Subunit structure of the galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica

The galactose and N-acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica is a cell surface protein which mediates parasite adherence to human colonic mucus, colonic epithelial cells, and other target cells. The amebic lectin was purified in 100-micrograms quantities from detergent-solubilized trophozoites by monoclonal antibody affinity chromatography. The adherence lectin was purified 500-fold as judged by radioimmunoassay. The nonreduced lectin had a molecular mass of 260 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of pH 6.2. The amebic lectin reduced with beta-mercaptoethanol consisted of 170- and 35-kDa subunits. Both subunits could be labeled on the cell surface with 125I, and both were metabolically labeled with [3H]glucosamine. The amino termini of the subunits had unique amino acid sequences, and polyclonal antisera to the heavy subunit did not cross-react with the light subunit. The yield of phenylthiohydantoin derivatives from the second and third positions in the sequence of the heavy and light subunits gave a molar ratio of one 170- to one 35-kDa subunit. Antibodies directed to the heavy subunit inhibited amebic adherence to Chinese hamster ovary cells by 100%, suggesting that the heavy subunit is predominantly responsible for mediating amebic adherence.     

Recognition of 29 kDa surface-associated adhesive molecule of Entamoeba histolytica by monoclonal antibodies

Abstract Monoclonal antibodies have been developed and used as specific probe to locate and identify a 29-kDa molecule of axenic Entamoeba histolytica trophozoites. Monoclonal antibody produced by clone C8 (MoAb C8) strongly agglutinated the amoebic trophozoites. THe immunofluorescence of live E. histolytica trophozoites and surface fluorescence of acetone-fixed trophozoites by MoAb C8 indicated existence of a 29-kDa molecule on surface-associated plasma membrane of E. histolytica . The monoclonal antibody belonged to IgG₁ isotype. The prior treatment of E. histolytica trophozoites with MoAb C8 resulted in significant ( P < 0.01) reduction in adherence of amoebic trophozoites to cultured Chinese Hamster Ovary cells and significant ( P < 0.01) reduction in cytotoxicity to cultured Baby Hamster Kidney cells. Pretreatment of amoebic trophozoites with MoAb C8 prior to cultivation in TPS-1 medium resulted in significant ( P < 0.01) reduction in growth of the parasite. Thus, the data suggested that the surface-exposed 29-kDa molecule may be one of the receptors involved in E. histolytica host cell interactions and may possibly modulate amoebic disease processes.     

Recognition of 29 kDa surface-associated adhesive molecule of Entamoeba histolytica by monoclonal antibodies

Monoclonal antibodies have been developed and used as specific probe to locate and identify a 29-kDa molecule of axenic Entamoeba histolytica trophozoites. Monoclonal antibody produced by clone C8 (MoAb C8) strongly agglutinated the amoebic trophozoites. The immunofluorescence of live E. histolytica trophozoites and surface fluorescence of acetone-fixed trophozoites by MoAb C8 indicated existence of a 29-kDa molecule on surface-associated plasma membrane of E. histolytica. The monoclonal antibody belonged to IgG1 isotype. The prior treatment of E. histolytica trophozoites with MoAb C8 resulted in significant (P less than 0.01) reduction in adherence of amoebic trophozoites to cultured Chinese Hamster Ovary cells and significant (P less than 0.01) reduction in cytotoxicity to cultured Baby Hamster Kidney cells. Pretreatment of amoebic trophozoites with MoAb C8 prior to cultivation in TPS-1 medium resulted in significant (P less than 0.01) reduction in growth of the parasite. Thus, the data suggested that the surface-exposed 29-kDa molecule may be one of the receptors involved in E. histolytica host cell interactions and may possibly modulate amoebic disease processes.     

Interaction of Eimeria tenella with intestinal mucin in vitro

The mucus gel layer overlying the gastrointestinal epithelium plays an important role in host-pathogen interactions. The initial interaction between the coccidian parasite Eimeria tenella and host cells of the intestinal epithelium must occur across this mucus interface. In this study, we examined the relationship between E. tenella and avian mucin, in particular the effect of purified intestinal regional mucin on parasite adherence and invasion in vitro. Secreted mucin from the chicken duodenum and cecum was purified by density gradient centrifugation and gel chromatography. Parasite invasion studies were performed in the Madin-Darby bovine kidney cell model. Eimeria tenella adherence to chicken duodenal mucin was detected, whereas adherence to cecal or bovine mucin was not shown. Parasite invasion into epithelial cells was not influenced by bovine mucin, whereas chicken mucin purified from the duodenum and cecum significantly inhibited invasion. Inhibition of E. tenella invasion into cells by mucin from the duodenum was marginally greater than that of the cecum, but this was not significant. This study demonstrated E. tenella interaction with native chicken intestinal mucin, which in turn inhibited parasite invasion into epithelial cells in vitro.     

Mucus blocks probiotics but increases penetration of motile pathogens and induces TNF-α and IL-8 secretion

The mucosal barrier in combination with innate immune system are the first line of defense against luminal bacteria at the intestinal mucosa. Dysfunction of the mucus layer and bacterial infiltration are linked to tissue inflammation and disease. To study host–bacterial interactions at the mucosal interface, we created an experimental model that contains luminal space, a mucus layer, an epithelial layer, and suspended immune cells. Reconstituted porcine small intestinal mucus formed an 880 ± 230 µm thick gel layer and had a porous structure. In the presence of mucus, sevenfold less probiotic and nonmotile VSL#3 bacteria transmigrated across the epithelial barrier compared to no mucus. The higher bacterial transmigration caused immune cell differentiation and increased the concentration of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α; p < .01). Surprisingly, the mucus layer increased transmigration of pathogenic Salmonella and increased secretion of TNF-α and IL-8 (p < .05). Nonmotile, flagella knockout Salmonella had lower transmigration and caused lower IL-8 and TNF-α secretion (p < .05). These results demonstrate that motility enables pathogenic bacteria to cross the mucus and epithelial layers, which could lead to infection. Using an in vitro coculture platform to understand the interactions of bacteria with the intestinal mucosa has the potential to improve the treatment of intestinal diseases.     

Entamoeba histolytica: identification of a distinct beta2 integrin-like molecule with a potential role in cellular adherence

Entamoeba histolytica infection causes dysentery, intestinal colitis, and hepatic abscess in an estimated 50 million people worldwide. Attachment of E. histolytica trophozoites to intestinal epithelium and vascular endothelium during liver metastasis results in an inflammatory process. We report the identification of a distinct amebic beta2 integrin (CD18)-like molecule which affords adherence to TNF-alpha-activated endothelial cells. Data from flow cytometry and indirect immunofluorescence assays suggest the amebic beta2 integrin was localized to focal adhesion plates and was present in both E. histolytica and Entamoeba dispar. The amebic beta2 integrin appeared to be distinct from the amebic Gal/GalNAc lectin based on recombinant expression, amebic colocalization, and ELISA studies. Trophozoite adherence to endothelial cells expressing ICAM-1 (CD54) following activation with TNF-alpha or ICAM-1-transfected CHO cells was specifically inhibited with anti-CD18 or anti-CD54 MAbs. In summary, evidence in support of a distinct beta2 integrin-like molecule participating in amebic adherence to TNF-alpha-activated endothelial cells expressing ICAM-1 is presented. The presence of integrin-dependent binding may allow trophozoites to opportunistically adhere to activated intestinal epithelium or vascular endothelium expressing ICAM-1 during amebic colitis or hepatic abscess.     

Cytopathogenicity of Entamoeba histolytica to human intestinal epithelial cells: inhibition by monoclonal antibodies and sera from amoebic patients.

Interactions of pathogenic Entamoeba histolytica (HM 1) with human intestinal epithelial cells (Henle-407) were investigated. The E. histolytica trophozoites adhered and cytolysed 87% of cultured epithelial cell monolayers. A significant (P less than 0.001) inhibition of cytopathic effect of amoebic trophozoites pretreated with monoclonal antibodies to a 29 kDa surface associated protein suggested utilization of the 29 kDa surface protein in recognition and cytolysis of epithelial target cells. The polyclonal sera from treated patients of amoebic liver abscess and anti-amoebic hyperimmune serum inhibited cytopathogenicity to a greater degree (P less than 0.001) than did the monoclonal antibodies. The data thus suggest involvement of several amoebic molecules in exercising cytopathogenicity to epithelial cells.     

Dynamics of endocytic traffic of Entamoeba histolytica revealed by confocal microscopy and flow cytometry

Entamoeba histolytica, the protozoan parasite of humans, manifests constitutive endocytosis to obtain nutrients and, when induced to express invasive behavior, as a means of ingesting and processing host cells and tissue debris. E. histolytica trophozoites were grown in liquid axenic medium that contained fluorescently labeled fluid-phase markers, so that the kinetics of uptake, the transit of loaded endosomes through the cytoplasm, and the time of release of the markers could be monitored by flow cytometry. Confocal microscopy of live trophozoites revealed uptake of fluid by avid macropinocytosis and the occurrence of fusion between young and older endosomes, as well as between pinosomes and phagosomes containing bacteria. Endosomes were rapidly acidified, then gradually neutralized; finally, indigestible material was released. Transit of endosomes containing fluid-phase markers required about 2 h. Uptake and release of fluid-phase markers were impaired by drugs that inhibited actin dynamics and actin-myosin interaction; uptake was also impaired by inhibition of PI 3-kinase. A striking feature of the trophozoites was the great heterogeneity of their endocytic behavior.     

The role of Mycobacterium avium complex fibronectin attachment protein in adherence to the human respiratory mucosa

Mycobacterium avium complex (MAC) are opportunistic respiratory pathogens that infect non-immunocompromised patients with established lung disease, although they can also cause primary infections. The ability to bind fibronectin is conserved among many mycobacterial species. We have investigated the adherence of a sputum isolate of MAC to the mucosa of organ cultures constructed with human tissue and the contribution of M. avium fibronectin attachment protein (FAP) to the process. MAC adhered to fibrous, but not globular mucus, and to extracellular matrix (ECM) in areas of epithelial damage, but not to intact extruded cells and collagen fibres. Bacteria occasionally adhered to healthy unciliated epithelium and to cells that had degenerated exposing their contents, but never to ciliated cells. The results obtained with different respiratory tissues were similar. Two ATCC strains of MAC gave similar results. There was a significant reduction (P < 0.05) in the number of bacteria adhering to ECM after preincubation of bacteria with fibronectin and after preincubation of the tissue with M. avium FAP in a concentration-dependant manner. The number of bacteria adhering to fibrous mucus was unchanged. Immunogold labelling demonstrated fibronectin in ECM as well as in other areas of epithelial damage, but only ECM bound FAP. A Mycobacterium smegmatis strain had the same pattern of adherence to the mucosa as MAC. When the FAP gene was deleted, the strain demonstrated reduced adherence to ECM, and adherence was restored when the strain was transfected with an M. avium FAP expression construct. We conclude that MAC adheres to ECM in areas of epithelial damage via FAP and to mucus with a fibrous appearance via another adhesin. Epithelial damage exposing ECM and poor mucus clearance will predispose to MAC airway infection.     

Entamoeba dispar: infrastructure, Surface Properties and Cytopathic Effect

The cytological features of Entamoeba dispar , recently recognized by biochemical and molecular biology criteria as a distinct species, were compared to those of Entamoeba histolytica. When cultured under axenic conditions, living trophozoites of E. dispar strain SAW 760RR clone A were more elongated in form, had a single frontal pseudopodium, and showed a noticeable uroid. In sections of E. dispar trophozoites stained with Toluidine blue, characteristic areas of cytoplasmic metachromasia were seen due to the presence of large deposits of glycogen, seldom found in E. histolytica strain HM1:IMSS. Under the light microscope the periphery of the nucleus in E. dispar was lined by finer, more regularly distributed dense granules. With transmission electron microscopy the surface coat of E. dispar was noticeable thinner. In addition, E. dispar had a lower sensitivity to agglutinate with concanavalin A and a higher negative surface charge, measured by cellular microelectrophoresis. The cytopathic effect of E. dispar was much slower, analyzed by the gradual loss of transmural electrical resistance of MDCK epithelial cell monolayers mounted in Ussing chambers. Whereas in E. histolytica phagocytosis of epithelial cells plays an important role in its cytopathic effect, E. dispar trophozoites placed in contact with MDCK cells showed only rare evidence of phagocytosis. The results demonstrate that the morphology of E. dispar is different to that of E. histolytica , both at the light microscopical and the ultrastructural levels. In addition, they show that E. dispar in axenic culture has a moderate cytopathic effect on epithelial cell monolayers. However, when compared to E. histolytica, the in vitro lytic capacity of E. dispar is much slower and less intense.     

Bacteria Penetrate the Inner Mucus Layer before Inflammation in the Dextran Sulfate Colitis Model

Background

Protection of the large intestine with its enormous amount of commensal bacteria is a challenge that became easier to understand when we recently could describe that colon has an inner attached mucus layer devoid of bacteria (Johansson et al. (2008) Proc. Natl. Acad. Sci. USA 105, 15064–15069). The bacteria are thus kept at a distance from the epithelial cells and lack of this layer, as in Muc2-null mice, allow bacteria to contact the epithelium. This causes colitis and later on colon cancer, similar to the human disease Ulcerative Colitis, a disease that still lacks a pathogenetic explanation. Dextran Sulfate (DSS) in the drinking water is the most widely used animal model for experimental colitis. In this model, the inflammation is observed after 3–5 days, but early events explaining why DSS causes this has not been described.

Principal Findings

When mucus formed on top of colon explant cultures were exposed to 3% DSS, the thickness of the inner mucus layer decreased and became permeable to 2 µm fluorescent beads after 15 min. Both DSS and Dextran readily penetrated the mucus, but Dextran had no effect on thickness or permeability. When DSS was given in the drinking water to mice and the colon was stained for bacteria and the Muc2 mucin, bacteria were shown to penetrate the inner mucus layer and reach the epithelial cells already within 12 hours, long before any infiltration of inflammatory cells.

Conclusion

DSS thus causes quick alterations in the inner colon mucus layer that makes it permeable to bacteria. The bacteria that reach the epithelial cells probably trigger an inflammatory reaction. These observations suggest that altered properties or lack of the inner colon mucus layer may be an initial event in the development of colitis.     

Giardia lamblia: stimulation of growth by human intestinal mucus and epithelial cells in serumfree medium

Giardia lamblia trophozoites specifically colonize the upper human small intestine which is normally serumfree but have been grown in vitro only in medium supplemented with serum or serum fractions. Recently, we demonstrated that biliary lipids will support the growth of G. lamblia without added serum. Now, we report that human duodenal jejunal mucus stimulates growth of Giardia in medium with biliary lipids. Stimulation by mucus was enhanced by inclusion of chymotrypsin or crude pancreatic proteases. Coculture of trophozoites with human intestinal epithelial cells also promoted growth, especially in the presence of mucus and/or biliary lipids. With biliary lipids alone, the mean increase in cell number was 3.2 fold and in the presence of mucus 8 fold (P less than 0.01) in 24 serial subcultures. Our demonstration that human intestinal mucus and epithelial cells promote serumfree growth of G. lamblia may help to explain specific colonization of the small intestine by G. lamblia.     

Mucus Properties and Goblet Cell Quantification in Mouse,Rat and Human Ileal Peyer's Patches

Peyer''s patches (PPs) are collections of lymphoid follicles in the small intestine, responsible for scanning the intestinal content for foreign antigens such as soluble molecules, particulate matter as well as intact bacteria and viruses. The immune cells of the patch are separated from the intestinal lumen by a single layer of epithelial cells, the follicle-associated epithelium (FAE). This epithelium covers the dome of the follicle and contains enterocyte-like cells and M cells, which are particularly specialized in taking up antigens from the gut. However, the presence and number of goblet cells as well as the presence of mucus on top of the FAE is controversial. When mouse ileal PPs were mounted in a horizontal Ussing-type chamber, we could observe a continuous mucus layer at mounting and new, easily removable mucus was released from the villi on the patch upon stimulation. Confocal imaging using fluorescent beads revealed a penetrable mucus layer covering the domes. Furthermore, immunostaining of FAE from mice, rats and humans with a specific antibody against the main component of intestinal mucus, the MUC2 mucin, clearly identify mucin-containing goblet cells. Transmission electron micrographs further support the identification of mucus releasing goblet cells on the domes of PPs in these species.     

In vitro and in vivo interaction of Entamoeba histolytica Gal/GalNAc lectin with various target cells: an immunocytochemical analysis

The Gal/GalNAc lectin of Entamoeba histolytica trophozoites plays an important role in adhesion. The distribution and final destiny of the lectin during the interaction with host cells are poorly understood. Using monoclonal and polyclonal antibodies against the lectin we studied by immunocytochemistry the in vitro and in vivo interaction of E. histolytica trophozoites with human and hamster hepatocytes. We also analyzed the presence and distribution of the lectin in a mouse model of intestinal amoebiasis. In all cases, trophozoites were highly labeled by anti-lectin antibodies. Cultured human and hamster hepatocytes in contact with, or localized at the vicinity of parasites were also labeled by anti-lectin antibodies. Most of the labeled hepatocytes showed variable degrees of cell damage. Hepatocytes distantly localized from the parasites were also stained with the anti-lectin antibodies. Immunolabeling of tissue sections from different stages of the development of experimental amoebic liver abscess in hamsters showed inflammatory foci containing lectin-labeled trophozoites, hepatocytes, and sinusoidal and inflammatory cells. Lectin-containing hepatocytes had vacuolated cytoplasm with some nuclei with a condensed appearance. Damaged intestinal epithelium also was labeled with anti-lectin antibodies in a mouse model of intestinal amoebiasis. Electron microscopy of axenically cultured trophozoites using gold-labeled monoclonal and polyclonal anti-lectin antibody showed that plasma membrane, vacuole membranes and areas of cell cytosol were labeled. Higher deposits of gold particles in plasma membrane suggestive of cell secretion were observed. Our results demonstrated that Gal/GalNAc lectin was bound and captured by different target cells, and that host cells containing the lectin showed signs of cell damage. The contribution of lectin transfer to host cells in adherence and cell injury remains to be determined.     

The EhADH112 recombinant polypeptide inhibits cell destruction and liver abscess formation by Entamoeba histolytica trophozoites

The Entamoeba histolytica EhCPADH complex, formed by a cysteine proteinase (EhCP112) and an adhesin (EhADH112), is involved in adherence, phagocytosis and cytolysis. This makes this complex an attractive candidate as a vaccine against amoebiasis. Here, we produced the recombinant polypeptide EhADH243, which includes the adherence epitope detected by a monoclonal antibody against the EhCPADH complex. EhADH243 was purified, and the effect of the polypeptide on in vitro and in vivo virulence was studied. Antibodies against EhADH243 reacted with the EhCPADH complex and with the recombinant polypeptide. EhADH243 and antibodies against this polypeptide inhibited adherence, phagocytosis and destruction of cell monolayers by live trophozoites, but had little effect on cell monolayer destruction by trophozoite extracts. EhADH243 recognized a 97 kDa protein in the MDCK membrane fraction that could be a putative receptor for E. histolytica trophozoites. Hamsters immunized with EhADH243 developed humoral response against EhCPADH, and animals were partially protected from amoebic liver abscess.     

Human IgA inhibits adherence of Acanthamoeba polyphaga to epithelial cells and contact lenses

Specific anti-Acanthamoeba IgA antibodies have been detected in the serum and tears of patients and healthy individuals. However, the role of human secretory IgA antibodies in inhibiting the adherence of Acanthamoeba had not been previously investigated. Therefore, the purpose of this study was to purify secretory IgA from human colostrum and analyze its effect on the adherence of Acanthamoeba trophozoites to contact lenses and Madin-Darby canine kidney (MDCK) cells. IgA antibodies to Acanthamoeba polyphaga in colostrum of healthy women as well as in saliva and serum of healthy subjects were analyzed by ELISA and Western blot analysis. In serum, saliva, and colostrum, we detected IgA antibodies that recognized several antigens of A. polyphaga. In addition, colostrum and IgA antibodies purified from it inhibited adherence of A. polyphaga trophozoites to contact lenses and MDCK cells. These results suggest that IgA antibodies may participate in the resistance to the amoebic infection, probably by inhibiting the adherence of the trophozoites to contact lenses and corneal epithelial cells.