Identification of O-glycan Structures from Chicken Intestinal Mucins Provides Insight into Campylobactor jejuni Pathogenicity.

The Gram-negative bacteria Campylobactor jejuni is the primary bacteria responsible for food poisoning in industrialized countries, and acute diarrheal illness is a leading cause of mortality among children in developing countries. C. jejuni are commensal in chickens. They are particularly abundant in the caecal crypts, and poultry products are commonly infected as a result of cross-contamination during processing. The interactions between C. jejuni and chicken intestinal tissues as well as the pathogenic molecular mechanisms of colonization in humans are unknown, but identifying these factors could provide potential targets to reduce the incidence of campylobacteriosis. Recently, purified chicken intestinal mucin was shown to attenuate adherence and invasion of C. jejuni in the human colorectal adenocarcinoma cell line HCT-8 in vitro, and this effect was attributed to mucin O-glycosylation. Mucins from different regions of the chicken intestine inhibited C. jejuni binding and internalization differentially, with large intestine>small intestine>caecum. Here, we use LC-MS to perform a detailed structural analysis of O-glycans released from mucins purified from chicken large intestine, small intestine, and caecum. The O-glycans identified were abundantly sulfated compared with the human intestines, and sulfate moieties were present throughout the chicken intestinal tract. Interestingly, alpha 1-2 linked fucose residues, which have a high binding affinity to C. jejuni, were identified in the small and large intestines. Additionally, N-glycolylneuraminic/N-acetylneuraminic acid containing structures present as Sd(a)-like epitopes were identified in large intestine samples but not small intestine or caecum. O-glycan structural characterization of chicken intestinal mucins provides insights into adherence and invasion properties of C. jejuni, and may offer prospective candidate molecules aimed at reducing the incidence of infection.

Modulation of expression in BEAS-2B airway epithelial cells of α-L-fucosidase A1 and A2 by Th1 and Th2 cytokines, and overexpression of α-L-fucosidase 2.

Chronic Th2-driven airway inflammation with excessive mucus production occurs in asthma. The regulation of FUCA1 and FUCA2 gene expression and enzyme activity in response to asthma-associated Th2 cytokines and, for contrast, Th1 cytokine IFN-γ, were investigated in a human airway cell line. BEAS-2B cells were supplemented with Th2-derived cytokines (IL-13, IL-4, IL-5) or/and IFN-γ. RNA and cell supernatants from stimulated and unstimulated cells were collected over a period of 3 h. Alpha-L-fucosidase A1 and A2 gene expression were assessed using real time RT-PCR, while enzymatic activities were measured using a fluorescent assay. To characterise α-L-fucosidase A2, CHO-K1 and BEAS-2B cell lines were transiently transfected, the FUCA2 gene was overexpressed, and the protein was immunoprecipitated. The transcription of FUCA1 was upregulated (p < 0.01) in response to IFN-γ, suggesting that FUCA1 transcription and fucosidase activity are regulated in a Th1-dependent manner. The gene expression was the highest for 30 min after IFN-γ stimulation (>twofold induction), whereas secreted enzyme activity in BEAS-2B cells was significantly increased 1 h after IFN-γ addition. IL-4, IL-5 and IL-13 had no effect on FUCA1 and FUCA2 expression and activity. The IFN-γ-induced increase in expression and activity was repressed by the presence of the Th2 cytokine IL-5. Enzymatically active α-L-fucosidase 2 was immunoprecipitated from BEAS-2B cells, with highest activity at pH 4.9. IL-13, IL-4 and IL-5 have no effect on the expression of FUCA1 and FUCA2, but its expression is upregulated by IFN-γ, a Th1 cytokine. Active α-L-fucosidase 2 was overexpressed in BEAS-2B cells.

The expression of mucin genes and the presence of mucin gene products in the equine endometrium.

In the equine reproductive tract, little is known about mucin gene expression and the role of mucins in barrier function and host-cell interaction. The aims of the study were to identify equine orthologs of mammalian mucin genes using available equine sequence data, to profile expression of equine orthologous mucin genes in the endometrium using reverse transcriptase polymerase chain reaction (RT-PCR), to determine spatial expression patterns of mucin genes using in situ hybridisation, and to confirm the presence of mucin gene products using Western blotting and equine-specific mucin antibodies during oestrus and dioestrus. While the mucin gene expression pattern in equine endometrium is similar to that of other mammals, several mucins appear to be uniquely expressed in this tissue (eqMUC3B, 7, 18, and 20) and one is hormonally regulated (eqMUC3B).

Construction of a natural mucin microarray and interrogation for biologically relevant glyco-epitopes.

Mucins are the principal components of mucus, and mucin glycosylation has important roles in defense, microbial adhesion, immunomodulation, inflammation, and cancer. Mucin expression and glycosylation are dynamic, responding to changes in local environment and disease. Potentially hundreds of heterogeneous glycans can substitute one mucin molecule, and it is difficult to identify biologically accessible glyco-epitopes. Thirty-seven mucins, from the reproductive and gastrointestinal (GI) tracts of six species (bovine, ovine, equine, porcine, chicken, and deer) and from two human-derived cell lines, were purified. Following optimization of mucin printing and construction of a novel mucin microarray, the glycoprofiles of the whole mucins on the microarray were compared using a panel of lectins and one antibody. Accessible glyco-motifs of GI mucins varied according to species and localization of mucin origin, with terminal fucose, the sialyl T-antigen, and N-linked oligosaccharides identified as potentially important. The occurrence of T- and sialyl T-antigen varied in bovine and ovine reproductive tract mucins, and terminal N-acetylgalactosamine (GalNAc) and sulfated carbohydrates were detected. This study introduces natural mucin microarrays as an effective tool for profiling mucin glyco-epitopes and highlights their potential for discovery of biologically important motifs in bacterial-host interactions and fertility.

Purified chicken intestinal mucin attenuates Campylobacter jejuni pathogenicity in vitro.

Campylobacter jejuni is a major causative agent of diarrhoeal disease worldwide in the human population. In contrast, heavy colonization of poultry typically does not lead to disease and colonized chickens are a major source of Campylobacter infections in humans. Previously, we have shown that chicken (but not human) intestinal mucus inhibits C. jejuni internalization. In this study, we test the hypothesis that chicken mucin, the main component of mucus, is responsible for this inhibition of C. jejuni virulence. Purified chicken intestinal mucin attenuated C. jejuni binding and internalization into HCT-8 cells depending on the site of origin of the mucin (large intestine>small intestine>caecum). C. jejuni invasion of HCT-8 cells was preferentially inhibited compared to bacterial binding to cells. Exposure of the mucin to sodium metaperiodate recovered bacterial invasion levels, suggesting a glycan-mediated effect. However, fucosidase or sialidase pre-treatment of mucin failed to abrogate the inhibition of C. jejuni pathogenicity. In conclusion, differences in the composition of chicken and human intestinal mucin may contribute to the differential outcome of Campylobacter infection of these hosts.