Amebic Virulence Research Articles

Characteristics of inflammatory reactions during development of liver abscess in hamsters inoculated with Entamoeba nuttalli

Background Entamoeba nuttalli is an intestinal protozoan with pathogenic potential that can cause amoebic liver abscess. It is highly prevalent in wild and captive macaques. Recently, cysts were detected in a caretaker of nonhuman primates in a zoo, indicating that E. nuttalli may be a zoonotic pathogen. Therefore, it is important to evaluate the pathogenicity of E. nuttalli in detail and in comparison with that of E. histolytica. Methodology/Principal findings Trophozoites of E. nuttalli GY4 and E. histolytica SAW755 strains were inoculated into liver of hamsters. Expression levels of proinflammatory factors of hamsters and virulence factors from E. histolytica and E. nuttalli were compared between the two parasites. Inoculations with trophozoites of E. nuttalli resulted in an average necrotic area of 24% in liver tissue in 7 days, whereas this area produced by E. histolytica was nearly 50%. Along with the mild liver tissue damage induced by E. nuttalli, expression levels of proinflammatory factors (TNF-α, IL-6 and IL-1β) and amebic virulence protein genes (lectins, cysteine proteases and amoeba pores) in local tissues were lower with E. nuttalli in comparison with E. histolytica. In addition, M2 type macrophages were increased in E. nuttalli-induced amebic liver abscesses in the late stage of disease progression and lysate of E. nuttalli trophozoites induced higher arginase expression than E. histolytica in vitro. Conclusions/Significance The results show that differential secretion of amebic virulence proteins during E. nuttalli infection triggered lower levels of secretion of various cytokines and had an impact on polarization of macrophages towards a M1/M2 balance. However, regardless of the degree of macrophage polarization, there is unambiguous evidence of an intense acute inflammatory reaction in liver of hamsters after infection by both Entamoeba species.

Characteristics of inflammatory reactions during development of liver abscess in hamsters inoculated with Entamoeba nuttalli.

BackgroundEntamoeba nuttalli is an intestinal protozoan with pathogenic potential that can cause amebic liver abscess. It is highly prevalent in wild and captive macaques. Recently, cysts were detected in a caretaker of nonhuman primates in a zoo, indicating that E. nuttalli may be a zoonotic pathogen. Therefore, it is important to evaluate the pathogenicity of E. nuttalli in detail and in comparison with that of E. histolytica.Methodology/Principal findingsTrophozoites of E. nuttalli GY4 and E. histolytica SAW755 strains were inoculated into liver of hamsters. Expression levels of proinflammatory factors of hamsters and virulence factors from E. histolytica and E. nuttalli were compared between the two parasites. Inoculations with trophozoites of E. nuttalli resulted in an average necrotic area of 24% in liver tissue in 7 days, whereas this area produced by E. histolytica was nearly 50%. Along with the mild liver tissue damage induced by E. nuttalli, expression levels of proinflammatory factors (TNF-α, IL-6 and IL-1β) and amebic virulence protein genes (lectins, cysteine proteases and amoeba pores) in local tissues were lower with E. nuttalli in comparison with E. histolytica. In addition, M2 type macrophages were increased in E. nuttalli-induced amebic liver abscesses in the late stage of disease progression and lysate of E. nuttalli trophozoites induced higher arginase expression than E. histolytica in vitro.Conclusions/SignificanceThe results show that differential secretion of amebic virulence proteins during E. nuttalli infection triggered lower levels of secretion of various cytokines and had an impact on polarization of macrophages towards a M1/M2 balance. However, regardless of the degree of macrophage polarization, there is unambiguous evidence of an intense acute inflammatory reaction in liver of hamsters after infection by both Entamoeba species.

Toll-like Receptor Signaling Activation by Entamoeba histolytica Induces Beta Defensin 2 in Human Colonic Epithelial Cells: Its Possible Role as an Element of the Innate Immune Response

Background Entamoeba histolytica, a protozoan parasite of humans, produces dysenteric diarrhea, intestinal mucosa damage and extraintestinal infection. It has been proposed that the intestinal microbiota composition could be an important regulatory factor of amebic virulence and tissue invasion, particularly if pathogenic bacteria are present. Recent in vitro studies have shown that Entamoeba histolytica trophozoites induced human colonic CaCo2 cells to synthesize TLR-2 and TLR-4 and proinflammatory cytokines after binding to the amebic Gal/GalNac lectin carbohydrate recognition domain. The magnitude of the inflammatory response induced by trophozoites and the subsequent cell damage were synergized when cells had previously been exposed to pathogenic bacteria.Methodology/Principal FindingsWe show here that E. histolytica activation of the classic TLR pathway in CaCo2 cells is required to induce β defensin-2 (HBD2) mRNA expression and production of a 5-kDa cationic peptide with similar properties to the antimicrobial HBD2 expressed by CaCo2 cells exposed to enterotoxigenic Escherichia coli. The induced peptide showed capacity to permeabilize membranes of bacteria and live trophozoites. This activity was abrogated by inhibition of TLR2/4-NFκB pathway or by neutralization with an anti-HBD2 antibody.Conclusions/Significance Entamoeba histolytica trophozoites bind to human intestinal cells and induce expression of HBD2; an antimicrobial molecule with capacity to destroy pathogenic bacteria and trophozoites. HDB2's possible role as a modulator of the course of intestinal infections, particularly in mixed ameba/bacteria infections, is discussed.

Exploring the Possible Role of Lysine Acetylation onEntamoeba histolyticaVirulence: A Focus on the Dynamics of the Actin Cytoskeleton

Cytoskeleton remodeling can be regulated, among other mechanisms, by lysine acetylation. The role of acetylation on cytoskeletal and other proteins of Entamoeba histolytica has been poorly studied. Dynamic rearrangements of the actin cytoskeleton are crucial for amebic motility and capping formation, processes that may be effective means of evading the host immune response. Here we report the possible effect of acetylation on the actin cytoskeleton dynamics and in vivo virulence of E. histolytica. Using western blot, immunoprecipitation, microscopy assays, and in silico analysis, we show results that strongly suggest that the increase in Aspirin-induced cytoplasm proteins acetylation reduced cell movement and capping formation, likely as a consequence of alterations in the structuration of the actin cytoskeleton. Additionally, intrahepatic inoculation of Aspirin-treated trophozoites in hamsters resulted in severe impairment of the amebic virulence. Taken together, these results suggest an important role for lysine acetylation in amebic invasiveness and virulence.

The Antiretroviral Lectin Cyanovirin-N Targets Well-Known and Novel Targets on the Surface of Entamoeba histolytica Trophozoites

Entamoeba histolytica, the protist that causes amebic dysentery and liver abscess, has a truncated Asn-linked glycan (N-glycan) precursor composed of seven sugars (Man(5)GlcNAc(2)). Here, we show that glycoproteins with unmodified N-glycans are aggregated and capped on the surface of E. histolytica trophozoites by the antiretroviral lectin cyanovirin-N and then replenished from large intracellular pools. Cyanovirin-N cocaps the Gal/GalNAc adherence lectin, as well as glycoproteins containing O-phosphodiester-linked glycans recognized by an anti-proteophosphoglycan monoclonal antibody. Cyanovirin-N inhibits phagocytosis by E. histolytica trophozoites of mucin-coated beads, a surrogate assay for amebic virulence. For technical reasons, we used the plant lectin concanavalin A rather than cyanovirin-N to enrich secreted and membrane proteins for mass spectrometric identification. E. histolytica glycoproteins with occupied N-glycan sites include Gal/GalNAc lectins, proteases, and 17 previously hypothetical proteins. The latter glycoproteins, as well as 50 previously hypothetical proteins enriched by concanavalin A, may be vaccine targets as they are abundant and unique. In summary, the antiretroviral lectin cyanovirin-N binds to well-known and novel targets on the surface of E. histolytica that are rapidly replenished from large intracellular pools.

Insights Into Entamoeba histolytica Virulence Modulation

Entamoeba histolytica is able to invade human tissues by means of several molecules and biological properties related to the virulence. Pathogenic amebas use three major virulence factors, Gal/GalNAc lectin, amebapore and proteases, for lyse, phagocytose, kill and destroy a variety of cells and tissues in the host. Responses of the parasite to host components such as mucins and bacterial flora influence the behavior of pathogenic amebas altering their expression of virulence factors. The relative virulence of different strains of E. histolytica has been shown to vary as a consequence of changes in conditions of in vitro cultivation which implies substantial changes in basic metabolic aspects and factors directly and indirectly related to amebic virulence. Comparison of E. histolytica strains with different virulence phenotypes and under different conditions of growth will help to identify new virulence factor candidates and define the interplay between virulence factors and invasive phenotype. Virulence attenuate mutants of E. histolytica are useful also to uncover novel virulence determinants. The comparison of biological properties and virulence factors between E. histolytica and E. dispar, a non-pathogenic species, has been a useful approach to investigate the key factors involved in the experimental presentation of amebiasis and its complex regulation. The molecular mechanisms that regulate these variations in virulence are not yet known. Their elucidation will help us to better understand the gene expression plasticity that enables the effective adaptation of the ameba to changes in growth culture conditions and host factors.

Regulation of Virulence of Entamoeba histolytica by the URE3-BP Transcription Factor

ABSTRACTIt is not understood why only some infections with Entamoeba histolytica result in disease. The calcium-regulated transcription factor upstream regulatory element 3-binding protein (URE3-BP) was initially identified by virtue of its role in regulating the expression of two amebic virulence genes, the Gal/GalNac lectin and ferredoxin. Here we tested whether this transcription factor has a broader role in regulating virulence. A comparison of in vivo to in vitro parasite gene expression demonstrated that 39% of in vivo regulated transcripts contained the URE3 motif recognized by URE3-BP, compared to 23% of all promoters (P < 0.0001). Amebae induced to express a dominant positive mutant form of URE3-BP had an increase in an elongated morphology (30% ± 6% versus 14% ± 5%; P = 0.001), a 2-fold competitive advantage at invading the intestinal epithelium (P = 0.017), and a 3-fold increase in liver abscess size (0.1 ± 0.1 g versus 0.036 ± 0.1 g; P = 0.03). These results support a role for URE3-BP in virulence regulation.

Molecular nature of virulence in Entamoeba histolytica

For many years virulence of pathogenic Entamoeba histolytica has been attributed to the capacity of the parasite to destroy tissues through the expression and/or secretion of various molecules. Such view is supported mainly by in vitro experimentation, whereas data obtained by using animal models of the disease have clearly demonstrated that the host's inflammatory response is primarily responsible for tissue damage. This review analyzes the content and/or activity of some of the presumed toxic amebic molecules present in amebic strains with different degrees of virulence compared to various parasite in vitro functions that are supposed to correlate with in vivo virulence. The analysis suggests that amebic virulence is primarily determined by the parasite's capacity to adapt and survive the aerobic conditions present in animal tissues. This initial episode in the host–parasite relationship is an absolute requirement for the further development of tissue lesions, which result from the concerted action of many molecules derived from both, the host and the parasite.

Nitric oxide inhibits cysteine proteinases and alcohol dehydrogenase 2 of Entamoeba histolytica.

Nitric oxide is involved in the neutrophil and macrophage killing of the protozoan parasite Entamoeba histolytica. In the present study, we found that cysteine proteinases, significant contributors to amebic virulence and alcohol dehydrogenase 2, an enzyme absolutely required for the survival of the parasite, are both significantly inhibited by S-nitroso-glutathione, a physiological nitric oxide donor, within the concentration range 0.5-2.0 m M.

Entamoeba histolytica: sequence conservation of the Gal/GalNAc lectin from clinical isolates

The Gal/GalNAc lectin gene of Entamoeba histolytica is a major amebic virulence protein responsible for interaction with host tissues. We investigated sequence differences in the Gal/GalNAc lectin heavy subunit in three isolates from Bangladesh and one isolate from Georgia, each of which was determined to be genetically distinct by SREHP AluI digestion. Interestingly, we observed only slight genetic diversity in the lectin gene as compared with the HM1:IMSS laboratory strain, originally a clinical isolate from Mexico. Genetic conservation of the Gal/GalNAc lectin between isolates may reflect that the lectin is under strong functional selection or possibly, that E. histolytica is a clonal population. Sequence conservation of the lectin indicates that immune responses against it should be cross-protective. Index Descriptors and Abbreviations: Entamoeba histolytica, Entamoeba dispar, Protozoa, Amebiasis, Gal/GalNAc lectin, Serine-rich E. histolytica protein (SREHP), Polymerase chain reaction (PCR), Carbohydrate recognition domain (CRD).

Lipophosphopeptidoglycan of Entamoeba histolytica Induces an Antiinflammatory Innate Immune Response and Downregulation of Toll-Like Receptor 2 (TLR-2) Gene Expression in Human Monocytes

Introduction Bacterial infection results in activation of the innate im-mune system. The recognition of a pathogen is mediated bya set of germline-encoded receptors that are referred to aspattern-recognition receptors (PRRs) that can directly rec-ognize invariant molecular structures known as pathogen-associated molecular patterns (PAMPs), which are sharedby large groups of microorganisms. One PAMP recognizedby PRRs is lipopolysaccharide (LPS), a mayor componentof the outer membrane of Gram-negative bacteria. LPSstimulates host cells and makes them produce various proin-flammatory cytokines, such as TNF- a , IL-1 a , and IL-6.LPS is captured by LPS-binding protein (LBP), which de-livers to CD14 on the cell surface. Because CD14 lacks anintracellular signaling domain, a transmembrane coreceptorhas been postulated in LPS-responsive cells that transducesthe LPS signaling into the cytoplasm. In Drosophila , theToll receptor participates in establishing the dorsoventralpatterns in the embryo and inducing antifungal immune re-sponse in the adult fly. Toll is a type I transmembrane pro-tein with a cytoplasmic domain with sequence homology tothe interleukin-1 receptor (IL-1R). Activation of Toll leadsto induction of genes through the transcription factor NF-kBpathway (1). Recently, several Toll-like receptors (TLRs)have been identified in macrophages and monocytes, basedon homology to Drosophila protein. Other studies havevariably suggested that TLR-2 and TLR-4 serve as the mainmediators of responses to LPS (2). In addition, TLR-2 mayalso act as a signaling receptor for other common bacterialstructures: peptidoglycan (PGN) and lipoteichoic acid(LTA) from Gram-positive bacteria and lipoarabinomannan(LAM) from Mycobacterium. Lipophosphopeptidoglycan (LPPG) antigens from Ent-amoeba histolytica were described for the first time by Isi-basi et al., and were later used to study its expression underdifferent culture conditions and in different E. histolytica strains using monoclonal antibodies. One antibody againstLPPG was able to inhibit adhesion of amebas and cytotoxic-ity to target cells. Recently, the expression of LPPG wascorrelated to amebic virulence. In addition, the protectionagainst invasive amebiasis by a single monoclonal antibodydirected against LPPG has been studied (3). We studiedLPPG, and found that, due to its polysaccharide nature, thisameba surface molecule behaves like a PAMP. LPPG isprobably recognized by TLR-2, but instead of inducing apro-inflammatory response, it induced production of inter-leukin-10 and downregulation of tlr -2 gene expression. Materials and Methods Isolation of LPPG. Trophozoites from E. histolytica strainHM-1 were cultivated under axenic conditions, and wereused to extract LPPG using the phenol–water method de-scribed by Westphal and Jann and modified by Isibasi et al.Contaminating LPS in LPPG preparations were determinedusing quantitative Limulus lysate assay (BioWhittaker). Inall preparations, 0.020 ng of LPS/ m g of carbohydrate wasdetected. Monocyte isolation and LPPG/LPS stimulation assay. Periph-eral blood mononuclear cells were isolated on Ficoll-paquegradients (Pharmacia LKB Biotecnology) and cultured (5 3 10

Novel Thermo-Stable Serine-Metallo Proteinase of Entamoeba histolytica

Proteases of Entamoeba histolytica are considered to be an important virulence factor in the pathogenesis of amebiasis. Cysteine proteinases have been suggested to play a key role in tissue invasion and modulation of cell-mediated immune response. Several cysteine proteinases with molecular masses in the range of 16–96 kDa have been observed in extracts of E. histolytica (1). Up to six different cysteine proteinase genes have been identified, and inhibition of their expression by antisense RNA has been shown to affect amebic virulence (2,3). The presence of a collagenase in E. histolytica trophozoites has been reported (4). Very little is known, however, about serine and metallo proteinases in this parasite, and no function has been attributed to such proteases. The present study was undertaken to investigate the presence and characterization of serine and metallo proteinases of E. histolytica.

Specific and Reversible Inhibition of Entamoeba histolytica Cysteine-Proteinase Activities by Zn 2+: Implications for Adhesion and Cell Damage

Background Cysteine-proteinases are thought to play an important role in E. histolytica pathogenicity. Although effective blockage of proteolytic activities can be obtained with several known inhibitors, the high cellular toxicity of most of the inhibitors precludes experimentation with live cells or animal models. Specific cysteine-proteinase inhibitors that could be utilized in studies of virulence are of great need in the field of amebiasis. Methods Cysteine-proteinase activities were determined in trophozoite lysates by azocasein degradation and after PAGE and gelatin zymograms. Inhibition of the activities was assessed in the presence of 0.01–2.5 mM concentrations of divalent cations of the IIB and VIII series such as Zn, Cd, Hg, Ni, and Co. Reversibility was induced with 25 mM L-cysteine or 50 mM L-histidine and by metal chelation with 5 mM phenantroline. The inhibitory effect of ZnCl 2 was tested with live cells in fibronectin-binding and cytotoxicity assays. Results ZnCl 2 specifically inhibited cysteine-proteinase activities in trophozoite lysates in a concentration-dependent manner. Additionally, 1.0–2.5 mM ZnCl 2 blocked proteolysis in more than 70%. This inhibition was completely reverted by L-cysteine, L-histidine, or phenantroline. Similar results were obtained by analyzing individual cysteine-proteinase activities separated in gelatin zymograms. At these concentrations, ZnCl 2 significantly interfered with trophozoite adhesion, thus making amebas deficient in substrate degradation and cell damage. Conclusions ZnCl 2 is an effective inhibitor of amebic cysteine-proteinases. Its low toxicity at relatively high concentrations, high solubility, and low cost make it adequate for live cell experimentation and animal models of amebic virulence.

Allicin from garlic strongly inhibits cysteine proteinases and cytopathic effects of Entamoeba histolytica.

The ability of Entamoeba histolytica trophozoites to destroy monolayers of baby hamster kidney cells is inhibited by allicin, one of the active principles of garlic. Cysteine proteinases, an important contributor to amebic virulence, as well as alcohol dehydrogenase, are strongly inhibited by allicin.

Modulation of a surface antigen of Entamoeba histolytica in response to bacteria

Changes in the cell surface of Entamoeba histolytica, a human intestinal parasite and the causative agent of amebic dysentery, were examined with a monoclonal antibody, 2D7.10, which selectively recognizes carbohydrate epitopes in some axenic amebic strains. While high-level expression of this epitope was observed in axenic amebae, it was either absent or present only in small amounts in xenic amebae. Furthermore, reassociation of the axenic amebae with intestinal flora resulted in loss of the 2D7.10 epitope. Our data suggest that surface antigens of E. histolytica can be modulated in response to bacteria and may provide an explanation for the observed influence of bacteria on amebic virulence.

Virulence of Entamoeba histolytica trophozoites. Effects of bacteria, microaerobic conditions, and metronidazole.

The association of axenically grown trophozoites of Entamoeba histolytica strains HK-9 or HM-1:IMSS with various types of gram-negative bacteria for relatively short periods markedly increased their virulence, as evidenced by their ability to destroy monolayers of tissue-cultured cells. Interaction of trophozoites with bacteria that were heat inactivated, glutaraldehyde fixed, or disrupted by sonication, or bacteria treated with inhibitors of protein synthesis, did not augment amebic virulence. Lethally irradiated bacteria, however, retained their stimulative properties and trophozoites that ingested bacteria were protected from the toxic effects of added hydrogen peroxide. An increase in virulent properties of amebae was also found in experiments carried out under microaerobic conditions (5% O2, 10% CO2). The augmentation of amebic virulence due to association with bacteria was specifically blocked by metronidazole, but not by tetracycline or aminoglycosides, and the rate of metronidazole uptake in stimulated trophozoites was two to three times higher. The results obtained suggest that virulence of axenically grown E. histolytica trophozoites may depend to a considerable extent on the cell's reducing power. Both microaerobic conditions and the association with bacteria apparently stimulate the electron transport system of the ameba. Bacteria may function as broad range scavengers for oxidized molecules and metabolites through the contribution of enzymatic systems, components, or products.

Role of bacteria in modifying virulence of Entamoeba histolytica. Studies of amebae from axenic cultures.

Abstract Strains of E. histolytica from axenic cultures were studied with regard to their virulence and pathogenicity. Inoculation of amebae from axenic cultures into the liver of weanling hamsters did not result in hepatic abscesses. Reassociation of amebae with various bacterial strains resulted in hepatic abscess regularly when amebae and bacteria were reassociated for an interval of about 6 to 12 hours before inoculation into the liver. Loss of virulence upon axenization, however, was found to decline slowly over a period of from 3 to 5 months. In order to determine if there was some factor elaborated by, or associated with, bacteria that would cause increased amebic virulence, we added filtered bacterial cultures, bacteria killed by heat or X-radiation, or ground or sonicated bacteria to amebae from axenic cultures, which were then inoculated into livers of hamsters or guinea pigs. Hepatic abscess could not be produced by these methods. Similarly, growth of axenic amebae and bacteria in parabiotic chambers separated by a semipermeable membrane did not result in enhanced amebic virulence. These experiments suggest that increased virulence is associated only with contact of amebae with live bacteria. There may be the transfer of a factor from the bacteria to the amebae that alters virulence.