Regulated virulence controls the ability of a pathogen to compete with the gut microbiota

Abstract

The virulence mechanisms that allow pathogens to colonize the intestine remain unclear. Here, we show that germ-free animals are unable to eradicate Citrobacter rodentium, a model for human infections with attaching and effacing bacteria. Early in infection, virulence genes were expressed and required for pathogen growth in conventionally raised mice but not germ-free mice. Virulence gene expression was down-regulated during the late phase of infection, which led to relocation of the pathogen to the intestinal lumen where it was outcompeted by commensals. The ability of commensals to outcompete C. rodentium was determined, at least in part, by the capacity of the pathogen and commensals to grow on structurally similar carbohydrates. Thus, pathogen colonization is controlled by bacterial virulence and through competition with metabolically related commensals.

Fig. 1. The microbiota is required for eradication of C. rodentium

A,B, SPF and GF mice (n= 7) were infected orally with 1×10⁹ cfu of C. rodentium and pathogen load in feces (A) and mouse survival (B) were determined over the indicated time. Data points are given as mean ± SD. Results are representative of at least 3 independent experiments. ***; p<0.001 by Mann-Whitney U test.

Fig. 2. Expression and role of ler during C. rodentium infection in SPF and GF mice

A, ler mRNA levels were determined by qPCR in fecal pellets of SPF and GF mice infected with C. rodentium at the indicated days post-infection. Expression was normalized to that of the kanamycin resistance gene carried by the C. rodentium strain. Control experiments were performed by determining ler mRNA levels in C. rodentium grown under inducing (DMEM) and repressing (LB) in vitro culture conditions (4, 10). Data represent mRNA expression relative to that in C. rodentium cultured in LB medium. Results are given as mean ± SD of individual mice (n=3). Results are representative of at least 2 experiments. *; p<0.05, ***; p<0.001 by Dunnett's multiple comparison test. B, Expression of ler in fecal pellets of SPF and GF mice infected with the reporter ler-lux C. rodentium strain at the indicated day post-infection. Results show luminescence (relative light units) and cfu of ler-lux C. rodentium in the same samples. Data expressed as mean ± SD of individual mice (n=4). Results are representative of at least 2 experiments. C, Bioluminescent imaging of ler expression in the intestines of SPF and GF mice infected with the ler-lux C. rodentium strain. Imaging was performed on day 5 and 14 post-infection and the signal was quantified based on the color scale shown below. Results are representative of 3 individual mice. D, SPF and GF mice (n= 5) were infected orally with 10⁹cfu of WT and Δler mutant C. rodentium, and pathogen load in feces was determined over the indicated time. Data points are given as mean ± SD. Results are representative of at least 2 experiments. E, GF mice were infected with WT and Δler mutant C. rodentium. At day 3 or day 21 post infection, mice were co-housed with SPF mice (1:1). Pathogen load was determined in feces on indicated days after co-housing. Dots represent individual mice. Results are representative of at least 3 experiments. N.S., not significant, *; p<0.05, **; p<0.01 by Dunn's test.

Fig. 3. Localization of C. rodentium to intestinal niches is mediated by LEE-encoded virulence factors

A, Dual FISH staining using DNA probes that label virtually all true bacteria (EUB338, red) and the γ-Proteobacteria class to which C. rodentium belongs (GAM42a, green). Pathogenic bacteria (i.e. EUB338⁺/GAM42a⁺ cells) are yellow. Lower panels show high magnification images corresponding to boxed areas (upper panels). Note the yellow color on all the bacteria stained in the cecum infected with the Δler mutant; indicating all bacteria in the lumen are C. rodentium. Scale bar: 50μm (upper panels), 20μm (lower panels), Results are representative of 2 experiments. B, Transmission Electron Micrographs of cecum from infected GF mice at day 5 and day 21 post infection with WT C. rodentium and at day 5 with the Δler mutant. Original magnification: 3,400× (left panel) and 13,500× (right panel). Scale bar: 2 μm (left panel) and 500 nm (right panel). Results are representative of 2 experiments. C, GF mice were infected orally with WT C. rodentium carrying the ler-lux fusion. Cecum and colonic tissues were collected at the indicated day and then washed with PBS to remove non-adherent bacteria. Bioluminescent imaging of ler expression of C. rodentium attached to the cecum (top) and colon (bottom). Results are representative of 2 experiments using 4 different mice.

Fig. 4. Similar catabolic preferences for saccharides may determine the competing ability of commensal bacteria with the enteric pathogen

A, GF mice were infected with WT C. rodentium (Cr). At day 21 post infection, E. coli (Ec) or B. thetaiotaomicron (Bt) or B. vulgatus (Bv) were inoculated. As a second inoculation, mixture of B. thetaiotaomicron and B. vulgatus was inoculated into the E. coli harboring group, and E. coli was inoculated into the Bacteroides harboring group, respectively. Pathogen load was determined in feces on the indicated days after inoculation of commensal bacteria. Dots represent individual mice from 2 independent experiments. B, Total Enterobacteria (C. rodentium and E. coli) and Bacteroides culture in feces at day 0 and day 14 (first inoculation in panel A). Data are given as mean ± SD (n=4). Results are representative of at least 2 experiments. C, Number of C. rodentium (kanamycin resistant) in the total Enterobacteria (kanamycin sensitive) are indicated as a percentage. Data are given as mean ± SD (n=4). Results are representative of at least 2 experiments. D, Carbohydrate catabolic profiles of C. rodentium and commensal bacteria strains. Robust growth under supplementation of monosaccharides (MSs) or polysaccharides (PSs) indicated as red, and no growth indicated as yellow. Raw data is provided in Table S1. E, GF mice were infected with WT C. rodentium (Cr). At day 21 post infection, B. thetaiotaomicron (Bt) was inoculated. On day 7 post-colonization with Bt, the mice were divided into two groups that were fed a conventional maintenance diet (C) or a simple sugar diet (ss). Pathogen load was determined in feces on day 3 after diet switching. Results are given as mean ± SD of individual mice (initially n=10, and divided into 2 groups n=5 each). Results are representative of 2 experiments. F, C. rodentium mono-associated GF mice (day 21) were fed with a simple sugar diet (ssDiet) for 7 days. Pathogen load was compared before and after switching to ssDiet from conventional diet (cDiet). Dots represent individual mice and representative of 3 independent experiments **; p<0.01. N.S., denotes not significant by Mann-Whitney U test.