Application of a gut-immune co-culture system for the study of N-glycan-dependent host–pathogen interactions of Campylobacter jejuni

Abstract

An in vitro gut-immune co-culture model with apical and basal accessibility, designed to more closely resemble a human intestinal microenvironment, was employed to study the role of the N-linked protein glycosylation pathway in Campylobacter jejuni pathogenicity. The gut-immune co-culture (GIC) was developed to model important aspects of the human small intestine by the inclusion of mucin-producing goblet cells, human enterocytes and dendritic cells, bringing together a mucus-containing epithelial monolayer with elements of the innate immune system. The utility of the system was demonstrated by characterizing host–pathogen interactions facilitated by N-linked glycosylation, such as host epithelial barrier functions, bacterial invasion and immunogenicity. Changes in human intestinal barrier functions in the presence of 11168 C. jejuni (wildtype) strains were quantified using GICs. The glycosylation-impaired strain 11168 ΔpglE was 100-fold less capable of adhering to and invading this intestinal model in cell infectivity assays. Quantification of inflammatory signaling revealed that 11168ΔpglE differentially modulated inflammatory responses in different intestinal microenvironments, suppressive in some but activating in others. Virulence-associated outer membrane vesicles produced by wildtype and 11168ΔpglE C. jejuni were shown to have differential composition and function, with both leading to immune system activation when provided to the gut-immune co-culture model. This analysis of aspects of C. jejuni infectivity in the presence and absence of its N-linked glycome is enabled by application of the gut-immune model, and we anticipate that this system will be applicable to further studies of C. jejuni and other enteropathogens of interest.

Identifying the targets and functions of N-linked protein glycosylation in Campylobacter jejuni

“…Packaging of PglB into OMVs may enable glycosylation of sites not typically found in the membrane; however despite this, we and others have observed no such cytoplasmic N-glycosites, even at low levels, which may imply that PglB does not occur in OMVs, or that OMVs are not induced (or collected) under the culture conditions employed in the N-glycosite discovery studies conducted thus far. C. jejuni OMV composition is however, altered in pgl-negative compared with wild-type C. jejuni, 113 suggesting N-glycosylation does impact protein packaging, although no differences were observed in the ability of OMVs from either pgl positive or negative bacteria to induce an immune response. 113 Putative functions of C. jejuni protein N-glycosylation Deletion of genes from the pgl cluster (except pglI) results in C. jejuni that are poorly able to colonize chickens and display reduced adherence to, and invasion of, human epithelial cells.…”

“…C. jejuni OMV composition is however, altered in pgl-negative compared with wild-type C. jejuni, 113 suggesting N-glycosylation does impact protein packaging, although no differences were observed in the ability of OMVs from either pgl positive or negative bacteria to induce an immune response. 113 Putative functions of C. jejuni protein N-glycosylation Deletion of genes from the pgl cluster (except pglI) results in C. jejuni that are poorly able to colonize chickens and display reduced adherence to, and invasion of, human epithelial cells. 114,115 Additional recent modelling of C. jejuni virulence in a human small intestine-like gut-immune co-culture model also revealed that pgl-negative C. jejuni (in this case, pglE deletion) were vastly deficient (B100 times less) in adherence and invasion.…”

“…114,115 Additional recent modelling of C. jejuni virulence in a human small intestine-like gut-immune co-culture model also revealed that pgl-negative C. jejuni (in this case, pglE deletion) were vastly deficient (B100 times less) in adherence and invasion. 113 Therefore, N-glycosylation is considered a fundamental virulence determinant in this organism. Despite this, until recently 116,117 there have been very few studies that have broadly characterized pgl-associated phenotypes, and the function(s) of the N-glycosylation system in general, and even more so the role of the N-glycan on individual proteins, remains almost completely unknown.…”

The Role of Organoids as a Novel Platform for Modeling of Inflammatory Bowel Disease

“…Interestingly, duodenal, ileal and colonic organoid cultures derived from different donors demonstrate a differential response to infection and differing patterns of bacterial adhesion, possibly due to the genetic variability based on the tissue of origin ( 89 ). A co-culture model developed to study the host-pathogen interactions of C. jejuni incorporates intestinal enterocytes, mucin-secreting goblet cells and dendritic cells, thus combining a mucus-secreting epithelial layer with cellular elements of the intestinal innate immune system ( 92 ).…”

Reactive oxygen species limit intestinal mucosa-bacteria homeostasis in vitro

“…To date, the majority of reported long-term (up to 2 weeks) co-cultures with live bacteria in contact with epithelial cells have been in microfluidic devices 12 , 13 , although primarily short-term (< 4 h) co-cultures have been reported in static systems 14 – 18 , with some recent medium-term (24 h) exceptions 19 . Notably, reports of static cultures incorporating immune components, specifically dendritic cells 15 , 19 or macrophage simulating differentiated THP-1 cells 20 , and bacteria result in high accumulation of dead epithelial cells and significant increases in paracellular permeability when compared to control cultures, even when cultured with bacteria for as little as 2 h. This suggests that flow is an important factor in preserving epithelial health and barrier function. It has been proposed that flow is important for preventing the accumulation of microbial waste and microbial overgrowth, thus aiding in establishing a steady-state microenvironment 12 .…”