A51 Utilizing Mucin-Derived Sugars Confers a Fitness Advantage to the Enteric Pathogen Citrobacter Rodentium That Promotes Intestinal Colonization

Abstract

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