Differences in morphology of phagosomes and kinetics of acidification and degradation in phagosomes between the pathogenicEntamoeba histolytica and the non-pathogenicEntamoeba dispar

Keio University, Edo, Tokyo, Japan
Cell Motility and the Cytoskeleton (Impact Factor: 4.19). 10/2005; 62(2):84-99. DOI: 10.1002/cm.20087
Source: PubMed


Phagocytosis plays an important role in the pathogenicity of the intestinal protozoan parasite Entamoeba histolytica. We compared the morphology of phagosomes and the kinetics of phagosome maturation using conventional light and electron microscopy and live imaging with video microscopy between the virulent E. histolytica and the closely-related, but non-virulent E. dispar species. Electron micrographs showed that axenically cultivated trophozoites of the two Entamoeba species revealed morphological differences in the number of bacteria contained in a single phagosome and the size of phagosomes. Video microscopy using pH-sensitive fluorescein isothiocynate-conjugated yeasts showed that phagosome acidification occurs within 2 min and persists for >12 h in both species. The acidity of phagosomes significantly differed between two species (4.58 +/- 0.36 or 5.83 +/- 0.38 in E. histolytica or E. dispar, respectively), which correlated well with the differences in the kinetics of degradation of promastigotes of GFP-expressing Leishmania amazonensis. The acidification of phagosomes was significantly inhibited by a myosin inhibitor, whereas it was only marginally inhibited by microtubules or actin inhibitors. A specific inhibitor of vacuolar ATPase, concanamycin A, interrupted both the acidification and degradation in phagosomes in both species, suggesting the ubiquitous role of vacuolar ATPase in the acidification and degradation in Entamoeba. In contrast, inhibitors against microtubules or cysteine proteases (CP) showed distinct effects on degradation in phagosomes between these two species. Although depolymerization of microtubules severely inhibited degradation in phagosomes of E. histolytica, it did not affect degradation in E. dispar. Similarly, the inhibition of CP significantly reduced degradation in phagosomes of E. histolytica, but not in E. dispar. These data suggest the presence of biochemical or functional differences in the involvement of microtubules and proteases in phagosome maturation and degradation between the two species.

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    • "Amebic cathepsin-like CPs are also known to be targeted to lysosomes and phagosomes to digest endocytosed fluid and engulfed bacteria and host cells (Marion et al., 2005; Mitra et al., 2005; Okada et al., 2005; 2006). While the E. histolytica genome contains genes encoding CD-MPR, AP and retromer, it apparently lacks genes encoding CI-MPR, GGA, TIP47 and PACS-1 (Loftus et al., 2005; Nakada-Tsukui et al., 2005). "
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    ABSTRACT: The transport of lysosomal proteins is, in general, mediated by mannose 6-phosphate receptors via carbohydrate modifications. Here, we describe a novel class of receptors that regulate the transport of lysosomal hydrolases in the enteric protozoan Entamoeba histolytica, which is a good model organism to investigate membrane traffic. A novel 110 kDa cysteine protease (CP) receptor (CP-binding protein family 1, CPBF1) was initially discovered by affinity co-precipitation of the major CP (EhCP-A5), which plays a pivotal role in the pathogenesis of E. histolytica. We demonstrated that CPBF1 regulates EhCP-A5 transport from the endoplasmic reticulum to lysosomes and its binding to EhCP-A5 is independent of carbohydrate modifications. Repression of CPBF1 by gene silencing led to the accumulation of the unprocessed form of EhCP-A5 in the non-acidic compartment and the mis-secretion of EhCP-A5, suggesting that CPBF1 is involved in the trafficking and processing of EhCP-A5. The CPBF represents a new class of transporters that bind to lysosomal hydrolases in a carbohydrate-independent fashion and regulate their trafficking, processing and activation and, thus, regulate the physiology and pathogenesis of E. histolytica.
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    • "Entamoeba histolytica that have phagocytized bacteria have been shown to be more cytotoxic to host cells and have increased invasiveness in animal models (Wittner and Rosenbaum, 1970; Mirelman et al., 1983; Bracha and Mirelman, 1984; Galván-Moroyoqui et al., 2008). Entamoeba dispar, a related non-pathogenic species, also ingests bacteria, but contained fewer bacteria in phagosomes that are structurally dissimilar and mature at a slower rate than those of E. histolytica (Pimenta et al., 2002; Mitra et al., 2005). Furthermore, E. histolytica trophozoites regained virulence after co-culture with bacteria (Padilla-Vaca et al., 1999). "
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    • "It has been proposed that pathogenicity of E. histolytica is associated with phagocytosis of certain bacterial strains present in the intestine of the host, as it has been shown that trophozoites that ingest and immediately degrade bacteria become more virulent, while non-pathogenic amoebas allow bacterial survival and replication. (Bracha et al., 1982; Mirelman, 1987; Pimenta et al., 2002; Meza and Clarke, 2004; Mitra et al., 2005; Galván-Moroyoqui et al., 2008). Trophozoites' aggressive behaviour has been studied in animal models, responsive to infection, where amoebas were directly inoculated into the caecum or into the liver of the experimental animals (Shibayama et al., 1997; Pacheco-Yépez et al., 2010), and in vitro, under conditions where the parasite interacted with different types of cultured cells in the absence of any other pathogen (Franco et al., 1999; Pacheco-Yépez et al., 2010). "
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