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Defining enteric bacterial pathogenesis using organoids: Citrobacter rodentium uses EspC, an atypical mucinolytic protease, to penetrate mouse colonic mucus

ABSTRACT: Enteric bacterial pathogens pose significant threats to human health; however, the mechanisms by which they infect the mammalian gut in the face of daunting host defenses remain to be fully defined. For the attaching and effacing (A/E) bacterial family member and murine pathogen Citrobacter rodentium, its virulence strategy appears to involve penetration of the colonic mucus barrier, to reach the underlying epithelium. To better define these interactions, we grew colonoids under air-liquid interface (ALI) conditions, producing a thick mucus layer that mimicked in vivo mucus composition and glycosylation. C. rodentium’s penetration of ALI mucus was dramatically enhanced upon exposure to sialic acid, in concert with the secretion of two autotransporter (SPATE) proteins, Pic and EspC. Despite Pic being a class II SPATE, and already recognized as a mucinase, it was EspC, belonging to the class I SPATE family, and not previously shown to possess mucinase activity, that degraded the ALI-derived mucus. Confirming this finding, E. coli DH5 carrying a plasmid that expresses C. rodentium-derived EspC was able to degrade the mucus. Notably, recombinant EspC alone also displayed mucinolytic activity in a dose-dependent manner. Collectively, our results reveal the utility of ALI-derived mucus in modeling microbe-host interactions at the intestinal mucosal surface, as well as identify EspC as an atypical Class I SPATE that shows significant mucinolytic activity towards ALI-derived mucus.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Crypt Of Lieberkuhn Of Colon, Colon, Colonic Mucosa

DISEASE(S): Intestinal Infectious Disease

SUBMITTER: Dominique Levesque

LAB HEAD: Bruce A. Vallance

PROVIDER: PXD060170 | Pride | 2026-02-03

REPOSITORIES: Pride

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			Action	DRS
	DIA_mucus_01_Slot1-15_1_20220.d.7z	Other
	DIA_mucus_02_Slot1-16_1_20223.d.7z	Other
	DIA_mucus_03_Slot1-17_1_20226.d.7z	Other
	DIA_mucus_04_Slot1-5_1_20298.d.7z	Other
	DIA_mucus_05_Slot1-6_1_20301.d.7z	Other

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Project description:Transcription profiling of wild type E. coli MG1655, intestine-adapted E. coli MG1655star, and E. coli MG1655 flhD mutant grown on glucose, mannose, and mucus. We previously isolated a spontaneous mutant of E. coli K-12, strain MG1655, following passage through the streptomycin-treated mouse intestine, which has colonization traits superior to the wild-type parent strain (Leatham, et. al., 2005, Infect Immun 73:8039-49) The intestine-adapted strain (E. coli MG1655star) grew faster on several different carbon sources compared to the wild-type and was non-motile due to deletion of the flhD gene. To further characterize E. coli MG1655star, we used several high-throughput genomic approaches. Whole-genome pyrosequencing did not reveal any changes on its genome, aside from the deletion at the flhDC locus, that could explain the colonization advantage of E. coli MG1655star. Microarray analysis revealed modest, yet significant induction of catabolic gene systems across the genome in both E. coli MG1655star and the isogenic flhD mutant. Catabolome analysis with Biolog GN2 Microplates revealed an enhanced ability of both E. coli MG1655star and the isogenic flhD mutant to oxidize a wide variety of carbon sources. The results show that intestine-adapted E. coli MG1655star is more fit than the wild-type for intestinal colonization because loss of FlhD results in elevated expression of genes involved in carbon and energy metabolism, leading to more efficient carbon source utilization, which results in a higher population size in the intestine. Hence mutations that enhance metabolic efficiency confer a colonization advantage. Three strains were profiled: E. coli MG1655 wildtype, E. coli flhD, and an intestine adapted strain, MG1655star, derived from the wildtype and isolated from feces after 15 days in the streptomycin treated mouse intestine, which proved to be a better colonizer than the wildtype, were grown on MOPS minimal medium containing 0.2% glucose or mannose, or mucus (10 mg/ml) and RNA was extracted from logarithmic phase cultures, and also from mucus grown cells in late log phase.

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Defining enteric bacterial pathogenesis using organoids: Citrobacter rodentium uses EspC, an atypical mucinolytic protease, to penetrate mouse colonic mucus

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