Oral Delivery of Nisin in Resistant Starch Based Matrices Alters the Gut Microbiota in Mice.

There is a growing recognition of the role the gastrointestinal microbiota plays in health and disease. Ingested antimicrobial proteins and peptides have the potential to alter the gastrointestinal microbiota; particularly if protected from digestion. Nisin is an antimicrobial peptide that is used as a food preservative. This study examined the ability of nisin to affect the murine microbiota when fed to mice in two different starch based matrices; a starch dough comprising raw starch granules and a starch gel comprising starch that was gelatinized and retrograded. The effects of the two starch matrices by themselves on the microbiota were also examined. Following 16S rRNA compositional sequencing, beta diversity analysis highlighted a significant difference (p = 0.001, n = 10) in the murine microbiota between the four diet groups. The differences between the two nisin containing diets were mainly attributable to differences in the nisin release from the starch matrices while the differences between the carriers were mainly attributable to the type of resistant starch they possessed. Indeed, the differences in the relative abundance of several genera in the mice consuming the starch dough and starch gel diets, in particular Akkermansia, the relative abundance of which was 0.5 and 11.9%, respectively (p = 0.0002, n = 10), points to the potential value of resistance starch as a modulator of beneficial gut microbes. Intact nisin and nisin digestion products (in particular nisin fragment 22-31) were detected in the feces and the nisin was biologically active. However, despite a three-fold greater consumption of nisin in the group fed the nisin in starch dough diet, twice as much nisin was detected in the feces of the group which consumed the nisin in starch gel diet. In addition, the relative abundance of three times as many genera from the lower gastrointestinal tract (GIT) were significantly different (p < 0.001, n = 10) to the control for the group fed the nisin in starch gel diet, implying that the starch gel afforded a degree of protection from digestion to the nisin entrapped within it.

A Simple Method for the Purification of Nisin.

Nisin, an antimicrobial peptide showing activity against a broad range of Gram-positive bacteria, is widely used as a food preservative and has potential as a therapeutic for a range of infectious diseases. Here, we present a simple purification method, based on a salting-out approach, which can produce a powder containing ∼33% nisin, from a nisin-producing culture in a whey permeate-based medium. This process removes over 99% of the lactic acid, NaCl, lactose and non-nisin proteins from the cell-free culture supernatant. The approach can also enrich a commonly used commercial nisin preparation over 30-fold to a purity of ∼58%. These are higher purities than comparable published methods. The simplicity of this approach facilitates its use in research and also its scale-up.

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.

Divergent mechanisms of interaction of Helicobacter pylori and Campylobacter jejuni with mucus and mucins.

Helicobacter pylori and Campylobacter jejuni colonize the stomach and intestinal mucus, respectively. Using a combination of mucus-secreting cells, purified mucins, and a novel mucin microarray platform, we examined the interactions of these two organisms with mucus and mucins. H. pylori and C. jejuni bound to distinctly different mucins. C. jejuni displayed a striking tropism for chicken gastrointestinal mucins compared to mucins from other animals and preferentially bound mucins from specific avian intestinal sites (in order of descending preference: the large intestine, proximal small intestine, and cecum). H. pylori bound to a number of animal mucins, including porcine stomach mucin, but with less avidity than that of C. jejuni for chicken mucin. The strengths of interaction of various wild-type strains of H. pylori with different animal mucins were comparable, even though they did not all express the same adhesins. The production of mucus by HT29-MTX-E12 cells promoted higher levels of infection by C. jejuni and H. pylori than those for the non-mucus-producing parental cell lines. Both C. jejuni and H. pylori bound to HT29-MTX-E12 mucus, and while both organisms bound to glycosylated epitopes in the glycolipid fraction of the mucus, only C. jejuni bound to purified mucin. This study highlights the role of mucus in promoting bacterial infection and emphasizes the potential for even closely related bacteria to interact with mucus in different ways to establish successful infections.

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.