Fabian Rivera-Chávez

Phage-Mediated Acquisition of a Type III Secreted Effector Protein Boosts Growth of Salmonella by Nitrate Respiration

ABSTRACT 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. 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.

Depletion of Butyrate-Producing Clostridia from the Gut Microbiota Drives an Aerobic Luminal Expansion of Salmonella

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.

Salmonella Uses Energy Taxis to Benefit from Intestinal Inflammation

Chemotaxis enhances the fitness of Salmonella enterica serotype Typhimurium (S. Typhimurium) during colitis. However, the chemotaxis receptors conferring this fitness advantage and their cognate signals generated during inflammation remain unknown. Here we identify respiratory electron acceptors that are generated in the intestinal lumen as by-products of the host inflammatory response as in vivo signals for methyl-accepting chemotaxis proteins (MCPs). Three MCPs, including Trg, Tsr and Aer, enhanced the fitness of S. Typhimurium in a mouse colitis model. Aer mediated chemotaxis towards electron acceptors (energy taxis) in vitro and required tetrathionate respiration to confer a fitness advantage in vivo. Tsr mediated energy taxis towards nitrate but not towards tetrathionate in vitro and required nitrate respiration to confer a fitness advantage in vivo. These data suggest that the energy taxis receptors Tsr and Aer respond to distinct in vivo signals to confer a fitness advantage upon S. Typhimurium during inflammation by enabling this facultative anaerobic pathogen to seek out favorable spatial niches containing host-derived electron acceptors that boost its luminal growth.

Microbiota-activated PPAR-γ signaling inhibits dysbiotic Enterobacteriaceae expansion

Healthy guts exclude oxygen Normally, the lumen of the colon lacks oxygen. Fastidiously anaerobic butyrate-producing bacteria thrive in the colon; by ablating these organisms, antibiotic treatment removes butyrate. Byndloss et al. discovered that loss of butyrate deranges metabolic signaling in gut cells (see the Perspective by Cani). This induces nitric oxidase to generate nitrate in the lumen and disables β-oxidation in epithelial cells that would otherwise mop up stray oxygen before it enters the colon. Simultaneously, regulatory T cells retreat, and inflammation is unchecked, which contributes yet more oxygen species to the colon. Then, facultative aerobic pathogens, such as Escherichia coli and Salmonella enterica, can take advantage of the altered environment and outgrow any antibiotic-crippled and benign anaerobes. Science, this issue p. 570; see also p. 548 Butyrate-producing microbes contribute to synergism between epithelial cell metabolism and immune response regulation to maintain gut heath. Perturbation of the gut-associated microbial community may underlie many human illnesses, but the mechanisms that maintain homeostasis are poorly understood. We found that the depletion of butyrate-producing microbes by antibiotic treatment reduced epithelial signaling through the intracellular butyrate sensor peroxisome proliferator–activated receptor γ (PPAR-γ). Nitrate levels increased in the colonic lumen because epithelial expression of Nos2, the gene encoding inducible nitric oxide synthase, was elevated in the absence of PPAR-γ signaling. Microbiota-induced PPAR-γ signaling also limits the luminal bioavailability of oxygen by driving the energy metabolism of colonic epithelial cells (colonocytes) toward β-oxidation. Therefore, microbiota-activated PPAR-γ signaling is a homeostatic pathway that prevents a dysbiotic expansion of potentially pathogenic Escherichia and Salmonella by reducing the bioavailability of respiratory electron acceptors to Enterobacteriaceae in the lumen of the colon.

The Pyromaniac Inside You: Salmonella Metabolism in the Host Gut

A metabolically diverse microbial community occupies all available nutrient-niches in the lumen of the mammalian intestine, making it difficult for pathogens to establish themselves in this highly competitive environment. Salmonella serovars sidestep the competition by using their virulence factors to coerce the host into creating a novel nutrient-niche. Inflammation-derived nutrients available in this new niche support a bloom of Salmonella serovars, thereby ensuring transmission of the pathogen to the next susceptible host by the fecal-oral route. Here we review the anaerobic food chain that characterizes resident gut-associated microbial communities along with the winning metabolic strategy Salmonella serovars use to edge out competing microbes in the inflamed intestine.

Virulence factors enhance Citrobacter rodentium expansion through aerobic respiration

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.

The Periplasmic Nitrate Reductase NapABC Supports Luminal Growth of Salmonella enterica Serovar Typhimurium during Colitis

ABSTRACT 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.

Oxygen as a driver of gut dysbiosis.

Changes in the composition of gut-associated microbial communities may underlie many inflammatory and allergic diseases. However, the processes that help maintain a stable community structure are poorly understood. Here we review topical work elucidating the nutrient-niche occupied by facultative anaerobic bacteria of the family Enterobacteriaceae, whose predominance within the gut-associated microbial community is a common marker of dysbiosis. A paucity of exogenous respiratory electron acceptors limits growth of Enterobacteriaceae within a balanced gut-associated microbial community. However, recent studies suggest that the availability of oxygen in the large bowel is markedly elevated by changes in host physiology that accompany antibiotic treatment or infection with enteric pathogens, such as Salmonella serovars or attaching and effacing (AE) pathogens. The resulting increase in oxygen availability, alone or in conjunction with other electron acceptors, drives an uncontrolled luminal expansion of Enterobacteriaceae. Insights into the underlying mechanisms provide important clues about factors that control the balance between the host and its resident microbial communities.

Energy Taxis toward Host-Derived Nitrate Supports a Salmonella Pathogenicity Island 1-Independent Mechanism of Invasion

ABSTRACT Salmonella enterica serovar Typhimurium can cross the epithelial barrier using either the invasion-associated type III secretion system (T3SS-1) or a T3SS-1-independent mechanism that remains poorly characterized. Here we show that flagellum-mediated motility supported a T3SS-1-independent pathway for entering ileal Peyer’s patches in the mouse model. Flagellum-dependent invasion of Peyer’s patches required energy taxis toward nitrate, which was mediated by the methyl-accepting chemotaxis protein (MCP) Tsr. Generation of nitrate in the intestinal lumen required inducible nitric oxide synthase (iNOS), which was synthesized constitutively in the mucosa of the terminal ileum but not in the jejunum, duodenum, or cecum. Tsr-mediated invasion of ileal Peyer’s patches was abrogated in mice deficient for Nos2, the gene encoding iNOS. We conclude that Tsr-mediated energy taxis enables S. Typhimurium to migrate toward the intestinal epithelium by sensing host-derived nitrate, thereby contributing to invasion of Peyer’s patches. IMPORTANCE Nontyphoidal Salmonella serovars, such as S. enterica serovar Typhimurium, are a common cause of gastroenteritis in immunocompetent individuals but can also cause bacteremia in immunocompromised individuals. While the invasion-associated type III secretion system (T3SS-1) is important for entry, S. Typhimurium strains lacking a functional T3SS-1 can still cross the intestinal epithelium and cause a disseminated lethal infection in mice. Here we observed that flagellum-mediated motility and chemotaxis contributed to a T3SS-1-independent pathway for invasion and systemic dissemination to the spleen. This pathway required the methyl-accepting chemotaxis protein (MCP) Tsr and energy taxis toward host-derived nitrate, which we found to be generated by inducible nitric oxide synthase (iNOS) in the ileal mucosa prior to infection. Collectively, our data suggest that S. Typhimurium enhances invasion by actively migrating toward the intestinal epithelium along a gradient of host-derived nitrate emanating from the mucosal surface of the ileum. Nontyphoidal Salmonella serovars, such as S. enterica serovar Typhimurium, are a common cause of gastroenteritis in immunocompetent individuals but can also cause bacteremia in immunocompromised individuals. While the invasion-associated type III secretion system (T3SS-1) is important for entry, S. Typhimurium strains lacking a functional T3SS-1 can still cross the intestinal epithelium and cause a disseminated lethal infection in mice. Here we observed that flagellum-mediated motility and chemotaxis contributed to a T3SS-1-independent pathway for invasion and systemic dissemination to the spleen. This pathway required the methyl-accepting chemotaxis protein (MCP) Tsr and energy taxis toward host-derived nitrate, which we found to be generated by inducible nitric oxide synthase (iNOS) in the ileal mucosa prior to infection. Collectively, our data suggest that S. Typhimurium enhances invasion by actively migrating toward the intestinal epithelium along a gradient of host-derived nitrate emanating from the mucosal surface of the ileum.

Colonization resistance: The deconvolution of a complex trait

Carbapenemase-producing Enterobacteriaceae are an emerging threat to hospitals worldwide, and antibiotic exposure is a risk factor for developing fecal carriage that may lead to nosocomial infection. Here, we review how antibiotics reduce colonization resistance against Enterobacteriaceae to pinpoint possible control points for curbing their spread. Recent work identifies host-derived respiratory electron acceptors as a critical resource driving a post-antibiotic expansion of Enterobacteriaceae within the large bowel. By providing a conceptual framework for colonization resistance against Enterobacteriaceae, these mechanistic insights point to the metabolism of epithelial cells as a possible target for intervention strategies.