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Louis Pasteur's experiments on tartaric acid laid the foundation for our understanding of molecular chirality, but major questions remain. By comparing the optical activity of naturally-occurring tartaric acid with chemically-synthesized paratartaric acid, Pasteur realized that naturally-occurring tartaric acid contained only L-tartaric acid while paratartaric acid consisted of a racemic mixture of D- and L-tartaric acid. Curiously, D-tartaric acid has no known natural source, yet several gut bacteria specifically degrade D-tartaric acid. Here, we investigated the oxidation of monosaccharides by inflammatory reactive oxygen and nitrogen species. We found that this reaction yields an array of alpha hydroxy carboxylic acids, including tartaric acid isomers. Utilization of inflammation- derived D- and L-tartaric acid enhanced colonization by Salmonella Typhimurium and E. coli in murine models of gut inflammation. Our findings suggest that byproducts of inflammatory radical metabolism, such as tartrate and other alpha hydroxy carboxylic acids, create transient nutrient niches for enteric pathogens and other potentially harmful bacteria. Furthermore, this work illustrates that inflammatory radicals generate a zoo of molecules, some of which may erroneously presumed to be xenobiotics.
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Salmonella enterica serovar Typhimurium induces intestinal inflammation to create a niche that fosters the outgrowth of the pathogen over the gut microbiota. Under inflammatory conditions, Salmonella utilizes terminal electron acceptors generated as byproducts of intestinal inflammation to generate cellular energy through respiration. However, the electron donating reactions in these electron transport chains are poorly understood. Here, we investigated how formate utilization through the respiratory formate dehydrogenase-N (FdnGHI) and formate dehydrogenase-O (FdoGHI) contribute to gut colonization of Salmonella. Both enzymes fulfilled redundant roles in enhancing fitness in a mouse model of Salmonella-induced colitis, and coupled to tetrathionate, nitrate, and oxygen respiration. The formic acid utilized by Salmonella during infection was generated by its own pyruvate-formate lyase as well as the gut microbiota. Transcription of formate dehydrogenases and pyruvate-formate lyase was significantly higher in bacteria residing in the mucus layer compared to the lumen. Furthermore, formate utilization conferred a more pronounced fitness advantage in the mucus, indicating that formate production and degradation occurred predominantly in the mucus layer. Our results provide new insights into how Salmonella adapts its energy metabolism to the local microenvironment in the gut. IMPORTANCE Bacterial pathogens must not only evade immune responses but also adapt their metabolism to successfully colonize their host. The microenvironments encountered by enteric pathogens differ based on anatomical location, such as small versus large intestine, spatial stratification by host factors, such as mucus layer and antimicrobial peptides, and distinct commensal microbial communities that inhabit these microenvironments. Our understanding of how Salmonella populations adapt its metabolism to different environments in the gut is incomplete. In the current study, we discovered that Salmonella utilizes formate as an electron donor to support respiration, and that formate oxidation predominantly occurs in the mucus layer. Our experiments suggest that spatially distinct Salmonella populations in the mucus layer and the lumen differ in their energy metabolism. Our findings enhance our understanding of the spatial nature of microbial metabolism and may have implications for other enteric pathogens as well as commensal host-associated microbial communities.
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Liasas , Salmonelosis Animal , Animales , Ratones , Salmonella typhimurium/metabolismo , Serogrupo , Salmonelosis Animal/microbiología , Bacterias , Inflamación , Formiatos/metabolismo , Moco , Piruvatos/metabolismo , Liasas/metabolismoRESUMEN
Serine hydrolases have important roles in signaling and human metabolism, yet little is known about their functions in gut commensal bacteria. Using bioinformatics and chemoproteomics, we identify serine hydrolases in the gut commensal Bacteroides thetaiotaomicron that are specific to the Bacteroidetes phylum. Two are predicted homologs of the human dipeptidyl peptidase 4 (hDPP4), a key enzyme that regulates insulin signaling. Our functional studies reveal that BT4193 is a true homolog of hDPP4 that can be inhibited by FDA-approved type 2 diabetes medications targeting hDPP4, while the other is a misannotated proline-specific triaminopeptidase. We demonstrate that BT4193 is important for envelope integrity and that loss of BT4193 reduces B. thetaiotaomicron fitness during in vitro growth within a diverse community. However, neither function is dependent on BT4193 proteolytic activity, suggesting a scaffolding or signaling function for this bacterial protease.
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Bacteroides thetaiotaomicron , Diabetes Mellitus Tipo 2 , Humanos , Dipeptidil Peptidasa 4/genética , SerinaRESUMEN
Bacterial populations that originate from a single bacterium are not strictly clonal. Often, they contain subgroups with distinct phenotypes. Bacteria can generate heterogeneity through phase variation: a preprogrammed, reversible mechanism that alters gene expression levels across a population. One well studied type of phase variation involves enzyme-mediated inversion of specific intergenic regions of genomic DNA. Frequently, these DNA inversions flip the orientation of promoters, turning ON or OFF adjacent coding regions within otherwise isogenic populations. Through this mechanism, inversion can affect fitness, survival, or group dynamics. Here, we develop and apply bioinformatic approaches to discover thousands of previously undescribed phase-variable regions in prokaryotes using long-read datasets. We identify 'intragenic invertons', a surprising new class of invertible elements found entirely within genes, in bacteria and archaea. To date, inversions within single genes have not been described. Intragenic invertons allow a gene to encode two or more versions of a protein by flipping a DNA sequence within the coding region, thereby increasing coding capacity without increasing genome size. We experimentally characterize specific intragenic invertons in the gut commensal Bacteroides thetaiotaomicron, presenting a 'roadmap' for investigating this new gene-diversifying phenomenon.
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BACKGROUND: Intestinal inflammation disrupts the microbiota composition leading to an expansion of Enterobacteriaceae family members (dysbiosis). Associated with this shift in microbiota composition is a profound change in the metabolic landscape of the intestine. It is unclear how changes in metabolite availability during gut inflammation impact microbial and host physiology. RESULTS: We investigated microbial and host lactate metabolism in murine models of infectious and non-infectious colitis. During inflammation-associated dysbiosis, lactate levels in the gut lumen increased. The disease-associated spike in lactate availability was significantly reduced in mice lacking the lactate dehydrogenase A subunit in intestinal epithelial cells. Commensal E. coli and pathogenic Salmonella, representative Enterobacteriaceae family members, utilized lactate via the respiratory L-lactate dehydrogenase LldD to increase fitness. Furthermore, mice lacking the lactate dehydrogenase A subunit in intestinal epithelial cells exhibited lower levels of inflammation in a model of non-infectious colitis. CONCLUSIONS: The release of lactate by intestinal epithelial cells during gut inflammation impacts the metabolism of gut-associated microbial communities. These findings suggest that during intestinal inflammation and dysbiosis, changes in metabolite availability can perpetuate colitis-associated disturbances of microbiota composition. Video Abstract.
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Colitis , Microbioma Gastrointestinal , Ratones , Animales , Disbiosis , Escherichia coli/metabolismo , Ácido Láctico/metabolismo , Lactato Deshidrogenasa 5 , Ratones Endogámicos C57BL , Inflamación/patología , Colitis/patología , Enterobacteriaceae/metabolismoRESUMEN
Structural variation in bacterial genomes is an important evolutionary driver. Genomic rearrangements, such as inversions, duplications, and insertions, can regulate gene expression and promote niche adaptation. Importantly, many of these variations are reversible and preprogrammed to generate heterogeneity. While many tools have been developed to detect structural variation in eukaryotic genomes, variation in bacterial genomes and metagenomes remains understudied. However, recent advances in genome sequencing technology and the development of new bioinformatic pipelines hold promise in further understanding microbial genomics.
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Genoma Bacteriano , Genómica , Evolución Biológica , Biología Computacional , Reordenamiento GénicoRESUMEN
The composition of gut-associated microbial communities changes during intestinal inflammation, including an expansion of Enterobacteriaceae populations. The mechanisms underlying microbiota changes during inflammation are incompletely understood. Here, we analyzed previously published metagenomic datasets with a focus on microbial hydrogen metabolism. The bacterial genomes in the inflamed murine gut and in patients with inflammatory bowel disease contained more genes encoding predicted hydrogen-utilizing hydrogenases compared to communities found under non-inflamed conditions. To validate these findings, we investigated hydrogen metabolism of Escherichia coli, a representative Enterobacteriaceae, in mouse models of colitis. E. coli mutants lacking hydrogenase-1 and hydrogenase-2 displayed decreased fitness during colonization of the inflamed cecum and colon. Utilization of molecular hydrogen was in part dependent on respiration of inflammation-derived electron acceptors. This work highlights the contribution of hydrogenases to alterations of the gut microbiota in the context of non-infectious colitis.
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Ciego/microbiología , Colitis/inducido químicamente , Colitis/microbiología , Colon/microbiología , Infecciones por Escherichia coli/microbiología , Escherichia coli/metabolismo , Microbioma Gastrointestinal , Hidrógeno/metabolismo , Animales , Ciego/metabolismo , Ciego/patología , Colitis/metabolismo , Colitis/patología , Colon/metabolismo , Colon/patología , Bases de Datos Genéticas , Sulfato de Dextran , Modelos Animales de Enfermedad , Disbiosis , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/patología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Femenino , Humanos , Hidrogenasas/genética , Hidrogenasas/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Masculino , Metagenoma , Metagenómica , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , PiroxicamRESUMEN
The intestinal epithelium separates host tissue and gut-associated microbial communities. During inflammation, the host releases reactive oxygen and nitrogen species as an antimicrobial response. The impact of these radicals on gut microbes is incompletely understood. We discovered that the cryptic appBCX genes, predicted to encode a cytochrome bd-II oxidase, conferred a fitness advantage for E. coli in chemical and genetic models of non-infectious colitis. This fitness advantage was absent in mice that lacked epithelial NADPH oxidase 1 (NOX1) activity. In laboratory growth experiments, supplementation with exogenous hydrogen peroxide enhanced E. coli growth through AppBCX-mediated respiration in a catalase-dependent manner. We conclude that epithelial-derived reactive oxygen species are degraded in the gut lumen, which gives rise to molecular oxygen that supports the aerobic respiration of E. coli. This work illustrates how epithelial host responses intersect with gut microbial metabolism in the context of gut inflammation.
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Complejo IV de Transporte de Electrones/fisiología , Escherichia coli/fisiología , Inflamación/metabolismo , Mucosa Intestinal/metabolismo , NADPH Oxidasa 1/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Aerobiosis , Animales , Colitis/inducido químicamente , ADN Bacteriano , Modelos Animales de Enfermedad , Proteínas de Escherichia coli/fisiología , Femenino , Microbioma Gastrointestinal , Interacciones Microbiota-Huesped , Peróxido de Hidrógeno/metabolismo , Inflamación/microbiología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microbiota , NADPH Oxidasa 1/genética , Oxígeno/metabolismoRESUMEN
During short-lived perturbations, such as inflammation, the gut microbiota exhibits resilience and reverts to its original configuration. Although microbial access to the micronutrient iron is decreased during colitis, pathogens can scavenge iron by using siderophores. How commensal bacteria acquire iron during gut inflammation is incompletely understood. Curiously, the human commensal Bacteroides thetaiotaomicron does not produce siderophores but grows under iron-limiting conditions using enterobacterial siderophores. Using RNA-seq, we identify B. thetaiotaomicron genes that were upregulated during Salmonella-induced gut inflammation and were predicted to be involved in iron uptake. Mutants in the xusABC locus (BT2063-2065) were defective for xenosiderophore-mediated iron uptake in vitro. In the normal mouse gut, the XusABC system was dispensable, while a xusA mutant colonized poorly during colitis. This work identifies xenosiderophore utilization as a critical mechanism for B. thetaiotaomicron to sustain colonization during inflammation and suggests a mechanism of how interphylum iron metabolism contributes to gut microbiota resilience.
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Bacteroides thetaiotaomicron/metabolismo , Colitis/microbiología , Enterobacteriaceae/genética , Microbioma Gastrointestinal , Hierro/metabolismo , Sideróforos/genética , Animales , Bacteroides thetaiotaomicron/genética , Femenino , Genes Bacterianos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , RNA-Seq , SimbiosisRESUMEN
The Shigella species are Gram-negative, facultative intracellular pathogens that invade the colonic epithelium and cause significant diarrheal disease. Despite extensive research on the pathogen, a comprehensive understanding of how Shigella initiates contact with epithelial cells remains unknown. Shigella maintains many of the same Escherichia coli adherence gene operons; however, at least one critical gene component in each operon is currently annotated as a pseudogene in reference genomes. These annotations, coupled with a lack of structures upon microscopic analysis following growth in laboratory media, have led the field to hypothesize that Shigella is unable to produce fimbriae or other traditional adherence factors. Nevertheless, our previous analyses have demonstrated that a combination of bile salts and glucose induces both biofilm formation and adherence to colonic epithelial cells. The goal of this study was to perform transcriptomic and genetic analyses to demonstrate that adherence gene operons in Shigella flexneri strain 2457T are functional, despite the gene annotations. Our results demonstrate that at least three structural genes facilitate S. flexneri 2457T adherence for epithelial cell contact and biofilm formation. Furthermore, our results demonstrate that host factors, namely, glucose and bile salts at their physiological concentrations in the small intestine, offer key environmental stimuli required for adherence factor expression in S. flexneri This research may have a significant impact on Shigella vaccine development and further highlights the importance of utilizing in vivo-like conditions to study bacterial pathogenesis.IMPORTANCE Bacterial pathogens have evolved to regulate virulence gene expression at critical points in the colonization and infection processes to successfully cause disease. The Shigella species infect the epithelial cells lining the colon to result in millions of cases of diarrhea and a significant global health burden. As antibiotic resistance rates increase, understanding the mechanisms of infection is vital to ensure successful vaccine development. Despite significant gains in our understanding of Shigella infection, it remains unknown how the bacteria initiate contact with the colonic epithelium. Most pathogens harbor multiple adherence factors to facilitate this process, but Shigella was thought to have lost the ability to produce these factors. Interestingly, we have identified conditions that mimic some features of gastrointestinal transit and that enable Shigella to express adherence structural genes. This work highlights aspects of genetic regulation for Shigella adherence factors and may have a significant impact on future vaccine development.
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Adhesinas Bacterianas/biosíntesis , Adhesión Bacteriana , Células Epiteliales/microbiología , Regulación Bacteriana de la Expresión Génica , Shigella flexneri/crecimiento & desarrollo , Shigella flexneri/metabolismo , Adhesinas Bacterianas/genética , Ácidos y Sales Biliares/metabolismo , Biopelículas/crecimiento & desarrollo , Células Cultivadas , Perfilación de la Expresión Génica , Glucosa/metabolismo , Interacciones Huésped-Patógeno , Humanos , Operón , Shigella flexneri/efectos de los fármacosRESUMEN
During Salmonella enterica serovar Typhimurium infection, host inflammation alters the metabolic environment of the gut lumen to favor the outgrowth of the pathogen at the expense of the microbiota. Inflammation-driven changes in host cell metabolism lead to the release of l-lactate and molecular oxygen from the tissue into the gut lumen. Salmonella utilizes lactate as an electron donor in conjunction with oxygen as the terminal electron acceptor to support gut colonization. Here, we investigated transcriptional regulation of the respiratory l-lactate dehydrogenase LldD in vitro and in mouse models of Salmonella infection. The two-component system ArcAB repressed transcription of l-lactate utilization genes under anaerobic conditions in vitro The ArcAB-mediated repression of lldD transcription was relieved under microaerobic conditions. Transcription of lldD was induced by l-lactate but not d-lactate. A mutant lacking the regulatory protein LldR failed to induce lldD transcription in response to l-lactate. Furthermore, the lldR mutant exhibited reduced transcription of l-lactate utilization genes and impaired fitness in murine models of infection. These data provide evidence that the host-derived metabolites oxygen and l-lactate serve as cues for Salmonella to regulate lactate oxidation metabolism on a transcriptional level.
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Mucosa Intestinal/microbiología , Ácido Láctico/metabolismo , Infecciones por Salmonella/metabolismo , Salmonella typhimurium/crecimiento & desarrollo , Salmonella typhimurium/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Femenino , Regulación Bacteriana de la Expresión Génica , Humanos , Mucosa Intestinal/metabolismo , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Oxígeno/metabolismo , Infecciones por Salmonella/microbiología , Salmonella typhimurium/enzimología , Salmonella typhimurium/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
During Salmonella-induced gastroenteritis, mucosal inflammation creates a niche that favors the expansion of the pathogen population over the microbiota. Here, we show that Salmonella Typhimurium infection was accompanied by dysbiosis, decreased butyrate levels, and substantially elevated lactate levels in the gut lumen. Administration of a lactate dehydrogenase inhibitor blunted lactate production in germ-free mice, suggesting that lactate was predominantly of host origin. Depletion of butyrate-producing Clostridia, either through oral antibiotic treatment or as part of the pathogen-induced dysbiosis, triggered a switch in host cells from oxidative metabolism to lactate fermentation, increasing both lactate levels and Salmonella lactate utilization. Administration of tributyrin or a PPARγ agonist diminished host lactate production and abrogated the fitness advantage conferred on Salmonella by lactate utilization. We conclude that alterations of the gut microbiota, specifically a depletion of Clostridia, reprogram host metabolism to perform lactate fermentation, thus supporting Salmonella infection.
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Clostridium/crecimiento & desarrollo , Disbiosis/microbiología , Gastroenteritis/microbiología , Microbioma Gastrointestinal/efectos de los fármacos , Mucosa Intestinal/microbiología , Ácido Láctico/metabolismo , Salmonella typhimurium/metabolismo , Animales , Antibacterianos/farmacología , Ácido Butírico/metabolismo , Femenino , Fermentación , Gastroenteritis/patología , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos C57BL , PPAR gamma/agonistas , Infecciones por Salmonella/patología , Salmonella typhimurium/crecimiento & desarrollo , Triglicéridos/farmacologíaRESUMEN
The Shigella species cause millions of cases of watery or bloody diarrhea each year, mostly in children in developing countries. While many aspects of Shigella colonic cell invasion are known, crucial gaps in knowledge regarding how the bacteria survive, transit, and regulate gene expression prior to infection remain. In this study, we define mechanisms of resistance to bile salts and build on previous research highlighting induced virulence in Shigella flexneri strain 2457T following exposure to bile salts. Typical growth patterns were observed within the physiological range of bile salts; however, growth was inhibited at higher concentrations. Interestingly, extended periods of exposure to bile salts led to biofilm formation, a conserved phenotype that we observed among members of the Enterobacteriaceae Characterization of S. flexneri 2457T biofilms determined that both bile salts and glucose were required for formation, dispersion was dependent upon bile salts depletion, and recovered bacteria displayed induced adherence to HT-29 cells. RNA-sequencing analysis verified an important bile salt transcriptional profile in S. flexneri 2457T, including induced drug resistance and virulence gene expression. Finally, functional mutagenesis identified the importance of the AcrAB efflux pump and lipopolysaccharide O-antigen synthesis for bile salt resistance. Our data demonstrate that S. flexneri 2457T employs multiple mechanisms to survive exposure to bile salts, which may have important implications for multidrug resistance. Furthermore, our work confirms that bile salts are important physiological signals to activate S. flexneri 2457T virulence. This work provides insights into how exposure to bile likely regulates Shigella survival and virulence during host transit and subsequent colonic infection.
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Proteínas Bacterianas/metabolismo , Ácidos y Sales Biliares/farmacología , Biopelículas/efectos de los fármacos , Antígenos O/metabolismo , Shigella flexneri/efectos de los fármacos , Shigella flexneri/patogenicidad , Proteínas Bacterianas/genética , Perfilación de la Expresión Génica , Células HT29 , Células HeLa , Humanos , Microscopía Electrónica , Mutación , Antígenos O/genética , Análisis de Secuencia de ARN , Shigella flexneri/genética , Virulencia/genéticaRESUMEN
Bacterial pathogens have coevolved with humans in order to efficiently infect, replicate within, and be transmitted to new hosts to ensure survival and a continual infection cycle. For enteric pathogens, the ability to adapt to numerous host factors under the harsh conditions of the gastrointestinal tract is critical for establishing infection. One such host factor readily encountered by enteric bacteria is bile, an innately antimicrobial detergent-like compound essential for digestion and nutrient absorption. Not only have enteric pathogens evolved to resist the bactericidal conditions of bile, but these bacteria also utilize bile as a signal to enhance virulence regulation for efficient infection. This review provides a comprehensive and up-to-date analysis of bile-related research with enteric pathogens. From common responses to the unique expression of specific virulence factors, each pathogen has overcome significant challenges to establish infection in the gastrointestinal tract. Utilization of bile as a signal to modulate virulence factor expression has led to important insights for our understanding of virulence mechanisms for many pathogens. Further research on enteric pathogens exposed to this in vivo signal will benefit therapeutic and vaccine development and ultimately enhance our success at combating such elite pathogens.
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Bilis/metabolismo , Enterobacteriaceae/fisiología , Animales , Modelos Animales de Enfermedad , Enterobacteriaceae/efectos de los fármacos , Enterobacteriaceae/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Interacciones Huésped-Patógeno , Humanos , Viabilidad Microbiana , Virulencia , Factores de Virulencia/biosíntesisRESUMEN
Inflammatory bowel disease (IBD) is a complex, multi-factorial disease thought to arise from an inappropriate immune response to commensal bacteria in a genetically susceptible person that results in chronic, cyclical, intestinal inflammation. Dietary and environmental factors are implicated in the initiation and perpetuation of IBD; however, a singular causative agent has not been identified. As of now, the role of environmental priming or triggers in IBD onset and pathogenesis are not well understood, but these factors appear to synergize with other disease susceptibility factors. In previous work, we determined that the polysaccharide dietary additive, maltodextrin (MDX), impairs cellular anti-bacterial responses and suppresses intestinal anti-microbial defense mechanisms. In this addendum, we review potential mechanisms for dietary deregulation of intestinal homeostasis, postulate how dietary and genetic risk factors may combine to result in disease pathogenesis, and discuss these ideas in the context of recent findings related to dietary interventions for IBD.