Entamoeba histolytica is the cause of amebic colitis and liver abscess. This parasite induces apoptosis in host cells and utilizes exposed ligands such as phosphatidylserine to ingest the apoptotic corpses and invade deeper into host tissue. The purpose of this work was to identify amebic proteins involved in the recognition and ingestion of dead cells. A member of the transmembrane kinase family, phagosome-associated TMK96 (PATMK), was identified in a proteomic screen for early phagosomal proteins. Anti-peptide affinity-purified antibody produced against PATMK demonstrated that it was a type I integral membrane protein that was expressed on the trophozoite surface, and that co-localized with human erythrocytes at the site of contact. The role of PATMK in erythrophagocytosis in vitro was demonstrated by: (i) incubation of ameba with anti-PATMK antibodies; (ii) PATMK mRNA knock-down using a novel shRNA expression system; and (iii) expression of a carboxy-truncation of PATMK (PATMKΔ932). Expression of the carboxy-truncation of PATMKΔ932 also caused a specific reduction in the ability of E. histolytica to establish infection in the intestinal model of amebiasis, however these amebae retained the ability to cause hepatic abscesses when directly injected in the liver. In conclusion, PATMK was identified as a member of the TMK family that participates in erythrophagocytosis and is uniquely required for intestinal infection.
References
[1]
WHO (1997) WHO/PAHO/UNESCO report. A consultation with experts on amoebiasis. Mexico City, Mexico 28–29 January, 1997. Epidemiol Bull 18: 13–14.
[2]
Gonzalez-Ruiz A, Haque R, Aguirre A, Castanon G, Hall A, et al. (1994) Value of microscopy in the diagnosis of dysentery associated with invasive Entamoeba histolytica. J Clin Pathol 47: 236–239.
[3]
Jimenez F (1981) Pathology of amebiasis. Bull N Y Acad Med 57: 217–223.
[4]
Maltz G, Knauer CM (1991) Amebic liver abscess: a 15-year experience. Am J Gastroenterol 86: 704–710.
[5]
Brandt H, Tamayo RP (1970) Pathology of human amebiasis. Hum Pathol 1: 351–385.
[6]
Aikat BK, Bhusnurmath SR, Pal AK, Chhuttani PN, Datta DV (1979) The pathology and pathogenesis of fatal hepatic amoebiasis–A study based on 79 autopsy cases. Trans R Soc Trop Med Hyg 73: 188–192.
[7]
Berninghausen O, Leippe M (1997) Necrosis versus apoptosis as the mechanism of target cell death induced by Entamoeba histolytica. Infect Immun 65: 3615–3621.
[8]
Huston CD, Houpt ER, Mann BJ, Hahn CS, Petri WA Jr. (2000) Caspase 3-dependent killing of host cells by the parasite Entamoeba histolytica. Cell Microbiol 2: 617–625.
[9]
Ragland BD, Ashley LS, Vaux DL, Petri WA Jr. (1994) Entamoeba histolytica: target cells killed by trophozoites undergo DNA fragmentation which is not blocked by Bcl-2. Exp Parasitol 79: 460–467.
[10]
Huston CD, Boettner DR, Miller-Sims V, Petri WA Jr. (2003) Apoptotic killing and phagocytosis of host cells by the parasite Entamoeba histolytica. Infect Immun 71: 964–972.
[11]
Boettner DR, Huston CD, Sullivan JA, Petri WA Jr. (2005) Entamoeba histolytica and Entamoeba dispar utilize externalized phosphatidylserine for recognition and phagocytosis of erythrocytes. Infect Immun 73: 3422–3430.
[12]
Orozco E, Guarneros G, Martinez-Palomo A, Sanchez T (1983) Entamoeba histolytica. Phagocytosis as a virulence factor. J Exp Med 158: 1511–1521.
[13]
Rodriguez MA, Orozco E (1986) Isolation and characterization of phagocytosis- and virulence-deficient mutants of Entamoeba histolytica. J Infect Dis 154: 27–32.
[14]
Katz U, Ankri S, Stolarsky T, Nuchamowitz Y, Mirelman D (2002) Entamoeba histolytica expressing a dominant negative N-truncated light subunit of its gal-lectin are less virulent. Mol Biol Cell 13: 4256–4265.
[15]
Hirata KK, Que X, Melendez-Lopez SG, Debnath A, Myers S, et al. (2006) A phagocytosis mutant of Entamoeba histolytica is less virulent due to deficient proteinase expression and release. Exp Parasitol 115: 192–199.
[16]
Yan L, Stanley SL Jr. (2001) Blockade of caspases inhibits amebic liver abscess formation in a mouse model of disease. Infect Immun 69: 7911–7914.
[17]
Scianimanico S, Pasquali C, Lavoie J, Huber LA, Gorvel JP, et al. (1997) Two-dimensional gel electrophoresis analysis of endovacuolar organelles. Electrophoresis 18: 2566–2572.
[18]
Garin J, Diez R, Kieffer S, Dermine JF, Duclos S, et al. (2001) The phagosome proteome: insight into phagosome functions. J Cell Biol 152: 165–180.
[19]
Gotthardt D, Blancheteau V, Bosserhoff A, Ruppert T, Delorenzi M, et al. (2006) Proteomic fingerprinting of phagosome maturation and evidence for the role of a Galpha during uptake. Mol Cell Proteomics 5: 2228–2243.
[20]
Kiss RS, Elliott MR, Ma Z, Marcel YL, Ravichandran KS (2006) Apoptotic cells induce a phosphatidylserine-dependent homeostatic response from phagocytes. Curr Biol 16: 2252–2258.
[21]
Wolf A, Schmitz C, Bottger A (2007) Changing story of the receptor for phosphatidylserine-dependent clearance of apoptotic cells. EMBO Rep 8: 465–469.
[22]
Gagnon E, Duclos S, Rondeau C, Chevet E, Cameron PH, et al. (2002) Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages. Cell 110: 119–131.
[23]
Touret N, Paroutis P, Terebiznik M, Harrison RE, Trombetta S, et al. (2005) Quantitative and dynamic assessment of the contribution of the ER to phagosome formation. Cell 123: 157–170.
[24]
Okada M, Huston CD, Mann BJ, Petri WA Jr., Kita K, et al. (2005) Proteomic analysis of phagocytosis in the enteric protozoan parasite Entamoeba histolytica. Eukaryot Cell 4: 827–831.
[25]
Marion S, Laurent C, Guillen N (2005) Signalization and cytoskeleton activity through myosin IB during the early steps of phagocytosis in Entamoeba histolytica: a proteomic approach. Cell Microbiol 7: 1504–1518.
[26]
Loftus B, Anderson I, Davies R, Alsmark UC, Samuelson J, et al. (2005) The genome of the protist parasite Entamoeba histolytica. Nature 433: 865–868.
[27]
Marion S, Guillen N (2006) Genomic and proteomic approaches highlight phagocytosis of living and apoptotic human cells by the parasite Entamoeba histolytica. Int J Parasitol 36: 131–139.
[28]
Okada M, Huston CD, Oue M, Mann BJ, Petri WA Jr., et al. (2006) Kinetics and strain variation of phagosome proteins of Entamoeba histolytica by proteomic analysis. Mol Biochem Parasitol 145: 171–183.
[29]
Hanks SK, Hunter T (1995) The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J 9: 576–596.
[30]
Gumienny TL, Brugnera E, Tosello-Trampont AC, Kinchen JM, Haney LB, et al. (2001) CED-12/ELMO, a novel member of the CrkII/Dock180/Rac pathway, is required for phagocytosis and cell migration. Cell 107: 27–41.
[31]
Scott RS, McMahon EJ, Pop SM, Reap EA, Caricchio R, et al. (2001) Phagocytosis and clearance of apoptotic cells is mediated by MER. Nature 411: 207–211.
[32]
Wu Y, Singh S, Georgescu MM, Birge RB (2005) A role for Mer tyrosine kinase in alphavbeta5 integrin-mediated phagocytosis of apoptotic cells. J Cell Sci 118: 539–553.
[33]
Hellberg A, Nickel R, Lotter H, Tannich E, Bruchhaus I (2001) Overexpression of cysteine proteinase 2 in Entamoeba histolytica or Entamoeba dispar increases amoeba-induced monolayer destruction in vitro but does not augment amoebic liver abscess formation in gerbils. Cell Microbiol 3: 13–20.
[34]
Zhang X, Zhang Z, Alexander D, Bracha R, Mirelman D, et al. (2004) Expression of amoebapores is required for full expression of Entamoeba histolytica virulence in amebic liver abscess but is not necessary for the induction of inflammation or tissue damage in amebic colitis. Infect Immun 72: 678–683.
[35]
Diamond LS, Harlow DR, Cunnick CC (1978) A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans R Soc Trop Med Hyg 72: 431–432.
[36]
Ravdin JI, Stanley P, Murphy CF, Petri WA Jr. (1989) Characterization of cell surface carbohydrate receptors for Entamoeba histolytica adherence lectin. Infect Immun 57: 2179–2186.
[37]
Bratosin D, Estaquier J, Petit F, Arnoult D, Quatannens B, et al. (2001) Programmed cell death in mature erythrocytes: a model for investigating death effector pathways operating in the absence of mitochondria. Cell Death Differ 8: 1143–1156.
[38]
Bratosin D, Leszczynski S, Sartiaux C, Fontaine O, Descamps J, et al. (2001) Improved storage of erythrocytes by prior leukodepletion: flow cytometric evaluation of stored erythrocytes. Cytometry 46: 351–356.
[39]
Wessel D, Flugge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138: 141–143.
[40]
Callahan MK, Halleck MS, Krahling S, Henderson AJ, Williamson P, et al. (2003) Phosphatidylserine expression and phagocytosis of apoptotic thymocytes during differentiation of monocytic cells. J Leukoc Biol 74: 846–856.
[41]
Beck DL, Boettner DR, Dragulev B, Ready K, Nozaki T, et al. (2005) Identification and gene expression analysis of a large family of transmembrane kinases related to the Gal/GalNAc lectin in Entamoeba histolytica. Eukaryot Cell 4: 722–732.
[42]
Gou D, Jin N, Liu L (2003) Gene silencing in mammalian cells by PCR-based short hairpin RNA. FEBS Lett 548: 113–118.
[43]
Ramakrishnan G, Zhao JL, Newton A (1991) The cell cycle-regulated flagellar gene flbF of Caulobacter crescentus is homologous to a virulence locus (lcrD) of Yersinia pestis. J Bacteriol 173: 7283–7292.
[44]
Saito-Nakano Y, Yasuda T, Nakada-Tsukui K, Leippe M, Nozaki T (2004) Rab5-associated vacuoles play a unique role in phagocytosis of the enteric protozoan parasite Entamoeba histolytica. J Biol Chem 279: 49497–49507.
[45]
Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, et al. (1992) Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 148: 2207–2216.
[46]
Houpt ER, Glembocki DJ, Obrig TG, Moskaluk CA, Lockhart LA, et al. (2002) The mouse model of amebic colitis reveals mouse strain susceptibility to infection and exacerbation of disease by CD4+ T cells. J Immunol 169: 4496–4503.
[47]
Hamano S, Asgharpour A, Stroup SE, Wynn TA, Leiter EH, et al. (2006) Resistance of C57BL/6 mice to amoebiasis is mediated by nonhemopoietic cells but requires hemopoietic IL-10 production. J Immunol 177: 1208–1213.
[48]
Chadee K, Meerovitch E (1985) Entamoeba histolytica: lymphoreticular changes in gerbils (Meriones unguiculatus) with experimentally induced cecal amebiasis. J Parasitol 71: 566–575.
[49]
Davis PH, Zhang Z, Chen M, Zhang X, Chakraborty S, et al. (2006) Identification of a family of BspA like surface proteins of Entamoeba histolytica with novel leucine rich repeats. Mol Biochem Parasitol 145: 111–116.
[50]
Temesvari LA, Harris EN, Stanley SL Jr., Cardelli JA (1999) Early and late endosomal compartments of Entamoeba histolytica are enriched in cysteine proteases, acid phosphatase and several Ras-related Rab GTPases. Mol Biochem Parasitol 103: 225–241.
