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Rab small GTPases regulate membrane traffic between distinct cellular compartments of all eukaryotes in a tempo‐spatially specific fashion. Rab small GTPases are also involved in the regulation of cytoskeleton and signalling. Membrane traffic and cytoskeletal regulation play pivotal role in the pathogenesis of Entamoeba histolytica, which is a protozoan parasite responsible for human amebiasis. E. histolytica is unique in that its genome encodes over 100 Rab proteins, containing multiple isotypes of conserved members (e.g., Rab7) and Entamoeba‐specific subgroups (e.g., RabA, B, and X). Among them, E. histolytica Rab7 is the most diversified group consisting of nine isotypes. While it was previously demonstrated that EhRab7A and EhRab7B are involved in lysosome and phagosome biogenesis, the individual roles of other Rab7 members and their coordination remain elusive. In this study, we characterised the third member of Rab7, Rab7D, to better understand the significance of the multiplicity of Rab7 isotypes in E. histolytica. Overexpression of EhRab7D caused reduction in phagocytosis of erythrocytes, trogocytosis (meaning nibbling or chewing of a portion) of live mammalian cells, and phagosome acidification and maturation. Conversely, transcriptional gene silencing of EhRab7D gene caused opposite phenotypes in phago/trogocytosis and phagosome maturation. Furthermore, EhRab7D gene silencing caused reduction in the attachment to and the motility on the collagen‐coated surface. Image analysis showed that EhRab7D was occasionally associated with lysosomes and prephagosomal vacuoles, but not with mature phagosomes and trogosomes. Finally, in silico prediction of structural organisation of EhRab7 isotypes identified unique amino acid changes on the effector binding surface of EhRab7D. Taken together, our data suggest that EhRab7D plays coordinated counteracting roles: a inhibitory role in phago/trogocytosis and lyso/phago/trogosome biogenesis, and an stimulatory role in adherence and motility, presumably via interaction with unique effectors. Finally, we propose the model in which three EhRab7 isotypes are sequentially involved in phago/trogocytosis.

Summary Phagocytosis is indispensable for the pathogenesis of the intestinal protozoan parasite Entamoeba histolytica. Here, we showed that in E. histolytica Rab8A, which is generally involved in trafficking from the trans-Golgi network to the plasma membrane in other organisms but was previously identified in phagosomes of the amoeba in the proteomic analysis, primarily resides in the endoplasmic reticulum (ER) and participates in phagocytosis. We demonstrated that down-regulation of EhRab8A by small antisense RNA-mediated transcriptional gene silencing remarkably reduced adherence and phagocytosis of erythrocytes, bacteria and carboxylated latex beads. Surface biotinylation followed by SDS-PAGE analysis revealed that the surface expression of several proteins presumably involved in target recognition was reduced in the EhRab8A gene-silenced strain. Further, overexpression of wild-type EhRab8A augmented phagocytosis, whereas expression of the dominant-negative form of EhRab8A resulted in reduced phagocytosis. These results indicated that EhRab8A regulates transport of surface receptor(s) for the prey from the ER to the plasma membrane. To our knowledge, this is the first report that the ER-resident Rab GTPase is involved in phagocytosis through the regulation of trafficking of a surface receptor, supporting a premise of direct involvement of the ER in phagocytosis.

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.

Summary The assembly of vital reactive iron-sulfur (Fe-S) cofactors in eukaryotes is mediated by proteins inherited from the original mitochondrial endosymbiont. Uniquely among eukaryotes, however, Entamoeba and Mastigamoeba lack such mitochondrial-type Fe-S cluster assembly proteins and possess instead an analogous bacterial-type system acquired by lateral gene transfer. Here we demonstrate, using immunomicroscopy and biochemical methods, that beyond their predicted cytosolic distribution the bacterial-type Fe-S cluster assembly proteins NifS and NifU have been recruited to function within the relict mitochondrial organelles (mitosomes) of Entamoeba histolytica. Both Nif proteins are 10-fold more concentrated within mitosomes compared with their cytosolic distribution suggesting that active Fe-S protein maturation occurs in these organelles. Quantitative immunoelectron microscopy showed that amoebal mitosomes are minute but highly abundant cellular structures that occupy up to 2% of the total cell volume. In addition, protein colocalization studies allowed identification of the amoebal hydroperoxide detoxification enzyme rubrerythrin as a mitosomal protein. This protein contains functional Fe-S centres and exhibits peroxidase activity in vitro. Our findings demonstrate the role of analogous protein replacement in mitochondrial organelle evolution and suggest that the relict mitochondrial organelles of Entamoeba are important sites of metabolic activity that function in Fe-S proteinmediated oxygen detoxification.

Summary Autophagy is one of two major bulk protein degradation systems and is conserved throughout eukaryotes. The protozoan E ntamoeba histolytica, which is a human intestinal parasite, possesses a restricted set of autophagy‐related (Atg) proteins compared with other eukaryotes and thus represents a suitable model organism for studying the minimal essential components and ancestral functions of autophagy. E. histolytica possesses two conjugation systems: Atg8 and Atg5/12, although a gene encoding Atg12 is missing in the genome. Atg8 is considered to be the central and authentic marker of autophagosomes, but recent studies have demonstrated that Atg8 is not exclusively involved in autophagy per se, but other fundamental mechanisms of vesicular traffic. To investigate this question in E . histolytica, we studied on Atg8 during the proliferative stage. Atg8 was constitutively expressed in both laboratory‐maintained and recently established clinical isolates and appeared to be lipid‐modified in logarithmic growth phase, suggesting a role of Atg8 in non‐stress and proliferative conditions. These findings are in contrast to those for E ntamoeba invadens, in which autophagy is markedly induced during an early phase of differentiation from the trophozoite into the cyst. The repression of Atg8 gene expression in En . histolytica by antisense small RNA‐mediated transcriptional gene silencing resulted in growth retardation, delayed endocytosis and reduced acidification of endosomes and phagosomes. Taken together, these results suggest that Atg8 and the Atg8 conjugation pathway have some roles in the biogenesis of endosomes and phagosomes in this primitive eukaryote.

Department of Parasitology , N ational Institute ofInfectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo162-8640, Japan.SummaryV esicular trafficking plays a pivotal role in the viru-lence of the enteric protozoan parasite Entamoebahistolytica . In the present study , w e showed that oneisotype of the small GTPase Rab1 1, Eh Rab1 1B, plays acentral role in the secretion of a m ajor virulence factor ,cysteine proteases. Eh Rab1 1B did not colocalize withmarkers for the endoplasmic reticulum, early endo-somes and lysosomes, but was partially associatedwith non-acidiÞed vesicles in the endocytic pathway ,likely recycling endosomes. Overexpression ofEh Rab1 1B resulted in a remarkable increase in bothintracellular and secreted cysteine protease activity ,concomitant with an augmentation of cytolytic activityas demonstrated by an increased ability to destroymammalian cells. The oversecretion of cysteine pro-teases with Eh Rab1 1B overexpression was neithersensitive to brefeldin A nor speciÞc to a certain cys-teine protease species (e.g. CP1, 2 o r 5), suggestingthat these three major cysteine proteases aretrafficked via an Eh Rab1 1B-associated secretorypathway ,w hich is distinct from the classical brefeldin-sensitive pathway . O verexpression of Eh Rab1 1B alsoenhanced exocytosis of the incorporated suid-phasemarker , supporting the notion that it is involved inrecycling. This is the Þrst report demonstrating thatRab1 1 plays a central role in the transport and secre-tion of pathogenic factors.IntroductionRab proteins are members of the Ras super-family ofsmall GTPases and essential to the regulation of vesiculartrafficking in the endocytic and exocytic/secretory path-ways in eukaryotic cells (Zerial and McBride, 2001). RabGTPases are localized to discrete membrane-bound com-partments, where they have a speciÞc role depending onthe kind and location of Rab (Novick and Zerial, 1997;Chavrier and Goud, 1999). From Þssion yeast tomammals and higher plants, the Rab family hasexpanded signiÞcantly; the diversity of these proteins isoften regarded as a resection of membrane traffickingcomplexity (Zerial and McBride, 2001). The humangenome contains more than 60 Rabs, while the genomesof the fruit sy , nematode and yeast have 26, 29 and 11Rabs, respectively (Bock et al., 2001). Some R abs, e.g.Rab5 and Rab7, are ubiquitous (Stenmark and Olkkonen,2001), while others, e.g. Rab3 (Schluter et al., 2002) andRab27A (Hume et al., 2001; Stinchcombe et al., 2001),are unique to particular cells, tissues, organs and species(Seabra et al., 2002). Rab1 1 is one of the ubiquitousRabs, distributed in the trans-Golgi network (TGN), post-Golgi vesicles and recycling endosomes (Urbe et al.,1993; Ullrich et al., 1996; Ren et al., 1998; McNiven andThompson, 2006). In Chinese hamster ovary (CHO) cells,Rab1 1 w as shown to regulate the recycling of the trans-ferrin receptor to the plasma m embrane (Ullrich et al.,1996; Ren et al., 1998). In parietal epithelial cells, Rab1 1was also shown to control the cell surface expression ofH

Rab7 small GTPase plays a crucial role in the regulation of trafficking to late endosomes, lysosomes and phagosomes. While most eukaryotes encode a single Rab7, the parasitic protist Entamoeba histolytica possesses nine Rab7. In this study, to understand the significance of the presence of multiple Rab7 isotypes, a role of two representative Rab7 isotypes, EhRab7A and EhRab7B, was investigated. EhRab7B was exclusively localized to acidic vacuoles containing lysosomal proteins, e.g. amoebapore-A and cysteine protease. This lysosome localization of EhRab7B was in good contrast to EhRab7A, localized to a non-acidic compartment in steady state, and only partially colocalized with lysosomal proteins. Overexpression of EhRab7B resulted in augmentation of late endosome/lysosome acidification, similar to the EhRab7A overexpression. Expression of EhRab7B-GTP mutant caused dominant-negative phenotypes including decrease in late endosome/lysosome acidification and missecretion of lysosomal proteins, while EhRab7A-GTP enhanced acidification but did not affect either intracellular or secreted cysteine protease activity. Expression of either EhRab7B or EhRab7B-GTP mutant caused defect in phagocytosis, concomitant with the disturbed formation and disassembly of prephagosomal vacuoles, the compartment previously shown to be linked to efficient ingestion. Altogether, these data indicate that the two Rab7 isotypes play distinct but co-ordinated roles in lysosome and phagosome biogenesis.