Entamoeba histolytica Cysteine Proteinase 5 Evokes Mucin Exocytosis from Colonic Goblet Cells via αvß3 Integrin.

Critical to the pathogenesis of intestinal amebiasis, Entamoeba histolytica (Eh) induces mucus hypersecretion and degrades the colonic mucus layer at the site of invasion. The parasite component(s) responsible for hypersecretion are poorly defined, as are regulators of mucin secretion within the host. In this study, we have identified the key virulence factor in live Eh that elicits the fast release of mucin by goblets cells as cysteine protease 5 (EhCP5) whereas, modest mucus secretion occurred with secreted soluble EhCP5 and recombinant CP5. Coupling of EhCP5-αvß3 integrin on goblet cells facilitated outside-in signaling by activating SRC family kinases (SFK) and focal adhesion kinase that resulted in the activation/phosphorlyation of PI3K at the site of Eh contact and production of PIP3. PKCδ was activated at the EhCP5-αvß3 integrin contact site that specifically regulated mucin secretion though the trafficking vesicle marker myristoylated alanine-rich C-kinase substrate (MARCKS). This study has identified that EhCP5 coupling with goblet cell αvß3 receptors can initiate a signal cascade involving PI3K, PKCδ and MARCKS to drive mucin secretion from goblet cells critical in disease pathogenesis.

CCR9+ T cells contribute to the resolution of the inflammatory response in a mouse model of intestinal amoebiasis.

Analysis of the mechanisms underlying the inflammatory response in amoebiasis is important to understand the immunopathology of the disease. Mucosal associated effector and regulatory T cells may play a role in regulating the inflammatory immune response associated to Entamoeba histolytica infection in the colon. A subpopulation of regulatory T cells has recently been identified and is characterized by the expression of the chemokine receptor CCR9. In this report, we used CCR9 deficient (CCR9(-/-)) mice to investigate the role of the CCR9(+) T cells in a murine model of E. histolytica intestinal infection. Intracecal infection of CCR9(+/+), CCR9(+/-) and CCR9(-/-) mice with E. histolytica trophozoites, revealed striking differences in the development and nature of the intestinal inflammatory response observed between these strains. While CCR9(+/+) and CCR9(+/-) mice were resistant to the infection and resolved the pathogen-induced inflammatory response, CCR9(-/-) mice developed a chronic inflammatory response, which was associated with over-expression of the cytokines IFN-γ, TNF-α, IL-4, IL-6 and IL-17, while IL-10 was not present. In addition, increased levels of CCL11, CCL20 and CCL28 chemokines were detected by qRT-PCR in CCR9(-/-) mice. E. histolytica trophozoites were identified in the lumen of the cecum of CCR9(-/-) mice at seven days post infection (pi), whereas in CCR9(+/+) mice trophozoites disappeared by day 1 pi. Interestingly, the inflammation observed in CCR9(-/-) mice, was associated with a delayed recruitment of CD4(+)CD25(+)FoxP3(+) T cells to the cecal epithelium and lamina propria, suggesting that this population may play a role in the early regulation of the inflammatory response against E. histolytica, likely through IL-10 production. In support of these data, CCR9(+) T cells were also identified in colon tissue sections obtained from patients with amoebic colitis. Our data suggest that a population of CCR9(+)CD4(+)CD25(+)FoxP3(+) T cells may participate in the control and resolution of the inflammatory immune response to E. histolytica infection.

Entamoeba histolytica cysteine proteinase 5 binds integrin on colonic cells and stimulates NFkappaB-mediated pro-inflammatory responses.

Integrins are important mammalian receptors involved in normal cellular functions and the pathogenesis of inflammation and disease. Entamoeba histolytica is a protozoan parasite that colonizes the gut, and in 10% of infected individuals, causes amebic colitis and liver abscess resulting in 10(5) deaths/year. E. histolytica-induced host inflammatory responses are critical in the pathogenesis of the disease, yet the host and parasite factors involved in disease are poorly defined. Here we show that pro-mature cysteine proteinase 5 (PCP5), a major virulent factor that is abundantly secreted and/or present on the surface of ameba, binds via its RGD motif to α(V)ß(3) integrin on Caco-2 colonic cells and stimulates NFκB-mediated pro-inflammatory responses. PCP5 RGD binding to α(V)ß(3) integrin triggered integrin-linked kinase(ILK)-mediated phosphorylation of Akt-473 that bound and induced the ubiquitination of NF-κB essential modulator (NEMO). As NEMO is required for activation of the IKKα-IKKß complex and NFκB signaling, these events markedly up-regulated pro-inflammatory mediator expressions in vitro in Caco-2 cells and in vivo in colonic loop studies in wild-type and Muc2(-/-) mice lacking an intact protective mucus barrier. These results have revealed that EhPCP5 RGD motif is a ligand for α(V)ß(3) integrin-mediated adhesion on colonic cells and represents a novel mechanism that E. histolytica trophozoites use to trigger an inflammatory response in the pathogenesis of intestinal amebiasis.

Association between TNF-alpha and Entamoeba histolytica diarrhea.

An association between tumor necrosis factor alpha (TNF-alpha) and Entamoeba histolytica diarrhea was assessed in a cohort of 138 non-related Bangladeshi children who have been prospectively followed since 2001. Peripheral blood mononuclear cells (PBMCs) obtained at study entry were purified, cultured, and stimulated with soluble amebic antigen before cytokine measurement from supernatant. Higher levels of TNF-alpha were associated with increased risk of first (P = 0.01) and recurrent E. histolytica-related diarrheal episodes (P = 0.005). Children who developed E. histolytica diarrhea had significantly higher TNF-alpha protein levels than those who experienced asymptomatic E. histolytica infection (P value = 0.027) or no infection (P value = 0.017). Microarray studies performed using RNA isolated from acute and convalescent whole blood and colon biopsy samples revealed higher but non-significant TNF-alpha messenger RNA (mRNA) levels in subjects with acute E. histolytica diarrhea compared with convalescence. We conclude that there is an association between higher TNF-alpha production and E. histolytica diarrhea.

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 E(2) (PGE(2)) as trophozoites treated with cyclooxygenase inhibitors inhibited the biosynthesis of PGE(2) 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 PGE(2) 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 PGE(2) 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 PGE(2) may be a mechanism that explains the initiation and amplification of acute inflammation associated with intestinal amebiasis.

Characterization of an Entamoeba histolytica high-mobility-group box protein induced during intestinal infection.

The unicellular eukaryote Entamoeba histolytica is a human parasite that causes amebic dysentery and liver abscess. A genome-wide analysis of gene expression modulated by intestinal colonization and invasion identified an upregulated transcript that encoded a putative high-mobility-group box (HMGB) protein, EhHMGB1. We tested if EhHMGB1 encoded a functional HMGB protein and determined its role in control of parasite gene expression. Recombinant EhHMGB1 was able to bend DNA in vitro, a characteristic of HMGB proteins. Core conserved residues required for DNA bending activity in other HMGB proteins were demonstrated by mutational analysis to be essential for EhHMGB1 activity. EhHMGB1 was also able to enhance the binding of human p53 to its cognate DNA sequence in vitro, which is expected for an HMGB1 protein. Confocal microscopy, using antibodies against the recombinant protein, confirmed its nuclear localization. Overexpression of EhHMGB1 in HM1:IMSS trophozoites led to modulation of 33 transcripts involved in a variety of cellular functions. Of these, 20 were also modulated at either day 1 or day 29 in the mouse model of intestinal amebiasis. Notably, four transcripts with known roles in virulence, including two encoding Gal/GalNAc lectin light chains, were modulated in response to EhHMGB1 overexpression. We concluded that EhHMGB1 was a bona fide HMGB protein with the capacity to recapitulate part of the modulation of parasite gene expression seen during adaptation to the host intestine.

Entamoeba histolytica phagocytosis of human erythrocytes involves PATMK, a member of the transmembrane kinase family.

Entamoeba histolytica is the cause of amebic colitis and liver abscess. This parasite induces apoptosis in host cells and utilizes exposed ligands such as phosphatidylserine to ingest the apoptotic corpses and invade deeper into host tissue. The purpose of this work was to identify amebic proteins involved in the recognition and ingestion of dead cells. A member of the transmembrane kinase family, phagosome-associated TMK96 (PATMK), was identified in a proteomic screen for early phagosomal proteins. Anti-peptide affinity-purified antibody produced against PATMK demonstrated that it was a type I integral membrane protein that was expressed on the trophozoite surface, and that co-localized with human erythrocytes at the site of contact. The role of PATMK in erythrophagocytosis in vitro was demonstrated by: (i) incubation of ameba with anti-PATMK antibodies; (ii) PATMK mRNA knock-down using a novel shRNA expression system; and (iii) expression of a carboxy-truncation of PATMK (PATMK(delta932)). Expression of the carboxy-truncation of PATMK(delta932) also caused a specific reduction in the ability of E. histolytica to establish infection in the intestinal model of amebiasis, however these amebae retained the ability to cause hepatic abscesses when directly injected in the liver. In conclusion, PATMK was identified as a member of the TMK family that participates in erythrophagocytosis and is uniquely required for intestinal infection.

[The role of oxygen-nitrogen stress in pathogenesis of amoebiasis].

The goal of present investigation was determining peculiarities of oxidation metabolism in the case amoebiasis. The activity of pro- and anti-oxidative systems and content of NO in the blood of the patients with abscess, which developed in hepatic amoebiasis, has been studied. It was shown that in the patients' organism an increased generation of the free radicals of oxygen and nitrogen does occur. Intensification of peroxidation of the lipids (POL) and inactivation of antioxidation system of the organism were observed as well. Intensifying of the oxygen-nitrogen stress in the organism of the patients with amoebiasis, is induced by the activated neutrophyls accumulated in the focus of injury, which developed during invasion of amoebas. Low efficiency of the neutrophyl-induced oxidation explosion in amoebiasis, partly is determined by the highly active anti-oxidative system of these parasites. Their sensitivity to NO, allows suggesting of implementation of preparations--NO donors and modulators of NO production in the host organism, in treatment of the parasitic infections.

Entamoeba histolytica Gal/GalNAc lectin depletes antioxidant defences of target epithelial cells.

Among the variety of virulence factors of Entamoeba histolytica, an adherence lectin (Gal/GalNAc, 260 kDa) is known to mediate colonization and subsequent host responses. Gal/GalNAc lectin is universally recognized by the immune sera of patients with amoebic liver abscess. It plays a crucial role in cytolysis and phagocytosis of human and rat colonic mucin glycoproteins. The objective of the present study was to elucidate the role of antioxidants in E. histolytica Gal/GalNAc lectin-induced signals in the target epithelial cells. We have attempted to define a pathway in target cells, Henle-407 cells (human intestinal epithelial cell line), that could link this immunodominant antigen to a known biological pathway for target cell activation and triggering of subsequent disease pathology/parasite survival. Since several workers have demonstrated that cAMP and cGMP may act as important cellular signals for altering ion transport, so in the present study, cAMP and cGMP levels were measured in Henle-407 cells which showed significant increase at 15 min after stimulation. Elevated cAMP and cGMP levels are implicated in altered electrolyte transport and conductance. Results showed that there were increased levels of ROS and RNI which led to reduced activities of antioxidant enzymes--catalase, superoxide dismutase and glutathione peroxidase. Despite the increased glutathione (reduced) levels, the enzymes were not able to combat the damage caused by ROS and RNI. Thus, there was an increased local concentration of the free radicals and reduced activities of all the three enzymes which could damage the target cell in terms of cytoskeleton and permeability changes.

Studies on the development of oral colon targeted drug delivery systems for metronidazole in the treatment of amoebiasis.

The aim of the present study is to develop colon targeted drug delivery systems for metronidazole using guar gum as a carrier. Matrix, multilayer and compression coated tablets of metronidazole containing various proportions of guar gum were prepared. All the formulations were evaluated for the hardness, drug content uniformity, and were subjected to in vitro drug release studies. The amount of metronidazole released from tablets at different time intervals was estimated by high performance liquid chromatography method. Matrix tablets and multilayer tablets of metronidazole released 43-52% and 25-44% of the metronidazole, respectively, in the physiological environment of stomach and small intestine depending on the proportion of guar gum used in the formulation. Both the formulations failed to control the drug release within 5 h of the dissolution study in the physiological environment of stomach and small intestine. The compression coated formulations released less than 1% of metronidazole in the physiological environment of stomach and small intestine. When the dissolution study was continued in simulated colonic fluids, the compression coated tablet with 275 mg of guar gum coat released another 61% of metronidazole after degradation by colonic bacteria at the end of 24 h of the dissolution study. The compression coated tablets with 350 and 435 mg of guar gum coat released about 45 and 20% of metronidazole, respectively, in simulated colonic fluids indicating the susceptibility of the guar gum formulations to the rat caecal contents. The results of the study show that compression coated metronidazole tablets with either 275 or 350 mg of guar gum coat is most likely to provide targeting of metronidazole for local action in the colon owing to its minimal release of the drug in the first 5 h. The metronidazole compression coated tablets showed no change either in physical appearance, drug content or in dissolution pattern after storage at 40 degrees C/75% RH for 6 months.

Dose- and time-dependent functional and structural damage to the colon mucosa by Entamoeba histolytica trophozoite lysates.

Analysis of the initial interaction between Entamoeba histolytica trophozoites and the large intestine is impossible in humans and very difficult in experimental animals. To circumvent this obstacle we treated the luminal side of full-thickness rabbit colon segments mounted in Ussing-type chambers with trophozoite lysates of the E. histolytica HM1 virulent strain. Exposure to lysates for up to 90 min produced dose- and time-dependent effects on the colon, consisting of (a) increased decay rates for potential difference, short-circuit current, and transmural resistance and (b) mucosal damage ranging from vacuolation at the bases and shortening of epithelial cells to the loss of intercellular junctions, destruction of microvilli, and necrosis of interglandular epithelial zones. This acute model of intestinal amebiasis is sensitive, fast, and reliable.

Biochemical characterization of rat colonic mucins secreted in response to Entamoeba histolytica.

Invasion of the colonic mucosa by Entamoeba histolytica trophozoites is preceded by colonic mucus depletion. The aim of our studies was to determine whether E. histolytica caused a differential secretion of mucin species in a rat colonic loop model. Mucus secretion in response to amoebae was followed by release of acid-precipitable 3H-glucosamine metabolically labelled glycoproteins and in vitro labelling of glycoprotein secretion with NaB3H4. The secretory response consisted of high-Mr goblet cell mucins and an increase in the secretion of low-Mr nonmucin glycoproteins as determined by Sepharose 4B column chromatography. High-Mr mucins subfractionated by Cellex-E (ECTEOLA) ion-exchange chromatography demonstrated a minor neutral and a major acidic mucin (greater than 98%) species. Marked differences between the neutral and acidic mucin species were indicated by immunogenicity and amino acid compositions. Thin-section histochemistry of rat colons confirmed secretion of neutral and acidic mucins in response to E. histolytica and demonstrated secretory activity from goblet cells from both the crypts and interglandular epithelium. E. histolytica mucus secretagogue activity was generalized and may function to deplete the host's protective mucus layer, facilitating invasion by the parasites.

Influence of intestinal parasitism on lactose absorption in well-nourished African children.

Hydrogen breath tests were performed in Gabon (Central Africa) after a loading dose of lactose in 67 well-nourished African children (50 with intestinal parasites and 17 unparasitized) and in 18 unparasitized young adults. All had normal nutritional status, and none had diarrhea or digestive symptoms. Parasites that were found included Ascaris lumbricoides in 76% of the parasitized children, Trichuris trichiura in 58%, Giardia in 24%, Entamoeba histolytica in 20%, Schistosoma intercalatum in 16%, and Necator Americanus in 14%. A similar proportion of parasitized (64%) or unparasitized (62.8%) subjects were lactose malabsorbers. Giardia infection was associated with a higher, but not significantly different, proportion of lactose intolerance (10 of 12, 83.3%). The presence of infection with A. lumbricoides or T. trichiura did not increase the percentage of lactose malabsorption. These data indicate that a decrease of lactase activity in well-nourished African children is not related to the presence or the importance of Ascaris or other intestinal parasites if the nutritional status is normal.

Generation of reactive oxygen species by Kupffer cells and blood monocytes during intestinal amebiasis in guinea pigs.

The reactive oxygen species generation capacity of Kupffer cells and blood monocytes was studied through chemiluminescence (CL) in guinea pigs, infected intracecally with Entameba histolytica, on days 0, 3, 7, 14 and 21 post infection. There was an elevated response up to the 14th post-infection day. The CL response of these cells was diminished on the 21st post infection day though it was significantly higher than up the controls. The CL response increased exponentially with the severity of cecal lesions. A direct correlation was observed between the CL response of phagocytic cells and cecal lesions of animals. The role of oxygen species in causation of tissue injury during intestinal amebiasis is postulated.

Entamoeba histolytica: elevated nitroblue tetrazolium reduction activity in polymorphs during amoebic liver abscess.

Peripheral blood polymorphonuclear leucocytes (PMN) from patients with invasive amoebiasis, i.e. amoebic liver abscess (ALA) and acute amoebic dysentery, showed marked elevation of nitroblue tetrazolium dye (NBT) reduction. This dramatic change was not observed in PMN from patients with non-invasive amoebiasis, i.e. non-suppurative hepatic amoebiasis, or in asymptomatic Entamoeba histolytica cyst passers. A small number (12%) of patients with viral hepatitis displayed increased NBT reduction. 10 to 12 days after recovery following treatment, the majority (75%) of ALA patients failed to show increased NBT reduction. Our results suggest that the PMN-NBT reduction test could be useful as an aid to the diagnosis of ALA.

The iron status of Black subjects with amoebiasis.

Hepatic iron concentrations were measured in 60 Black patients who had died of amoebiasis. There were 18 infants and young children, 30 adult males and 12 adult females. The mean hepatic iron concentration in infants was normal (0,11% dry weight), while those in adult males and females were significantly raised (0,64% and 0,30% dry weight respectively). The figures for adults are somewhat higher than those previously found in Black subjects, suggesting that iron overload is more common in patients with amoebiasis than in the general population. The relevance of these findings to the pathogenesis of amoebiasis is not clear but the relationship may well not be a direct one, since hepatic iron stores were no larger in patients with liver abscesses than in subjects without.