Evidence of a continuous endoplasmic reticulum in the protozoan parasite Entamoeba histolytica

Entamoeba histolytica, the cause of amebiasis, is believed to have no continuous endoplasmic reticulum (ER), with ER functions occurring in vesicles. Here, using an ER-targeted green fluorescent protein fusion protein and fluorescence loss in photobleaching, we have unambiguously demonstrated the presence of a continuous ER compartment in living E. histolytica trophozoites.     

Proteomic analysis of phagocytosis in the enteric protozoan parasite Entamoeba histolytica

Proteomic analysis of phagosomes isolated from Entamoeba histolytica by liquid chromatography and mass spectrometry identified 85 proteins involved in surface recognition, actin cytoskeleton rearrangement, vesicular trafficking, and degradation. Phagosome localization of representative proteins was verified by immunofluorescence assay. This study should provide a basis for molecular identification and characterization of phagosome biogenesis.     

Delinking of S phase and cytokinesis in the protozoan parasite Entamoeba histolytica

The alternation of DNA replication in S phase and chromosome segregation in M phase is a hallmark in the cell cycle of most well-studied eukaryotes and ensures that the progeny do not have more than the normal complement of genes and chromosomes. An exception to this rule has been described in cancer cells that occasionally become polyploid as a result of failure to restrain S phase despite the failure to undergo complete mitosis. Here, we describe the cell division cycle of the human pathogen, Entamoeba histolytica, which routinely accumulates polyploid cells. We have studied DNA synthesis in freshly subcultured cells and show that, unlike most eukaryotes, Entamoeba cells reduplicate their genome several times before cell division occurs. Furthermore, polyploidy may occur without nuclear division so that single nuclei may contain 1-10 times or more genome contents. Multinucleated cells may also accumulate several genome contents in each nuclei of one cell. Thus, checkpoints that normally prevent DNA reduplication until after cytokinesis in most eukaryotes are not observed in E. histolytica.     

Two novel calcium-binding proteins from cytoplasmic granules of the protozoan parasite Entamoeba histolytica

We report on the molecular characterisation of two novel granule proteins of the protozoon and human pathogen Entamoeba histolytica. The proteins, which were named grainin 1 and 2, show a considerable structural similarity to calcium-binding proteins, particularly within EF-hand motifs. Each grainin possesses three of these putative calcium-binding sites. Based on careful inspection of known structures of protein families containing EF-hands, a domain of grainin 1 covering two EF-hand motifs was modeled by homology. Calcium-binding activity of grainins was demonstrated by two independent methods. These granule proteins may be implicated in functions vital for the primitive phagocyte and destructive parasite such as control of endocytotic pathways and granule discharge.     

Molecular cloning and characterization of a protein farnesyltransferase from the enteric protozoan parasite Entamoeba histolytica

Genes encoding alpha- and beta-subunits of a putative protein farnesyltransferase (FT) from the enteric protozoan parasite Entamoeba histolytica were obtained and their biochemical properties were characterized. Deduced amino acid sequences of the alpha- and beta-subunit of E. histolytica FT (EhFT) were 298- and 375-residues long with a molecular mass of 35.6 and 42.6 kDa, and a pI of 5.43 and 5.65, respectively. They showed 24% to 36% identity to and shared common signature domains and repeats with those from other organisms. Recombinant alpha- and beta-subunits, co-expressed in Escherichia coli, formed a heterodimer and showed activity to transfer farnesyl using farnesylpyrophosphate as a donor to human H-Ras possessing a C-terminal CVLS, but not a mutant H-Ras possessing CVLL. Among a number of small GTPases that belong to the Ras superfamily from this parasite, we identified EhRas4, which possesses CVVA at the C terminus, as a sole farnesyl acceptor for EhFT. This is in contrast to mammalian FT, which utilizes a variety of small GTPases that possess a C-terminal CaaX motif, where X is serine, methionine, glutamine, cysteine, or alanine. EhFT also showed remarkable resistance against a variety of known inhibitors of mammalian FT. These results suggest that remarkable biochemical differences in binding to substrates and inhibitors exist between amebic and mammalian FTs, which highlights this enzyme as a novel target for the development of new chemotherapeutics against amebiasis.     

Rab5-associated vacuoles play a unique role in phagocytosis of the enteric protozoan parasite Entamoeba histolytica

In mammals, Rab5 and Rab7 play a specific and coordinated role in a sequential process during phagosome maturation. Here, we report that Rab5 and Rab7 in the enteric protozoan parasite Entamoeba histolytica, EhRab5 and EhRab7A, are involved in steps that are distinct from those known for mammals. EhRab5 and EhRab7A were localized to independent small vesicular structures at steady state. Priming with red blood cells induced the formation of large vacuoles associated with both EhRab5 and EhRab7A ("prephagosomal vacuoles (PPV)") in the amoeba within an incubation period of 5-10 min. PPV emerged de novo physically and distinct from phagosomes. PPV were gradually acidified and matured by fusion with lysosomes containing a digestive hydrolase, cysteine proteinase, and a membrane-permeabilizing peptide amoebapore. After EhRab5 dissociated from PPV, 5-10 min later, the EhRab7A-PPV fused with phagosomes, and EhRab7A finally dissociated from the phagosomes. Immunoelectron and light micrographs showed that PPV contained small vesicle-like structures containing fluid-phase markers and amoebapores, which were not evenly distributed within PPV, suggesting that the mechanism was similar to multivesicular body formation in PPV generation. In contrast to Rab5 from other organisms, EhRab5 was involved exclusively in phagocytosis, but not in endocytosis. Overexpression of wild-type EhRab5 enhanced phagocytosis and the transport of amoebapore to phagosomes. Conversely, expression of an EhRab5Q67L GTP form mutant impaired the formation of PPV and phagocytosis. Altogether, we propose that the amoebic Rab5 plays an important role in the formation of unique vacuoles, which is essential for engulfment of erythrocytes and important for packaging of lysosomal hydrolases, prior to the targeting to phagosomes.     

Biosynthesis and catabolism of mannitol is developmentally regulated in the protozoan parasite Eimeria tenella

The mannitol cycle is a metabolic branch of the glycolytic pathway found in Eimeria tenella. In this paper, we describe the biosynthesis and consumption of mannitol during parasite development. Low micromolar levels of mannitol were detected in all of the asexual stages and mannitol production increased sharply during the sexual phase of the life cycle. Unsporulated oocysts had high mannitol content (300 mM or 25% of the oocyst mass). Mannitol-1-phosphate dehydrogenase (M1PDH), the first committed step of the mannitol cycle, was also elevated in sexual stages and this coincides with mannitol levels. Approximately 90% of the mannitol present in unsporulated oocysts was consumed in the first 15 hr of sporulation, and levels continued to drop until the sporulation process was complete at approximately 35 hr. Thus, mannitol appears to be the "fuel" for sporulation during the vegetative stage of the parasite life cycle. Evaluation of oocyst extracts from 6 additional Eimeria species for mannitol content and the presence of M1PDH indicated that the mannitol cycle was broadly present in this genus. This finding combined with the lack of mannitol metabolism in higher eukaryotes makes this pathway an attractive chemotherapeutic target.     

The intestinal protozoan parasite Entamoeba histolytica contains 20 cysteine protease genes,of which only a small subset is expressed during in vitro cultivation

Cysteine proteases are known to be important pathogenicity factors of the protozoan parasite Entamoeba histolytica. So far, a total of eight genes coding for cysteine proteases have been identified in E. histolytica, two of which are absent in the closely related nonpathogenic species E. dispar. However, present knowledge is restricted to enzymes expressed during in vitro cultivation of the parasite, which might represent only a subset of the entire repertoire. Taking advantage of the current E. histolytica genome-sequencing efforts, we analyzed databases containing more than 99% of all ameba gene sequences for the presence of cysteine protease genes. A total of 20 full-length genes was identified (including all eight genes previously reported), which show 10 to 86% sequence identity. The various genes obviously originated from two separate ancestors since they form two distinct clades. Despite cathepsin B-like substrate specificities, all of the ameba polypeptides are structurally related to cathepsin L-like enzymes. None of the previously described enzymes but 7 of the 12 newly identified proteins are unique compared to cathepsins of higher eukaryotes in that they are predicted to have transmembrane or glycosylphosphatidylinositol anchor attachment domains. Southern blot analysis revealed that orthologous sequences for all of the newly identified proteases are present in E. dispar. Interestingly, the majority of the various cysteine protease genes are not expressed in E. histolytica or E. dispar trophozoites during in vitro cultivation. Therefore, it is likely that at least some of these enzymes are required for infection of the human host and/or for completion of the parasite life cycle.     

Involvement of Src family tyrosine kinase in apoptosis of human neutrophils induced by protozoan parasite Entamoeba histolytica

Tyrosine kinases are one of the most important regulators for intracellular signal transduction related to inflammatory responses. However, there are no reports describing the effects of tyrosine kinases on neutrophil apoptosis induced by Entamoeba histolytica. In this study, isolated human neutrophils from peripheral blood were incubated with live trophozoites in the presence or absence of tyrosine kinase inhibitors. Entamoeba-induced receptor shedding of CD16 and PS externalization in neutrophils were inhibited by pre-incubation of neutrophils with the broad-spectrum tyrosine kinase inhibitor genistein or the Src family kinase inhibitor PP2. Entamoeba-induced ROS production was also inhibited by genistein or PP2. Moreover, genistein and PP2 blocked the phosphorylation of ERK and p38 MAPK in neutrophils induced by E. histolytica. These results suggest that Src tyrosine kinases may participate in the signaling event for ROS-dependent activation of MAPKs during neutrophil apoptosis induced by E. histolytica.     

Identification and characterization of EhCaBP2. A second member of the calcium-binding protein family of the protozoan parasite Entamoeba histolytica

Entamoeba histolytica, an early branching eukaryote, is the etiologic agent of amebiasis. Calcium plays a pivotal role in the pathogenesis of amebiasis by modulating the cytopathic properties of the parasite. However, the mechanistic role of Ca(2+) and calcium-binding proteins in the pathogenesis of E. histolytica remains poorly understood. We had previously characterized a novel calcium-binding protein (EhCaBP1) from E. histolytica. Here, we report the identification and partial characterization of an isoform of this protein, EhCaBP2. Both EhCaBPs have four canonical EF-hand Ca(2+) binding domains. The two isoforms are encoded by genes of the same size (402 bp). Comparison between the two genes showed an overall identity of 79% at the nucleotide sequence level. This identity dropped to 40% in the 75-nucleotide central linker region between the second and third Ca(2+) binding domains. Both of these genes are single copy, as revealed by Southern hybridization. Analysis of the available E. histolytica genome sequence data suggested that the two genes are non-allelic. Homology-based structural modeling showed that the major differences between the two EhCaBPs lie in the central linker region, normally involved in binding target molecules. A number of studies indicated that EhCaBP1 and EhCaBP2 are functionally different. They bind different sets of E. histolytica proteins in a Ca(2+)-dependent manner. Activation of endogenous kinase was also found to be unique for the two proteins and the Ca(2+) concentration required for their optimal functionality was also different. In addition, a 12-mer peptide was identified from a random peptide library that could differentially bind the two proteins. Our data suggest that EhCaBP2 is a new member of a class of E. histolytica calcium-binding proteins involved in a novel calcium signal transduction pathway.     

Rab11B small GTPase regulates secretion of cysteine proteases in the enteric protozoan parasite Entamoeba histolytica

Vesicular trafficking plays a pivotal role in the virulence of the enteric protozoan parasite Entamoeba histolytica. In the present study, we showed that one isotype of the small GTPase Rab11, EhRab11B, plays a central role in the secretion of a major virulence factor, cysteine proteases. EhRab11B did not colocalize with markers for the endoplasmic reticulum, early endosomes and lysosomes, but was partially associated with non-acidified vesicles in the endocytic pathway, likely recycling endosomes. Overexpression of EhRab11B resulted in a remarkable increase in both intracellular and secreted cysteine protease activity, concomitant with an augmentation of cytolytic activity as demonstrated by an increased ability to destroy mammalian cells. The oversecretion of cysteine proteases with EhRab11B overexpression was neither sensitive to brefeldin A nor specific to a certain cysteine protease species (e.g. CP1, 2 or 5), suggesting that these three major cysteine proteases are trafficked via an EhRab11B-associated secretory pathway, which is distinct from the classical brefeldin-sensitive pathway. Overexpression of EhRab11B also enhanced exocytosis of the incorporated fluid-phase marker, supporting the notion that it is involved in recycling. This is the first report demonstrating that Rab11 plays a central role in the transport and secretion of pathogenic factors.     

Differential use of multiple replication origins in the ribosomal DNA episome of the protozoan parasite Entamoeba histolytica

The factors that control the initiation of eukaryotic DNA replication from defined origins (oris) on the chromosome remain incompletely resolved. Here we show that the circular rDNA episome of the human pathogen Entamoeba histolytica contains multiple potential oris, which are utilized in a differential manner. The primary ori in exponentially growing cells was mapped close to the promoter of rRNA genes in the upstream intergenic spacer (IGS) by two-dimensional gel electrophoresis. Replication initiated predominantly from the upstream IGS and terminated in the downstream IGS. However, when serum-starved cells were allowed to resume growth, the early oris which became activated were located in other parts of the molecule. Later the ori in the upstream IGS became activated, with concomitant silencing of the early oris. When the upstream IGS was located ectopically in an artificial plasmid, it again lost ori activity, while other parts of the rDNA episome could function as oris in this system. Therefore, the activation or silencing of the ori in this episome is context dependent, as is also the case with many eukaryotic replicons. This is the first replication origin to be mapped in this primitive protozoan and will provide an opportunity to define the factors involved in differential ori activity, and their comparison with metazoans.     

Hsp60 is targeted to a cryptic mitochondrion-derived organelle ("crypton") in the microaerophilic protozoan parasite Entamoeba histolytica

Entamoeba histolytica is a microaerophilic protozoan parasite in which neither mitochondria nor mitochondrion-derived organelles have been previously observed. Recently, a segment of an E. histolytica gene was identified that encoded a protein similar to the mitochondrial 60-kDa heat shock protein (Hsp60 or chaperonin 60), which refolds nuclear-encoded proteins after passage through organellar membranes. The possible function and localization of the amebic Hsp60 were explored here. Like Hsp60 of mitochondria, amebic Hsp60 RNA and protein were both strongly induced by incubating parasites at 42 degreesC. 5' and 3' rapid amplifications of cDNA ends were used to obtain the entire E. histolytica hsp60 coding region, which predicted a 536-amino-acid Hsp60. The E. histolytica hsp60 gene protected from heat shock Escherichia coli groEL mutants, demonstrating the chaperonin function of the amebic Hsp60. The E. histolytica Hsp60, which lacked characteristic carboxy-terminal Gly-Met repeats, had a 21-amino-acid amino-terminal, organelle-targeting presequence that was cleaved in vivo. This presequence was necessary to target Hsp60 to one (and occasionally two or three) short, cylindrical organelle(s). In contrast, amebic alcohol dehydrogenase 1 and ferredoxin, which are bacteria-like enzymes, were diffusely distributed throughout the cytosol. We suggest that the Hsp60-associated, mitochondrion-derived organelle identified here be named "crypton," as its structure was previously hidden and its function is still cryptic.