The food-borne pathogen Salmonella enterica serovar Typhimurium benefits from acute inflammation in part by using host-derived nitrate to respire anaerobically and compete successfully with the commensal microbes during growth in the intestinal lumen. The S. Typhimurium genome contains three nitrate reductases, encoded by the narGHI, narZYV, and napABC genes. Work on homologous genes present in Escherichia coli suggests that nitrate reductase A, encoded by the narGHI genes, is the main enzyme promoting growth on nitrate as an electron acceptor in anaerobic environments. Using a mouse colitis model, we found, surprisingly, that S. Typhimurium strains with defects in either nitrate reductase A (narG mutant) or the regulator inducing its transcription in the presence of high concentrations of nitrate (narL mutant) exhibited growth comparable to that of wild-type S. Typhimurium. In contrast, a strain lacking a functional periplasmic nitrate reductase (napA mutant) exhibited a marked growth defect in the lumen of the colon. In E. coli, the napABC genes are transcribed maximally under anaerobic growth conditions in the presence of low nitrate concentrations. Inactivation of narP, encoding a response regulator that activates napABC transcription in response to low nitrate concentrations, significantly reduced the growth of S. Typhimurium in the gut lumen. Cecal nitrate measurements suggested that the murine cecum is a nitrate-limited environment. Collectively, our results suggest that S. Typhimurium uses the periplasmic nitrate reductase to support its growth on the low nitrate concentrations encountered in the gut, a strategy that may be shared with other enteric pathogens.

Our long-standing evolutionary association with gut-associated microbial communities has given rise to an intimate relationship, which affects many aspects of human health. Recent studies on the mechanisms that link these microbial communities to immune education, nutrition, and protection against pathogens point to microbiota-derived metabolites as key players during these microbe-host interactions. A disruption of gut-associated microbial communities by antibiotic treatment can result in a depletion of microbiota-derived metabolites, thereby enhancing pathogen susceptibility, impairing immune homeostasis, and contributing to the rise of certain chronic inflammatory diseases. Here, we highlight some of the recently elucidated mechanisms that showcase the impacts of microbiota-derived metabolites on human health.

Thionitrous acid (HSNO), a potential key intermediate in biological signaling pathways, has been proposed to link NO and H2S biochemistries, but its existence and stability in vivo remain controversial. We establish that HSNO is spontaneously formed in high concentration when NO and H2S gases are mixed at room temperature in the presence of metallic surfaces. Our measurements reveal that HSNO is formed by the reaction H2S + N2O3 → HSNO + HNO2, where N2O3 is a product of NO disproportionation. These studies also suggest that further reaction of HSNO with H2S may form HNO and HSSH. The length of the S-N bond has been derived to high precision and is found to be unusually long: 1.84 Å, the longest S-N bond reported to date for an R-SNO compound. The present structural and, particularly, reactivity investigations of this elusive molecule provide a firm foundation to better understand its potential physiological chemistry and propensity to undergo S-N bond cleavage in vivo.

Changes in the gut microbiota may underpin many human diseases, but the mechanisms that are responsible for altering microbial communities remain poorly understood. Antibiotic usage elevates the risk of contracting gastroenteritis caused by Salmonella enterica serovars, increases the duration for which patients shed the pathogen in their faeces, and may on occasion produce a bacteriologic and symptomatic relapse. These antibiotic-induced changes in the gut microbiota can be studied in mice, in which the disruption of a balanced microbial community by treatment with the antibiotic streptomycin leads to an expansion of S. enterica serovars in the large bowel. However, the mechanisms by which streptomycin treatment drives an expansion of S. enterica serovars are not fully resolved. Here we show that host-mediated oxidation of galactose and glucose promotes post-antibiotic expansion of S. enterica serovar Typhimurium (S. Typhimurium). By elevating expression of the gene encoding inducible nitric oxide synthase (iNOS) in the caecal mucosa, streptomycin treatment increased post-antibiotic availability of the oxidation products galactarate and glucarate in the murine caecum. S. Typhimurium used galactarate and glucarate within the gut lumen of streptomycin pre-treated mice, and genetic ablation of the respective catabolic pathways reduced S. Typhimurium competitiveness. Our results identify host-mediated oxidation of carbohydrates in the gut as a mechanism for post-antibiotic pathogen expansion.

Information on how emerging pathogens can invade and persist and spread within host populations remains sparse. In the 1980s, a multidrug-resistant Salmonella enterica serotype Typhimurium clone lysogenized by a bacteriophage carrying the sopE virulence gene caused an epidemic among cattle and humans in Europe. Here we show that phage-mediated horizontal transfer of the sopE gene enhances the production of host-derived nitrate, an energetically highly valuable electron acceptor, in a mouse colitis model. In turn, nitrate fuels a bloom of S. Typhimurium in the gut lumen through anaerobic nitrate respiration while suppressing genes for the utilization of energetically inferior electron acceptors such as tetrathionate. Through this mechanism, horizontal transfer of sopE can enhance the fitness of S. Typhimurium, resulting in its significantly increased abundance in the feces. IMPORTANCE During gastroenteritis, Salmonella enterica serotype Typhimurium can use tetrathionate respiration to edge out competing microbes in the gut lumen. However, the concept that tetrathionate respiration confers a growth benefit in the inflamed gut is not broadly applicable to other host-pathogen combinations because tetrathionate respiration is a signature trait used to differentiate Salmonella serotypes from most other members of the family Enterobacteriaceae. Here we show that by acquiring the phage-carried sopE gene, S. Typhimurium can drive the host to generate an additional respiratory electron acceptor, nitrate. Nitrate suppresses genes for the utilization of energetically inferior electron acceptors such as tetrathionate while enhancing the luminal growth of S. Typhimurium through anaerobic nitrate respiration. Pathways for anaerobic nitrate respiration are widely conserved among members of the family Enterobacteriaceae, thereby making our observations relevant to other enteric pathogens whose relative abundance in the intestinal lumen increases during infection.

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Treatment with streptomycin enhances the growth of human commensal Escherichia coli isolates in the mouse intestine, suggesting that the resident microbial community (microbiota) can inhibit the growth of invading microbes, a phenomenon known as "colonization resistance." However, the precise mechanisms by which streptomycin treatment lowers colonization resistance remain obscure. Here we show that streptomycin treatment rendered mice more susceptible to the development of chemically induced colitis, raising the possibility that the antibiotic might lower colonization resistance by changing mucosal immune responses rather than by preventing microbe-microbe interactions. Investigation of the underlying mechanism revealed a mild inflammatory infiltrate in the cecal mucosa of streptomycin-treated mice, which was accompanied by elevated expression of Nos2, the gene that encodes inducible nitric oxide synthase. In turn, this inflammatory response enhanced the luminal growth of E. coli by nitrate respiration in a Nos2-dependent fashion. These data identify low-level intestinal inflammation as one of the factors responsible for the loss of resistance to E. coli colonization after streptomycin treatment.

Importance

Our intestine is host to a complex microbial community that confers benefits by educating the immune system and providing niche protection. Perturbation of intestinal communities by streptomycin treatment lowers "colonization resistance" through unknown mechanisms. Here we show that streptomycin increases the inflammatory tone of the intestinal mucosa, thereby making the bowel more susceptible to dextran sulfate sodium treatment and boosting the Nos2-dependent growth of commensal Escherichia coli by nitrate respiration. These data point to the generation of alternative electron acceptors as a by-product of the inflammatory host response as an important factor responsible for lowering resistance to colonization by facultative anaerobic bacteria such as E. coli.

The mammalian intestine is host to a microbial community that prevents pathogen expansion through unknown mechanisms, while antibiotic treatment can increase susceptibility to enteric pathogens. Here we show that streptomycin treatment depleted commensal, butyrate-producing Clostridia from the mouse intestinal lumen, leading to decreased butyrate levels, increased epithelial oxygenation, and aerobic expansion of Salmonella enterica serovar Typhimurium. Epithelial hypoxia and Salmonella restriction could be restored by tributyrin treatment. Clostridia depletion and aerobic Salmonella expansion were also observed in the absence of streptomycin treatment in genetically resistant mice but proceeded with slower kinetics and required the presence of functional Salmonella type III secretion systems. The Salmonella cytochrome bd-II oxidase synergized with nitrate reductases to drive luminal expansion, and both were required for fecal-oral transmission. We conclude that Salmonella virulence factors and antibiotic treatment promote pathogen expansion through the same mechanism: depletion of butyrate-producing Clostridia to elevate epithelial oxygenation, allowing aerobic Salmonella growth.

Citrobacter rodentium uses a type III secretion system (T3SS) to induce colonic crypt hyperplasia in mice, thereby gaining an edge during its competition with the gut microbiota through an unknown mechanism. Here, we show that by triggering colonic crypt hyperplasia, the C. rodentium T3SS induced an excessive expansion of undifferentiated Ki67-positive epithelial cells, which increased oxygenation of the mucosal surface and drove an aerobic C. rodentium expansion in the colon. Treatment of mice with the γ-secretase inhibitor dibenzazepine to diminish Notch-driven colonic crypt hyperplasia curtailed the fitness advantage conferred by aerobic respiration during C. rodentium infection. We conclude that C. rodentium uses its T3SS to induce histopathological lesions that generate an intestinal microenvironment in which growth of the pathogen is fueled by aerobic respiration.