Intestinal inflammation allows Salmonella to use ethanolamine to compete with the microbiota

Abstract

Conventional wisdom holds that microbes support their growth in vertebrate hosts by exploiting a large variety of nutrients. We show here that use of a specific nutrient (ethanolamine) confers a marked growth advantage on Salmonella enterica serovar Typhimurium (S. Typhimurium) in the lumen of the inflamed intestine. In the anaerobic environment of the gut, ethanolamine supports little or no growth by fermentation. However, S. Typhimurium is able to use this carbon source by inducing the gut to produce a respiratory electron acceptor (tetrathionate), which supports anaerobic growth on ethanolamine. The gut normally converts ambient hydrogen sulfide to thiosulfate, which it then oxidizes further to tetrathionate during inflammation. Evidence is provided that S. Typhimurium's growth advantage in an inflamed gut is because of its ability to respire ethanolamine, which is released from host tissue, but is not utilizable by competing bacteria. By inducing intestinal inflammation, S. Typhimurium sidesteps nutritional competition and gains the ability to use an abundant simple substrate, ethanolamine, which is provided by the host.

Fig. 1.

Anaerobic growth of S. Typhimurium on ethanolamine as the sole carbon source in vitro. (A) Schematic drawing of the eut locus in the chromosome of the S. Typhimurium strain LT2. The locations, orientations and sizes of genes are indicated by arrows. Mutation in strains PT100 (eutC::pPT5) and TT26355 (ΔeutC::FRT) are indicated. (B) Anaerobic growth curve of the S. Typhimurium wild-type (IR715; circles) and an eutC mutant (PT100; squares) with ethanolamine as the sole carbon source in the presence (open symbols) or absence (filled symbols) of the respiratory electron acceptor tetrathionate (S₄O₆²⁻). The number of viable bacteria was determined at the indicated time points. (C) Competitive growth of S. Typhimurium strains [wild-type (IR715) vs. eutC mutant (PT100); invA spiB mutant (SPN452) vs. invA spiB eutC mutant (PT102); ttrA mutant (SW661) vs. ttrA eutC mutant (PT106)]. Bacterial numbers were determined 24 h after anaerobic growth with ethanolamine as the sole carbon source in the presence (black bars) or absence (white bars) of tetrathionate. Data represent the geometric mean of three independent experiments ± SE. Statistical significance (P value) is indicated above the bars.

Fig. 2.

Ethanolamine utilization genes confer a growth advantage in the inflamed intestine. (A–D) Streptomycin-pretreated mice (mouse colitis model) were orally inoculated with equal mixtures of S. Typhimurium strains [wild-type (IR715) vs. eutC mutant (PT100); invA spiB mutant (SPN452) vs. invA spiB eutC mutant (PT102); ttrA mutant (SW661) vs. ttrA eutC mutant (PT106)] as indicated below each panel. Samples were collected 4 d after infection. The number of animals is indicated in A. (A) Blinded, combined histopathology score of cecal inflammation showing averages (lines) of individual scores (circles). (B) Representative images of H&E-stained cecal sections. (Scale bars, 100 μm.) (C) Expression of proinflammatory markers in the cecal mucosa. Relative mRNA levels of Kc (black) and Nos2 (white) were determined by quantitative real-time RT-PCR analysis. Data shown are fold-changes over mock-treated control animals. (D) Competitive indices of S. Typhimurium strains recovered from the colon content. Data represent the geometric mean ± SE. Statistical significance (P value) is indicated above the bars.

Fig. 3.

Quantification of intestinal ethanolamine concentrations in the mouse colitis model. Streptomycin-pretreated mice were orally infected with the S. Typhimurium wild-type (IR715), an eutC mutant (PT100), an invA spiB mutant (SPN452), or mock-treated, as indicated below each panel. Four days after infection, the ethanolamine concentration in colonic extracts was determined by LC-MS/MS. Data represent the geometric mean of four animals ± SE. Statistical significance (P value) is indicated above the bars.

Fig. 4.

Contribution of ethanolamine utilization to intestinal colonization in the mouse colitis model. (A–D) Streptomycin-pretreated mice were orally inoculated with the S. Typhimurium wild-type (IR715), an eutC mutant (PT100), an invA spiB mutant (SPN452), or mock-treated, as indicated below each panel. Samples were collected 4 d after infection. The number of animals is indicated in A. (A) Blinded histopathology scoring of cecal inflammation showing averages (lines) of individual scores (circles). (B) Representative images of H&E-stained cecal sections. (Scale bars, 100 μm.) (C) Expression of proinflammatory markers in the cecal mucosa. Relative mRNA expression of Kc (black) and Nos2 (white) was determined by quantitative real-time RT-PCR analysis. Data shown are fold-changes over mock-treated animals. (D) Recovery of S. Typhimurium from the colon content of infected mice. Data represent the geometric mean ± SE. Statistical significance (P value) is indicated above the bars.

Fig. 5.

Schematic of ethanolamine utilization and tetrathionate respiration in the inflamed gut.