Intestinal organoids have become indispensable tools for many gastrointestinal researchers, advancing their studies of nontransformed intestinal epithelial cells, and their roles in an array of diseases, including inflammatory bowel disease and colon cancer. In many cases. these diseases, as well as many enteric infections, reflect pathogenic interactions between bacteria and the gut epithelium. The complexity of studying this microbe–epithelial interface in vivo has led to significant focus on modeling this cross-talk using organoid models. Considering how quickly the organoid field is advancing, it can be difficult to keep up to date with the latest techniques, as well as their respective strengths and weaknesses. This review addresses the advantages of using organoids derived from adult stem cells and the recently identified differences that biopsy location and patient age can have on organoids and their interactions with microbes. Several approaches to introducing bacteria in a relevant (apical) manner (ie, microinjecting 3-dimensional spheroids, polarity-reversed organoids, and 2-dimensional monolayers) also are addressed, as are the key readouts that can be obtained using these models. Lastly, the potential for new approaches, such as air–liquid interface, to facilitate studying bacterial interactions with important but understudied epithelial subsets such as goblet cells and their products, is evaluated.
Background Citrobacter rodentium is an enteric murine pathogen used to model the human diarrheal pathogens. Following inoculation, C. rodentium colonizes the mouse cecum where it expands and ultimately spreads to the distal colon. During this process, C. rodentium has to compete with commensal microbes for available nutrients. Moreover, to spread throughout the gut, and infect the intestinal epithelium, C. rodentium has to cross through, and or dwell within the intestinal mucus layer which is composed of the heavily glycosylated protein Muc2. Muc-2 is glycosylated and coated by 5 distinct terminal sugar residues: galactose, N-acetylgalactosamine, N-acetylglucosamine, fucose, and sialic acid. Many commensal microbes have the ability to cleave and free these sugars from the Muc2 protein, releasing them for their own consumption, however pathogens appear to exploit this process. While studies have indicated that C. rodentium uses these terminal sugar residues as a nutritional source, their relative importance in the pathogenic strategy of C. rodentium (and other gut pathogens) remains unclear Aims Investigate the role played by mucin sugar residues in controlling C. rodentium pathogenesis Methods Deletions of agaW, nagE, mglB, galP, fucK, and nanT were generated on the chromosome of C. rodentium (Strepr) by overlap extension PCR. Growth assays were performed to examine the growth kinetics of mutants C. rodentium in minimal (M9) media supplemented with one of the 5 mucin sugars or M9 with whole mucin as control. Specific pathogen free (SPF) C57BL/6 mice, or germfree C57BL/6 mice were orally gavaged with wildtype C. rodentium (Strepr) or one of ΔagaW, ΔnagE, ΔmglB, ΔgalP, ΔfucK, or ΔnanT strains. Mice were euthanized at 6 days post-infection, and the cecum, colon, and spleen were collected and histologically scored for pathology and intestinal and systemic bacterial burden. Stool samples were collected throughout the 6 days to quantify C. rodentium burdens Results Growth assays confirmed that the specific sugar transporter/kinase mutant C. rodentium strains grew normally when placed in media supplemented with whole mucin, or with most sugars, only showing overt defects in growth when solely supplemented with the sugar for which they were impaired. Several of the C. rodentium mutants including ΔnanT showed overt defects in colonization/infection of SPF C57BL/6 mice, but their pathogenesis was normalized in germfree mice, or in mice treated with the antibiotic streptomycin at each day post-infection. These findings indicate that the impact of mucin sugar utilization on C. rodentium virulence is microbiota-dependent Conclusions C. rodentium uses mucin sugars as nutrient source in the mouse gut, and an inability to use these sugars impairs their ability to infect their hosts in a microbiota dependent manner Funding Agencies CAG, CCC, CIHR, NRC
Background The ability of enteric pathogens to colonize and expand within the mammalian gastrointestinal (GI) tract is determined by several factors, including the ability to find and acquire nutrients. The thick mucus layer that lines the inner surface of the large intestine is rich in sugars that can serve as nutrient sources for several members of the microbiota. Whether these sugars can also be used by invading bacterial pathogens to colonize the GI tract is still unclear, in particular for the family of attaching and effacing (A/E) bacterial pathogens, including the human diarrheal pathogens EHEC and EPEC. Aims To investigate the ability of the murine A/E pathogen Citrobacter rodentium to use mucin-derived sugars as a nutrient source, and the importance of these sugars in the virulence of C. rodentium during in-vivo infection. Methods To identify which sugar(s) are required for C. rodentium to colonize and grow in the murine GI tract, we generated mutants lacking single or multiple genes involved in the uptake and catabolism of mucin-derived O-glycan sugars. This was followed by in-vitro growth assays in minimal media supplemented with mucin sugars to investigate the growth properties of C. rodentium and the generated mutants on mucin sugars. Results We determined that C. rodentium was able to use three mucin O-glycan sugars: sialic acid, galactose, and N-acetylglucosamine (GlcNAc) as both carbon and nitrogen sources for in-vitro growth. C. rodentium exhibited the maximal growth rate and density on GlcNAc, followed by sialic acid, and finally galactose. A mutant C. rodentium strain carrying a deletion in the nagA gene was unable to grow on both GlcNAc and sialic acid, confirming that the breakdown pathways for these two sugars merge and are processed by shared suite of enzymes. As for galactose, combined deletions in the genes mglB and galP were required to abolish growth on this sugar. Notably, a mutant strain carrying simultaneous deletions in nagA, mglB, and galP was unable to grow on all three mucin sugars, as well as on purified mucin. Conclusions Our results demonstrate that intestinal mucin sugars serve as potential nutrient sources for C. rodentium and that C. rodentium can catabolize three of these sugars. Future work will examine whether these sugar pathways contribute to C. rodentium colonization of the murine GI tract. Funding Agencies CCC, CIHRCH.I.L.D. Fdn
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