Byproducts of inflammatory radical metabolism provide transient nutrient niches for microbes in the inflamed gut.

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.

Formate oxidation in the intestinal mucus layer enhances fitness of Salmonella enterica serovar Typhimurium.

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.

A Member of an Ancient Family of Bacterial Amino Acids Transporters Contributes to Chlamydia Nutritional Virulence and Immune Evasion.

Many obligate intracellular bacteria, including members of the genus Chlamydia, cannot synthesize a variety of amino acids de novo and acquire these from host cells via largely unknown mechanisms. Previously, we determined that a missense mutation in ctl0225, a conserved Chlamydia open reading frame of unknown function, mediated sensitivity to interferon gamma. Here, we show evidence that CTL0225 is a member of the SnatA family of neutral amino acid transporters that contributes to the import of several amino acids into Chlamydia cells. Further, we show that CTL0225 orthologs from two other distantly related obligate intracellular pathogens (Coxiella burnetii and Buchnera aphidicola) are sufficient to import valine into Escherichia coli. We also show that chlamydia infection and interferon exposure have opposing effects on amino acid metabolism, potentially explaining the relationship between CTL0225 and interferon sensitivity. Overall, we show that phylogenetically diverse intracellular pathogens use an ancient family of amino acid transporters to acquire host amino acids and provide another example of how nutritional virulence and immune evasion can be linked in obligate intracellular pathogens.

Colonocyte-derived lactate promotes E. coli fitness in the context of inflammation-associated gut microbiota dysbiosis.

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.

An rfuABCD-Like Operon and Its Relationship to Riboflavin Utilization and Mammalian Infectivity by Borrelia burgdorferi.

Riboflavin is an essential micronutrient, but its transport and utilization have remained largely understudied among pathogenic spirochetes. Here, we show that Borrelia burgdorferi, the zoonotic spirochete that causes Lyme disease, is able to import riboflavin via products of its rfuABCD-like operon as well as synthesize flavin mononucleotide and flavin adenine dinucleotide despite lacking canonical genes for their synthesis. Additionally, a mutant deficient in the rfuABCD-like operon is resistant to the antimicrobial effect of roseoflavin, a natural riboflavin analog, and is attenuated in a murine model of Lyme borreliosis. Our combined results indicate not only that are riboflavin and the maintenance of flavin pools essential for B. burgdorferi growth but also that flavin utilization and its downstream products (e.g., flavoproteins) may play a more prominent role in B. burgdorferi pathogenesis than previously appreciated.

Reshaping of bacterial molecular hydrogen metabolism contributes to the outgrowth of commensal E. coli during gut inflammation.

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.

Advances and Obstacles in the Genetic Dissection of Chlamydial Virulence.

Obligate intracellular pathogens in the family Chlamydiaceae infect taxonomically diverse eukaryotes ranging from amoebae to mammals. However, many fundamental aspects of chlamydial cell biology and pathogenesis remain poorly understood. Genetic dissection of chlamydial biology has historically been hampered by a lack of genetic tools. Exploitation of the ability of chlamydia to recombine genomic material by lateral gene transfer (LGT) ushered in a new era in chlamydia research. With methods to map mutations in place, genetic screens were able to assign functions and phenotypes to specific chlamydial genes. Development of an approach for stable transformation of chlamydia also provided a mechanism for gene delivery and platforms for disrupting chromosomal genes. Here, we explore how these and other tools have been used to test hypotheses concerning the functions of known chlamydial virulence factors and discover the functions of completely uncharacterized genes. Refinement and extension of the existing genetic tools to additional Chlamydia spp. will substantially advance understanding of the biology and pathogenesis of this important group of pathogens.

Beyond Tryptophan Synthase: Identification of Genes That Contribute to Chlamydia trachomatis Survival during Gamma Interferon-Induced Persistence and Reactivation.

Chlamydia trachomatis can enter a viable but nonculturable state in vitro termed persistence. A common feature of C. trachomatis persistence models is that reticulate bodies fail to divide and make few infectious progeny until the persistence-inducing stressor is removed. One model of persistence that has relevance to human disease involves tryptophan limitation mediated by the host enzyme indoleamine 2,3-dioxygenase, which converts l-tryptophan to N-formylkynurenine. Genital C. trachomatis strains can counter tryptophan limitation because they encode a tryptophan-synthesizing enzyme. Tryptophan synthase is the only enzyme that has been confirmed to play a role in interferon gamma (IFN-γ)-induced persistence, although profound changes in chlamydial physiology and gene expression occur in the presence of persistence-inducing stressors. Thus, we screened a population of mutagenized C. trachomatis strains for mutants that failed to reactivate from IFN-γ-induced persistence. Six mutants were identified, and the mutations linked to the persistence phenotype in three of these were successfully mapped. One mutant had a missense mutation in tryptophan synthase; however, this mutant behaved differently from previously described synthase null mutants. Two hypothetical genes of unknown function, ctl0225 and ctl0694, were also identified and may be involved in amino acid transport and DNA damage repair, respectively. Our results indicate that C. trachomatis utilizes functionally diverse genes to mediate survival during and reactivation from persistence in HeLa cells.

Interrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination.

UNLABELLED: Intracellular bacterial pathogens in the family Chlamydiaceae are causes of human blindness, sexually transmitted disease, and pneumonia. Genetic dissection of the mechanisms of chlamydial pathogenicity has been hindered by multiple limitations, including the inability to inactivate genes that would prevent the production of elementary bodies. Many genes are also Chlamydia-specific genes, and chlamydial genomes have undergone extensive reductive evolution, so functions often cannot be inferred from homologs in other organisms. Conditional mutants have been used to study essential genes of many microorganisms, so we screened a library of 4,184 ethyl methanesulfonate-mutagenized Chlamydia trachomatis isolates for temperature-sensitive (TS) mutants that developed normally at physiological temperature (37°C) but not at nonphysiological temperatures. Heat-sensitive TS mutants were identified at a high frequency, while cold-sensitive mutants were less common. Twelve TS mutants were mapped using a novel markerless recombination approach, PCR, and genome sequencing. TS alleles of genes that play essential roles in other bacteria and chlamydia-specific open reading frames (ORFs) of unknown function were identified. Temperature-shift assays determined that phenotypes of the mutants manifested at distinct points in the developmental cycle. Genome sequencing of a larger population of TS mutants also revealed that the screen had not reached saturation. In summary, we describe the first approach for studying essential chlamydial genes and broadly applicable strategies for genetic mapping in Chlamydia spp. and mutants that both define checkpoints and provide insights into the biology of the chlamydial developmental cycle. IMPORTANCE: Study of the pathogenesis of Chlamydia spp. has historically been hampered by a lack of genetic tools. Although there has been recent progress in chlamydial genetics, the existing approaches have limitations for the study of the genes that mediate growth of these organisms in cell culture. We used a genetic screen to identify conditional Chlamydia mutants and then mapped these alleles using a broadly applicable recombination strategy. Phenotypes of the mutants provide fundamental insights into unexplored areas of chlamydial pathogenesis and intracellular biology. Finally, the reagents and approaches we describe are powerful resources for the investigation of these organisms.