCoy, J.J. et at. (1994) Infect. Immun. 62,3045-3050 6 Ravdin, J.1. et al. (1986) J. Protozoot. 33,478-486 7 Ravdin, J.I. et al. (1985) J. Inject. Dis. 152,542-549 8 Reed, S.L. et al. (1989) J. Clin. Microbial. 27,2772-2777 9 Leippe, M. et al. (1994) Proc. Nat1 Acad. Sci. USA 91,2602-2606 10 Lohden-Bendinger, U. and Bakker-Grunwald, T. (1990) Z. Naturforsch. 45c, 229-232 11 Descoteaux, S. et al. (1994) Infect. lmmun. 62,3572-3575 12 Yu, Y. and Samuelson, J. (1994) Mol. Biochem. Purasitol. 66, 165-169 13 Strehler, E.E. (1991) J. Membr. Biol. 120,1-15 14 Bowman, E.J. et al. (1988) Proc. Nat1 Acad. Sci. USA 85,7972-7976 15 Li, E., Becker, A. and Stanley, S.L. (1989) Infect. bnmun. 57,8-12 16 Mellman, I. et al. (1986) Annu. Rev. Biochem. 55,663-700 17 Diamond, L.S. and Clark, C.G. (1993) I. Euk. Microbial. 40, 340-344 18 Irosen, E.M. ef al. (1992) Clin. Infect. Dis. 14,889-893 19 Chatteriee. D. et al. (1992) 1. Exv. Biol. 172,193-204 20 Anrak;, Y; et al. (1992) I. i&p. Viol. 172,67-81 21 Nelson, H. and Nelson, N. (1989) FEBS Lett. 247,147-153 22 Gogarten, JF. et al. (1989) Proc. Nut1 Acad. Sci. USA 86, 6661-6665 23 Cunningham, K.W. and Fink, G.R. (1994) J. Cell Biot. 124, 351363 24 Bakker-Grunwald, T. and I’arduhn, H. (1993) Mol. Biochem. Parasitol. 57, 167-170 25 Murakami, K. et al. (1990) J. Cell Sci. 97,487-495 26 Collier, R.J. (1990) in ADP-ribosylating Toxins and G Proteins: Insights into Signal Transduction (Moss, J. and Vaughan, M., eds), pp 3-19, American Society of Microbiology 27 Strom, T.B. et al. (1990) Immunol. Rev. 129,131-163 28 Pastan, I. et al. (1992) Annu. Rev. Biochem. 61,331-354 29 Norris, SM. and Ravdin, J.1. (1990) in Amebiusis (Ravdin, J.I., ed.), pp 734-755, John Wiley & Sons 30 Plaimauer, B. et al. (1993) DNA Cell Biol. 1,89-96 31 Aktories, K. et al. (1992) Curr. Top. Microb. lmmunol. 175, 115-131 32 Cobum, J. (1992) Curr. Top. Micro. lmmunol. 175,113-143 33 Shen, P-S. et al. (1994) Mol. Biochem. Purusitol. 64,111-120 34 Stanley, S.L. et al. (1990) Proc. Nat1 Acad. Sci. USA 87,4976-4980

Towards Effective Control Strongyloides stercorulis

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D-1. Conway, J.F. Lindo, R.D. Robinson and D.A.P. Bundy A widespread intestinal parasite of humans, Strongyloides stercoralis has long been considered very difficult lo confro1 in endemic communities. This situation is now changing. In fhis article, David Conway, John Linda, Ralph Robinson and Don Bundy review recent advances in diagnosis, chemotherapy and epidemiology of S. stercoralis infection, and highlight new options for control. David Conway IS at the Department of Medical Parasitology, London School of Hygiene and TropIcal Medlclne, London, UK WCI E 7HT. John Llndo IS at the Department of Mlcrobloloa, Univenity of The West Indies. Mona. Klngston 7, Jamaica. Ralph Robinson IS at the Department of Zoology University of The West Indies, Mona, Klngston 7, Jamaica. Don Bundy IS at the Department of Zoology, University of Oxford, South Parks Road, Oxford, UK OX I 3PS. Tel: +44 I71 636 8636, Fax: +44 I ‘II 636 8739, e-mail: [email protected] 420

The intestinal nematode Sfrongyloides sfercoralis is endemic throughout tropical and warm temperate regions of the world’. Faeces from infected people contain first-stage larvae that rapidly develop directly or through a single free-living cycle to become infective third-stage larvae capable of infecting the same person or other people by skin penetrationz. Although an undetermined proportion of infections appear to be asymptomatic3, a very diverse array of clinical symptoms is attributed to infection with S. sfercorulis; the most common symptoms include abdominal discomfort and diarrhoea, and pruritus of the peri-anal skin caused by autoinfective larvae. More dramatically, a syndrome of severe hyperinfection or potentially fatal disseminated infection can occur in patients receiving corticosteroids for kidney transplants or cancer therapy, and in some individuals with immunological disorderss.