Abstract: Recent work has revealed that enterohaemorrhagic Escherichia coli encodes a two-component system, termed FusKR, which responds to fucose and represses expression of virulence genes. Furthermore, a representative member of the microbiota appears to cleave fucose from host glycans, indicating that the microbiota and EHEC may act in concert to suppress virulence gene expression.
Abstract: Residing within the intestine is a large community of commensal organisms collectively termed the microbiota. This community generates a complex nutrient environment by breaking down indigestible food products into metabolites that are used by both the host and the microbiota. Both the invading intestinal pathogen and the microbiota compete for these metabolites, which can shape both the composition of the flora, as well as susceptibility to infection. After infection is established, pathogen mediated inflammation alters the composition of the microbiota, which further shifts the makeup of metabolites in the gastrointestinal tract. A greater understanding of the interplay between the microbiota, the metabolites they generate, and susceptibility to enteric disease will enable the discovery of novel therapies against infectious disease.
Abstract: Antibiotics are often used in the clinic to treat bacterial infections, but the effects of these drugs on microbiota composition and on intestinal immunity are poorly understood. Citrobacter rodentium was used as a model enteric pathogen to investigate the effect of microbial perturbation on intestinal barriers and susceptibility to colitis. Streptomycin and metronidazole were used to induce alterations in the composition of the microbiota prior to infection with C. rodentium. Metronidazole pretreatment increased susceptibility to C. rodentium-induced colitis over that of untreated and streptomycin-pretreated mice, 6 days postinfection. Both antibiotic treatments altered microbial composition, without affecting total numbers, but metronidazole treatment resulted in a more dramatic change, including a reduced population of Porphyromonadaceae and increased numbers of lactobacilli. Disruption of the microbiota with metronidazole, but not streptomycin treatment, resulted in an increased inflammatory tone of the intestine characterized by increased bacterial stimulation of the epithelium, altered goblet cell function, and thinning of the inner mucus layer, suggesting a weakened mucosal barrier. This reduction in mucus thickness correlates with increased attachment of C. rodentium to the intestinal epithelium, contributing to the exacerbated severity of C. rodentium-induced colitis in metronidazole-pretreated mice. These results suggest that antibiotic perturbation of the microbiota can disrupt intestinal homeostasis and the integrity of intestinal defenses, which protect against invading pathogens and intestinal inflammation.
Abstract: The gram-positive bacterial pathogen Listeria monocytogenes has evolved mechanisms to rapidly replicate in the host cytosol, implying efficient utilization of host-derived nutrients. However, the contribution of host nutrient scavenging versus that of bacterial biosynthesis toward rapid intracellular growth remains unclear. Nutrients that contribute to growth of L. monocytogenes include branched-chain fatty acids (BCFAs), amino acids, and other metabolic intermediates generated from acyl-coenzyme A, which is synthesized using lipoylated metabolic enzyme complexes. To characterize which biosynthetic pathways support replication of L. monocytogenes inside the host cytosol, we impaired lipoate-dependent metabolism by disrupting two lipoate ligase genes that are responsible for bacterial protein lipoylation. Interrupting lipoate-dependent metabolism modestly impaired replication in rich broth medium but strongly inhibited growth in defined medium and host cells and impaired the generation of BCFAs. Addition of short BCFAs and amino acids restored growth of the A1A2-deficient (A1A2-) mutant in minimal medium, implying that lipoate-dependent metabolism generates amino acids and BCFAs. BCFAs alone rescued intracellular growth and spread in L2 fibroblasts of the A1A2- mutant. Lipoate-dependent metabolism was also required in vivo, as a wild-type strain robustly outcompeted the lipoylation-deficient mutant in a murine model of listeriosis. The results of this study suggest that lipoate-dependent metabolism contributes to both amino acid and BCFA biosynthesis and that BCFA biosynthesis is preferentially required for intracellular growth of L. monocytogenes.
Abstract: Glucose is a major source of energy and carbon in promastigotes of Leishmania mexicana, and its uptake is mediated by three glucose transporters whose genes are encoded within a single cluster. A null mutant in which the glucose transporter gene cluster was deleted by homologous gene replacement was generated previously and shown to grow more slowly than wild type promastigotes but not to be viable as amastigotes in primary tissue culture macrophages or in axenic culture. Further phenotypic characterization demonstrates that the null mutant is unable to import glucose, mannose, fructose, or galactose and that each of the three glucose transporter isoforms, LmGT1, LmGT2, and LmGT3, is capable of transporting each of these hexoses. Complementation of the null mutant with each isoform is able to restore growth in each of the four hexoses to wild type levels. Null mutant promastigotes are reduced in size to about 2/3 the volume of wild type parasites. In addition, the null mutants are significantly more sensitive to oxidative stress than their wild type counterparts. These results underscore the importance of glucose transporters in the parasite life cycle and suggest reasons for their non-viability in the disease-causing amastigote stage.
Abstract: The bacterial pathogen Listeria monocytogenes replicates within the cytosol of mammalian cells. Mechanisms by which the bacterium exploits the host cytosolic environment for essential nutrients are poorly defined. L. monocytogenes is a lipoate auxotroph and must scavenge this critical cofactor, using lipoate ligases to facilitate attachment of the lipoyl moiety to metabolic enzyme complexes. Although the L. monocytogenes genome encodes two putative lipoate ligases, LplA1 and LplA2, intracellular replication and virulence require only LplA1. Here we show that LplA1 enables utilization of host-derived lipoyl peptides by L. monocytogenes. LplA1 is dispensable for growth in the presence of free lipoate, but necessary for growth on low concentrations of mammalian lipoyl peptides. Furthermore, we demonstrate that the intracellular growth defect of the DeltalplA1 mutant is rescued by addition of exogenous lipoic acid to host cells, suggesting that L. monocytogenes dependence on LplA1 is dictated by limiting concentrations of available host lipoyl substrates. Thus, the ability of L. monocytogenes and other intracellular pathogens to efficiently use host lipoyl peptides as a source of lipoate may be a requisite adaptation for life within the mammalian cell.
Abstract: The genome of enteropathogenic Escherichia coli (EPEC) encodes a global regulator, Ler (locus of enterocyte effacement [LEE]-encoded regulator), which activates expression of several polycistronic operons within the 35.6-kb LEE pathogenicity island, including the LEE2-LEE3 divergent operon pair containing overlapping -10 regions and the LEE5 (tir) operon. Ler is a predicted 15-kDa protein that exhibits amino acid similarity with the nucleoid protein H-NS. In order to study Ler-mediated activation of virulence operons in EPEC, we used a molecular approach to characterize the interactions of purified Ler protein with the upstream regulatory sequences of the LEE5 operon. We determined the cis-acting DNA sequences necessary for Ler binding at LEE5 by mobility shift and DNase I protection assays, demonstrating that Ler acts directly at LEE5 by binding sequences between positions -190 and -73 in relation to the transcriptional start site. Based on the molecular weight of Ler, the similarity to H-NS, and the extended region of protection observed in a DNase I footprint at LEE5, we hypothesized that multiple Ler proteins bind upstream of the LEE5 promoter to increase transcriptional activity from a distance. Using an hns deletion strain, we demonstrated that like the LEE2-LEE3 operon pair, H-NS represses LEE5 transcription. We describe a model in which Ler activates transcription at both divergent overlapping paired and single promoters by displacing H-NS, which results in the disruption of a repressing nucleoprotein complex.
