Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells

Entamoeba histolytica is the protozoan parasite that causes invasive amebiasis, which is endemic to many developing countries and characterized by dysentery and liver abscesses. The virulence of E. histolytica correlates with the degree of host cell engulfment, or phagocytosis, and E. histolytica phagocytosis alters amebic gene expression in a feed-forward manner that results in an increased phagocytic ability. Here, we used a streamlined RNA interference screen to silence the expression of 15 genes whose expression was upregulated in phagocytic E. histolytica trophozoites to determine whether these genes actually function in the phagocytic process. When five of these genes were silenced, amebic strains with significant decreases in the ability to phagocytose apoptotic host cells were produced. Phagocytosis of live host cells, however, was largely unchanged, and the defects were surprisingly specific for phagocytosis. Two of the five encoded proteins, which we named E. histolytica ILWEQ (EhILWEQ) and E. histolytica BAR (EhBAR), were chosen for localization via SNAP tag labeling and localized to the site of partially formed phagosomes. Therefore, both EhILWEQ and EhBAR appear to contribute to E. histolytica virulence through their function in phagocytosis, and the large proportion (5/15 [33%]) of gene-silenced strains with a reduced ability to phagocytose host cells validates the previously published microarray data set demonstrating feed-forward control of E. histolytica phagocytosis. Finally, although only limited conclusions can be drawn from studies using the virulence-deficient G3 Entamoeba strain, the relative specificity of the defects induced for phagocytosis of apoptotic cells but not healthy cells suggests that cell killing may play a rate-limiting role in the process of Entamoeba histolytica host cell engulfment.     

Participation of the serine-rich Entamoeba histolytica protein in amebic phagocytosis of apoptotic host cells

Entamoeba histolytica is an intestinal ameba that causes dysentery and liver abscesses. Cytotoxicity and phagocytosis of host cells characterize invasive E. histolytica infection. Prior to phagocytosis of host cells, E. histolytica induces apoptotic host cell death, using a mechanism that requires contact via an amebic galactose-specific lectin. However, lectin inhibition only partially blocks phagocytosis of already dead cells, implicating at least one additional receptor in phagocytosis. To identify receptors for engulfment of apoptotic cells, monoclonal antibodies against E. histolytica membrane antigens were screened for inhibition of phagocytosis. Of 43 antibodies screened, one blocked lectin-independent uptake of apoptotic cells, with >90% inhibition at a dose of 20 μg/ml (P < 0.0003 versus control). The same antibody also inhibited adherence to apoptotic lymphocytes and, to a lesser extent, adherence to and killing of viable lymphocytes. The antigen recognized by the inhibitory antibody was purified by affinity chromatography and identified by liquid chromatography-mass spectrometry as the serine-rich E. histolytica protein (SREHP). Consistent with this, the inhibitory antibody bound to recombinant SREHP present in bacterial lysates on immunoblots. The SREHP is an abundant immunogenic surface protein of unclear function. The results of this unbiased antibody screen strongly implicate the SREHP as a participant in E. histolytica phagocytosis and suggest that it may play an important role in adherence to apoptotic cells.     

Intestinal amoebiasis: 160 years of its first detection and still remains as a health problem in developing countries

Amoebiasis is a parasitic disease caused by Entamoeba histolytica (E. histolytica), an extracellular enteric protozoan. This infection mainly affects people from developing countries with limited hygiene conditions, where it is endemic. Infective cysts are transmitted by the fecal-oral route, excysting in the terminal ileum and producing invasive trophozoites (amoebae). E. histolytica mainly lives in the large intestine without causing symptoms; however, possibly as a result of so far unknown signals, the amoebae invade the mucosa and epithelium causing intestinal amoebiasis. E. histolytica possesses different mechanisms of pathogenicity for the adherence to the intestinal epithelium and for degrading extracellular matrix proteins, producing tissue lesions that progress to abscesses and a host acute inflammatory response. Much information has been obtained regarding the virulence factors, metabolism, mechanisms of pathogenicity, and the host immune response against this parasite; in addition, alternative treatments to metronidazole are continually emerging. An accesible and low-cost diagnostic method that can distinguish E. histolytica from the most nonpathogenic amoebae and an effective vaccine are necessary for protecting against amoebiasis. However, research about the disease and its prevention has been a challenge due to the relationship between E. histolytica and the host during the distinct stages of the disease is multifaceted. In this review, we analyze the interaction between the parasite, the human host, and the colon microbiota or pathogenic microorganisms, which together give rise to intestinal amoebiasis.     

Bacterial Expression of a Human Monoclonal Antibody-Alkaline Phosphatase Conjugate Specific for Entamoeba histolytica

We previously produced human monoclonal antibody Fab fragments specific to Entamoeba histolytica in Escherichia coli. In order to use these Fab fragments for diagnostic purposes, an expression vector to produce a fusion protein of Fab and alkaline phosphatase (PhoA) in E. coli was designed and constructed. The E. coli PhoA gene was fused to the 3′ terminus of the gene encoding the heavy-chain Fd region. The kappa and Fd genes from a previously prepared antibody clone, CP33, which is specific for the 260-kDa lectin of E. histolytica, were used as human antibody genes. When the fusion protein of CP33 and PhoA was incubated with paraformaldehyde-fixed trophozoites of E. histolytica and developed with a substrate, the trophozoites appeared to be stained. These results demonstrate the feasibility of bacterial expression of a human monoclonal antibody-PhoA conjugate specific for E. histolytica and that the antibody can be used to detect E. histolytica antigen without the use of chemically conjugated secondary antibodies.     

Entamoeba histolytica Induces Host Cell Death in Amebic Liver Abscess by a Non-Fas-Dependent, Non-Tumor Necrosis Factor Alpha-Dependent Pathway of Apoptosis

Amebic liver abscess is characterized by extensive areas of dead hepatocytes that form cavities surrounded by a thin rim of inflammatory cells and few Entamoeba histolytica trophozoites. E. histolytica produces pore-forming proteins and proteinases, but how trophozoites actually kill host cells has been unclear. Here, we report that E. histolytica induces apoptosis in both inflammatory cells and hepatocytes in a severe combined immunodeficient (SCID) mouse model of amebic liver abscess. By studying infection in C57/BL6.lpr and C57/BL6.gld mice, we found that E. histolytica-induced apoptosis does not require the Fas/Fas ligand pathway of apoptosis, and by using mice with a targeted deletion of the tumor necrosis factor receptor I gene, we have shown that E. histolytica-induced apoptosis is not mediated by tumor necrosis factor alpha. Our data indicate that apoptosis plays a prominent role in the host cell death seen in amebic liver abscess in a mouse model of disease and suggest that E. histolytica induces cell death without using two common pathways for apoptosis.     

Comparison of PCR, Isoenzyme Analysis, and Antigen Detection for Diagnosis of Entamoeba histolytica Infection

The diagnosis of amebiasis by microscopic identification of the parasite in stool is insensitive and unable to distinguish the invasive parasite Entamoeba histolytica from the commensal parasite E. dispar. In this study, we have tested a PCR technique for the detection of E. histolytica and compared it with isoenzyme analysis and the TechLab E. histolytica-specific antigen detection test. The nested-PCR test we used is based on amplification of the small subunit rRNA gene of E. histolytica and E. dispar followed by restriction digest analysis of the PCR product. Single stool samples were obtained from 98 patients from Dhaka, Bangladesh, with diarrhea: 88 patients diagnosed by microscopy and/or culture with E. histolytica and/or E. dispar infection and 10 patients without infection. Isoenzyme analysis identified 53 of the infections as E. histolytica and 28 as E. dispar. PCR and isoenzyme identification of E. histolytica agreed in 96% (51 of 53) of amebic cultures. PCR for E. histolytica was negative in all 10 samples that were negative for E. histolytica by isoenzyme and antigen detection. PCR and antigen detection had comparable sensitivities when performed directly on fresh stool specimens, identifying 87% (46 of 53) and 85% (45 of 53), respectively, of E. histolytica infections identified by isoenzyme analysis. The correlation of results by antigen detection and PCR for identification of E. histolytica in stool was 93% (45 of 48 cases). Mixed infections with E. histolytica and E. dispar were detected by PCR in 14% (12 of 88) of cases. In conclusion, all three techniques for specific identification of E. histolytica in fresh stool showed excellent correlation. Only the TechLab E. histolytica antigen detection test was both rapid and technically simple.     

The human macrophage mannose receptor is not a professional phagocytic receptor

The macrophage mannose receptor (MR) appears to play an important role in the binding and phagocytosis of several human pathogens, but its phagocytic property and signaling pathways have been poorly defined. The general strategy to explore such topics is to express the protein of interest in nonphagocytic cells, but in the case of MR, there are few reports using the full-length MR cDNA. When we searched to clone de novo the human MR (hMR) cDNA, problems were encountered, and full-length hMR cDNA was only obtained after devising a complex cloning strategy. Chinese hamster ovary cells, which have a fully functional phagocytic machinery when expressing professional phagocytic receptors, were stably transfected, and cell clones expressing hMR at quantitatively comparable levels than human macrophages or J774E cells were obtained. They exhibited a functional hMR-mediated endocytic capacity of a soluble ligand but failed to ingest classical particulate ligands of MR such as zymosan, Mycobacterium kansasii, or trimannoside bovine serum albumin-coated latex beads. Transient expression of hMR in two human cell lines did not provide a phagocytic capacity either. In conclusion, we show that MR is not a professional phagocytic receptor, as it does not possess the ability to promote particle ingestion in nonphagocytic cells on its own. We propose that MR is a binding receptor, which requires a partner to trigger phagocytosis in some specialized cells such as macrophages. Our new expression vector could represent a useful tool to study the receptor and its partnership further.     

Prostaglandin E2 produced by Entamoeba histolytica binds to EP4 receptors and stimulates interleukin-8 production in human colonic cells

Entamoeba histolytica pathogenesis in the colon occurs in a stepwise fashion. It begins with colonization of the mucin layer, which is followed by stimulation of a proinflammatory response that causes nonspecific tissue damage that may facilitate parasite invasion of the underlying colonic mucosa. Unfortunately, the parasite and/or host factors that stimulate a proinflammatory response in the gut are poorly understood. In this study, we found that live E. histolytica or secretory or proteins (SP) and soluble ameba components (SAP) can markedly increase interleukin-8 (IL-8) mRNA expression and protein production in colonic epithelial cells. The IL-8-stimulating molecule produced by live amebae was identified as prostaglandin E₂ (PGE₂) as trophozoites treated with cyclooxygenase inhibitors inhibited the biosynthesis of PGE₂ and eliminated IL-8 production induced by live parasites or ameba components. Moreover, using specific prostaglandin EP2 and EP4 receptor agonists and antagonists, we found that PGE₂ binds exclusively through EP4 receptors in colonic epithelial cells to stimulate IL-8 production. Silencing of EP4 receptors with EP4 small interfering RNA completely eliminated SP- and SAP-induced IL-8 production. These studies identified bioactive PGE₂ as a one of the major virulence factors produced by E. histolytica that can stimulate the potent neutrophil chemokine and activator IL-8, which can trigger an acute host inflammatory response. Thus, the induction of IL-8 production in response to E. histolytica-derived PGE₂ may be a mechanism that explains the initiation and amplification of acute inflammation associated with intestinal amebiasis.     

The Macrophage Migration Inhibitory Factor Homolog of Entamoeba histolytica Binds to and Immunomodulates Host Macrophages

The host inflammatory response contributes to the tissue damage that occurs during amebic colitis, with tumor necrosis factor alpha (TNF-α) being a key mediator of the gut inflammation observed. Mammalian macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that plays an important role in the exacerbation of a wide range of inflammatory diseases, including colitis. We identified a MIF gene homolog in the Entamoeba histolytica genome, raising the question of whether E. histolytica MIF (EhMIF) has proinflammatory activity similar to that of mammalian MIF. In this report, we describe the first functional characterization of EhMIF. Antibodies were prepared against recombinantly expressed EhMIF and used to demonstrate that EhMIF is expressed as a 12-kDa protein localized to the cytoplasm of trophozoites. In a manner similar to that of mammalian MIF, EhMIF interacted with the MIF receptor CD74 and bound to macrophages. EhMIF induced interleukin-6 (IL-6) production. In addition, EhMIF enhanced TNF-α secretion by amplifying TNF-α production by lipopolysaccharide (LPS)-stimulated macrophages and by inhibiting the glucocorticoid-mediated suppression of TNF-α secretion. EhMIF was expressed during human infection, as evidenced by the presence of anti-EhMIF antibodies in the sera of children living in an area where E. histolytica infection is endemic. Anti-EhMIF antibodies did not cross-react with human MIF. The ability of EhMIF to modulate host macrophage function may promote an exaggerated proinflammatory immune response and contribute to the tissue damage seen in amebic colitis.     

Antigen-Specific Gene Expression Profiles of Peripheral Blood Mononuclear Cells Do Not Reflect Those of T-Lymphocyte Subsets

Advances in microarray technology have allowed for the monitoring of thousands of genes simultaneously. This technology is of particular interest to immunologists studying infectious diseases, because it provides tremendous potential for investigating host-pathogen interactions at the level of immune gene expression. To date, many studies have focused either on cell lines, where the physiological relevance is questionable, or on mixed cell populations, where the contributions of individual subpopulations are unknown. In the present study, we perform an intrasubject comparison of antigen-stimulated immune gene expression profiles between a mixed population of peripheral blood mononuclear cells (PBMC) and the two predominant cell types found in PBMC, CD4⁺ and CD8⁺ T lymphocytes. We show that the microarray profiles of CD4⁺ and CD8⁺ T lymphocytes differ from each other as well as from that of the mixed cell population. The independence of the gene expression profiles of different cell types is demonstrated with a ubiquitous antigen (Candida albicans) as well as with a disease-specific antigen (human immunodeficiency virus p24). This study has important implications for microarray studies of host immunity and underscores the importance of profiling the expression of specific cell types.     

Germination of phagocytosed E. cuniculi spores does not significantly contribute to parasitophorous vacuole formation in J774 cells

The obligate intracellular microsporidia have developed a unique invasion mechanism to infect their host cells. Spores explosively evert a tube-like structure and extrude the infectious spore content through this organelle into the host cell. Spores from species of the genus Encephalitozoon were also shown to be efficiently internalized by phagocytosis, which led to the hypothesis that spore germination from inside a phagosome might contribute to the infection process. Here, we challenge this hypothesis by quantifying Encephalitozoon cuniculi infection rates of J774 cells that were incubated with the phagocytosis inhibitor cytochalasin D. We demonstrate that the invasion rate in cytochalasin D-treated cells is identical to untreated controls, although phagocytic uptake of E. cuniculi spores was less than 10% of control samples. This study suggests that germination of phagocytosed spores is not a significant infection mode for E. cuniculi.     

Localization of Phosphatidylinositol 4,5-Bisphosphate to Lipid Rafts and Uroids in the Human Protozoan Parasite Entamoeba histolytica

Entamoeba histolytica is an intestinal protozoan parasite and is the causative agent of amoebiasis. During invasive infection, highly motile amoebae destroy the colonic epithelium, enter the blood circulation, and disseminate to other organs such as liver, causing liver abscess. Motility is a key factor in E. histolytica pathogenesis, and this process relies on a dynamic actomyosin cytoskeleton. In other systems, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is known to regulate a wide variety of cellular functions, including signal transduction, actin remodeling, and cell motility. Little is known about the role of PI(4,5)P₂ in E. histolytica pathogenicity. In this study, we demonstrate that PI(4,5)P₂ is localized to cholesterol-rich microdomains, lipid rafts, and the actin-rich fractions of the E. histolytica membrane. Microscopy revealed that the trailing edge of polarized trophozoites, uroids, are highly enriched in lipid rafts and their constituent lipid, PI(4,5)P₂. Polarization and enrichment of uroids and rafts with PI(4,5)P₂ were enhanced upon treatment of E. histolytica cells with cholesterol. Exposure to cholesterol also increased intracellular calcium, which is a downstream effector of PI(4,5)P₂, with a concomitant increase in motility. Together, our data suggest that in E. histolytica, PI(4,5)P₂ may signal from lipid rafts and cholesterol may play a role in triggering PI(4,5)P₂-mediated signaling to enhance the motility of this pathogen.     

Molecular biology of the hexokinase isoenzyme pattern that distinguishes pathogenic Entamoeba histolytica from nonpathogenic Entamoeba dispar

The electrophoretic patterns of hexokinase and phosphoglucomutase have been widely used to distinguish Entamoeba histolytica from Entamoeba dispar isolates. Although E. histolytica and E. dispar, previously called pathogenic and nonpathogenic Entamoeba histolytica, differ clearly in sequences of many homologous genes, a conversion between the two has been reported by several laboratories, in each case showing the conversion of hexokinase (ATP, d-hexose 6-phosphotransferase, EC 2.7.1.1) isoenzyme patterns. An apparent mobility shift of this enzyme may either be due to posttranslational modification or processing, or to the appearance of a new isoform encoded by a second gene. In this study we observed that the four observed bands in the isoenzyme patterns of pathogenic and nonpathogenic forms of Entamoeba were correlated with four different cDNAs, and that the four recombinant hexokinases produced in Escherichia coli comigrated with their natural counterparts. Polymerase chain reaction (PCR) experiments did not reveal hidden genes which might be responsible for conversion phenomena. These results strongly support the redefinition of pathogenic and nonpathogenic Entamoeba histolytica as two closely related species Entamoeba histolytica and Entamoeba dispar.     

Effects of Panmede and various horse serum concentrations on the axenic cultivation ofEntamoeba histolytica strains in TPS-1 medium

We analyzed the influence of Panmede and horse serum concentrations on the growth of fiveEntamoeba histolytica strains (HK9, HM1, HM2, HM3 and HM38) axenically cultivated in TPS-1 medium. Panmede was evaluated by comparing the growth of strain HM1 in medium prepared with each of 15 Panmede lots; the yields ofE. histolytica trophozoites depended on the lot quality of Panmede, and their maximal values ranged from 8×10³ to 8.9×10⁴ amoebae/ml. The growth-promoting effect of eight lots of horse serum on strains HK9 and HM1 were studied using a single Panmede lot of good quality. Yields obtained with strain HK9 ranged from 8×10⁴ to 1.8×10⁵ amoebae/ml, whereas yields obtained with HM1 ranged from 3×10⁴ to 1.2×10⁵ amoebae/ml. Thus, the optimal serum concentration in TPS-1 medium that caused maximal growth ofE. histolytica cultures depended on the quality of the serum lot and proved to be specific for each of the fiveE. histolytica strains investigated. It ranged from 18% (v/v) for strain HM2 to 28% (v/v) for strain HM1. Our results reveal that the growth ofE. histolytica trophozoites in TPS-1 medium can be distinctly improved by selecting appropriate lots of Panmede and horse serum and using optimal serum concentrations.     

DNA characterization of simian <Emphasis Type="Italic">Entamoeba histolytica</Emphasis>-like strains to differentiate them from <Emphasis Type="Italic">Entamoeba histolytica</Emphasis>

Two simian Entamoeba histolytica-like strains, EHMfas1 and P19-061405, have been suggested to represent a new species based on genetic characterization. Sequence analyses of the hexokinase, glucose phosphate isomerase, and phosphoglucomutase genes supported the previous findings of isoenzyme analyses demonstrating a new zymodeme pattern. Phylogenetic studies of 18S rDNA, 5.8S rDNA, the chaperonin 60 gene, and the pyridine nucleotide transhydrogenase gene showed original clusters of simian E. histolytica-like strains below or near E. histolytica, respectively. Comparative studies of the chitinase and the serine-rich E. histolytica protein genes and locus 1–2 region revealed that most mutated units were shared among the simian E. histolytica-like strains. The similarities of each of the repeating units within the simian E. histolytica-like strains or E. histolytica and the differences of those between the both might be generated by concerted evolution. Our results indicate that EHMfas1 and P19-061405 should be considered to be the same species, despite that they were isolated from different monkey species and different habitats. Simian E. histolytica-like amebas may be endemic to macaque monkeys, as a counterpart to E. histolytica in humans, and should be differentiated from E. histolytica by the revival name Entamoeba nuttalli, as proposed for P19-061405.     

In vivo identification of novel regulators and conserved pathways of phagocytosis in A. gambiae

Anopheles gambiae uses effective immune responses, including phagocytosis, to fight microbial infection. We have developed a semiquantitative phagocytosis test and used it in conjunction with dsRNA gene silencing to test the in vivo roles of 71 candidate genes in phagocytosis of Escherichia coli and Staphylococcus aureus. Here, we show that inactivation of 26 genes changes the phagocytic activity by more than 45% and that two pathways similar to those that mediate apoptotic cell removal in Caenorhabditis elegans are used in A. gambiae for phagocytosis of microorganisms. Simultaneous inactivation of the identified regulators of phagocytosis and conserved components defining each signaling pathway permitted provisional assignment of the novel regulators to one or the other pathway. Pathway inactivation enhances at least three times the ability of E. coli and S. aureus to proliferate in the mosquito. Interestingly, mosquito survival is not compromised even if both pathways are perturbed simultaneously.