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. 2022 Jan 4;38(1):110180.
doi: 10.1016/j.celrep.2021.110180.

Salmonella enterica serovar Typhimurium uses anaerobic respiration to overcome propionate-mediated colonization resistance

Catherine D Shelton  1 , Woongjae Yoo  1 , Nicolas G Shealy  1 , Teresa P Torres  1 , Jacob K Zieba  1 , M Wade Calcutt  2 , Nora J Foegeding  1 , Dajeong Kim  3 , Jinshil Kim  4 , Sangryeol Ryu  4 , Mariana X Byndloss  5
Affiliations

Salmonella enterica serovar Typhimurium uses anaerobic respiration to overcome propionate-mediated colonization resistance

Catherine D Shelton et al. Cell Rep. .

Abstract

The gut microbiota benefits the host by limiting enteric pathogen expansion (colonization resistance), partially via the production of inhibitory metabolites. Propionate, a short-chain fatty acid produced by microbiota members, is proposed to mediate colonization resistance against Salmonella enterica serovar Typhimurium (S. Tm). Here, we show that S. Tm overcomes the inhibitory effects of propionate by using it as a carbon source for anaerobic respiration. We determine that propionate metabolism provides an inflammation-dependent colonization advantage to S. Tm during infection. Such benefit is abolished in the intestinal lumen of Salmonella-infected germ-free mice. Interestingly, S. Tm propionate-mediated intestinal expansion is restored when germ-free mice are monocolonized with Bacteroides thetaiotaomicron (B. theta), a prominent propionate producer in the gut, but not when mice are monocolonized with a propionate-production-deficient B. theta strain. Taken together, our results reveal a strategy used by S. Tm to mitigate colonization resistance by metabolizing microbiota-derived propionate.

Keywords: Salmonella; colonization resistance; gut microbiota; intestinal inflammation; microbial metabolism; propionate.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Propionate fuels S. Tm growth in the presence of nitrate in vitro
(A) Simplified model of propionate catabolism in S. Tm. Genes in the prpBCDE operon (red) metabolize propionate into pyruvate. prpE, propionyl-CoA synthase; prpC, methylcitrate synthase; prpD, methylcitrate dehydratase; prpB, 2-methylisocitrate lyase. (B) NCE minimal medium containing 40 mM of an alternative electron acceptor alone or 40 mM alternative electron acceptor + 10 mM propionate was inoculated with S. Tm and grown anaerobically for 24 h. Alternatively, S. Tm was grown in a hypoxic chamber under 0.8% oxygen in NCE minimal medium with or without 10 mM propionate. Fold change calculated by comparing growth on oxygen or alternative electron acceptor + 10 mM propionate with growth with oxygen or alternative electron acceptor alone. (C) NCE minimal medium containing increasing propionate concentration with or without 40 mM nitrate was inoculated with S. Tm. The OD600 of S. Tm was measured after 24 h of anaerobic growth. (D–F) Relative transcription of prpR (D), prpB (E), and prpC (F) in NCE minimal medium supplemented with propionate, nitrate, or both propionate and nitrate was determined by qRT-PCR. Transcription of target genes was normalized to gyrB rRNA. (G) NCE minimal medium containing propionate or propionate and nitrate was inoculated with WT S. Tm, ΔprpC, or a complemented strain of ΔprpC (−ΔprpC_pprpC). The medium was supplemented with 200 μM isopropyl β-D-1-thiogalactopyranoside to induce expression of prpC in the complemented mutant strain. The OD600 of each strain was measured after 24 h of anaerobic growth. Each dot represents one biological replicate (average of triplicate technical replicate per biological replicate). Bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not statistically significant. See also Figure S1.
Figure 2.
Figure 2.. Nitrate prevents inhibitory effects of propionate on S. Tm in vitro
(A–C) NCE minimal medium was adjusted to pH 7.0 (A), pH 6.5 (B), and pH 6.0 (C), respectively. The medium was supplemented with 10 mM propionate or 10 mM propionate + 40 mM nitrate and inoculated with WT S. Tm or ΔprpC. The culture was grown anaerobically for 14 h, and samples were taken every 2 h to plate for CFUs. n = 4. (D–F) Cultures grown in (A–C) were used to calculate the generation time of WT S. Tm or ΔprpC at pH 7.0 (D), pH 6.5 (E), and pH 6.0 (F). Each dot represents one biological replicate (average of triplicate technical replicate per biological replicate). Bars represent mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001. See also Figure S2.
Figure 3.
Figure 3.. Propionate utilization confers an advantage to S. Tm in an inflammation-dependent mechanism
(A) Streptomycin-pretreated C57BL/6 mice were inoculated with an equal mixture of WT S. Tm and ΔprpC. The competitive index in the colon content and homogenized samples from the liver or spleen was determined 4 days after infection. (B) Streptomycin-pretreated C57BL/6 mice were inoculated with an equal mixture of the indicated S. Tm strains. The competitive index in the colonic content was determined 4 days after infection. (C) Combined histopathology score of pathological lesions in the cecum of mice from (B) (n = 5). (D) Representative images of hematoxylin and eosin-stained cecal tissue of mice from (C). Scale bars, 200 μm. (E) Propionate concentration in the cecal content was determined by liquid chromatography/mass spectrometry (LC/MS) 2 days after infection. (F) Streptomycin-pretreated C57BL/6 mice were inoculated with an equal mixture of WT S. Tm and ΔprpC. Mice received propionate supplementation (20 mM) in the drinking water for the duration of the experiment. The competitive index in the colonic content was determined 4 days after infection. Each dot represents data from one animal (biological replicate). Bars represent mean ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001. See also Figure S3.
Figure 4.
Figure 4.. Inflammation generated nitrate required for propionate catabolism by S. Tm
(A) Streptomycin-pretreated C57BL/6 mice were inoculated with an equal mixture of the indicated S. Tm strains. The competitive index in the colon content was determined 4 days after infection. (B) Streptomycin-pretreated C57BL/6 mice were inoculated with WT S. Tm, ΔinvA ΔspiB, or mock treated. mRNA levels of Nos2 were measured in the cecal mucosa 4 days post-infection and normalized to β-actin mRNA levels. (C–E) Streptomycin-pretreated C57BL/6 WT mice and Nos2-deficient mice were inoculated with an equal mixture of the WT S. Tm and ΔprpC mutant. One group was treated with aminoguanidine (AG) as indicated. (C) The competitive index in the cecal content was determined 4 days post-infection. (D) Nitrate concentration in the cecal and colonic mucus layer was determined by a modified Griess assay. (E) Combined histopathology score of pathological lesions in the cecum of mice from (C) (n = 5). (F) Propionate concentration in the cecal content of mice from (C) was determined by liquid chromatography/mass spectrometry (LC/MS) 2 days after infection. (F and G) Streptomycin-pretreated C57BL/6 mice were inoculated with an equal mixture of WT S. Tm and ΔprpC in the indicated S. Tm strain backgrounds. The competitive index in the cecal content (F) and nitrate concentration in the cecal and colonic mucous layer (G) were determined 4 days post-infection. Each dot represents data from one animal (biological replicate). Bars represent mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001. See also Figure S4.
Figure 5.
Figure 5.. Bacteroides-produced propionate is a carbon source for S. Tm if nitrate is present
(A–C) Mucin broth was inoculated with WT B. thetaiotaomicron (WT) or B. thetaiotaomicron BT1686-89 (BT1686-89). B. theta BT1686-89 was cultured anaerobically for 4 days and WT B. theta was cultured for 2 days. (A) Propionate concentration in the digested mucin broth from WT or BT1686-89 B. theta culture (supplemented or not with propionate) was determined by liquid chromatography/mass spectrometry (LC/MS). (B) Filter-sterilized WT B. theta or B. theta BT1686-89-digested mucin broth was inoculated with an equal mixture of WT S. Tm and ΔprpC. Nitrate and propionate (40 and 10 mM, respectively) were added where indicated. Cultures were plated after 16 h of anaerobic growth. (C) Competitive index was calculated from CFU counts in (A). Nitrate and propionate were added as indicated. Each dot represents one biological replicate (average of triplicate technical replicate per biological replicate). Bars represent mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S5.
Figure 6.
Figure 6.. S. Tm utilization of microbiota-derived propionate provides an advantage during infection
(A) Schematic representation of the experiment and groups used. Germ-free mice were colonized with either WT B. theta (WT) or B. theta BT1686-89 for 7 days before infection with an equal mixture of WT S. Tm and a ΔprpC mutant. (B) Propionate concentration in the feces was measured after 7 days of colonization with different strains of Bacteroides and propionate supplementation. (C) Before infection, fecal samples were collected from monocolonized mice and plated on blood agar to confirm equal colonization between different Bacteroides strains. (D) Monocolonized mice were infected with an equal mixture of the WT S. Tm and ΔprpC mutant. The abundance of each strain in the colon content was determined by selective plating 3 days post-infection. (E) Competitive index was calculated from CFU counts in (D). (F) Combined histopathology score of pathological lesions in the cecum of mice from (D). Each dot represents data from one animal (biological replicate). Bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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