Project description:Transcriptional profiling of three sequenced S. enterica strains: S. Typhimurium LT2, S. Typhi CT18, and S. Typhi Ty2 in PhoP-inducing and non-inducing conditions in vitro, and compared these results to profiles of phoP-/Q- mutants derived from S. Typhimurium LT2 and S. Typhi Ty2.
Project description:Transcriptional profiling of three sequenced S. enterica strains: S. Typhimurium LT2, S. Typhi CT18, and S. Typhi Ty2 in PhoP-inducing and non-inducing conditions in vitro, and compared these results to profiles of phoP-/Q- mutants derived from S. Typhimurium LT2 and S. Typhi Ty2. Two-condition experiment: Each strain above was grown in PhoP-inducing (Low Magnesium concentration) and PhoP non-inducing conditions (High Magnesium concentration) with 1 dye reversal.
Project description:SdiA is a LuxR-type protein found in some Enterobacteriaceae. SdiA encoding bacteria do not encode a luxI homolog and rely on foreign bacteria for the production of N-acyl homoserine lactones (AHLs), SdiA's ligand. The regulon of Salmonella SdiA is largely unknown. In this study, we measured the sdiA dependent transcriptional changes of two serovars of Salmonella, Typhimurium and Typhi, exposed to synthetic AHLs. This was evaluated in two experiments. First, the wild-type and sdiA mutant were grown in the presence of AHLs. In the second, sdiA mutants harboring an arabinose inducible copy of SdiA on a plasmid and vector control were grown with AHLs and arabinose. From this a putative regulon was established and confirmed with subsequent characterization experiments.
Project description:BACKGROUND:S. Typhi, a human-restricted Salmonella enterica serovar, causes a systemic intracellular infection in humans (typhoid fever). In comparison, S. Typhimurium causes gastroenteritis in humans, but causes a systemic typhoidal illness in mice. The PhoP regulon is a well studied two component (PhoP/Q) coordinately regulated network of genes whose expression is required for intracellular survival of S. enterica. METHODOLOGY/PRINCIPAL FINDINGS:Using high performance liquid chromatography mass spectrometry (HPLC-MS/MS), we examined the protein expression profiles of three sequenced S. enterica strains: S. Typhimurium LT2, S. Typhi CT18, and S. Typhi Ty2 in PhoP-inducing and non-inducing conditions in vitro and compared these results to profiles of phoP(-)/Q(-) mutants derived from S. Typhimurium LT2 and S. Typhi Ty2. Our analysis identified 53 proteins in S. Typhimurium LT2 and 56 proteins in S. Typhi that were regulated in a PhoP-dependent manner. As expected, many proteins identified in S. Typhi demonstrated concordant differential expression with a homologous protein in S. Typhimurium. However, three proteins (HlyE, STY1499, and CdtB) had no homolog in S. Typhimurium. HlyE is a pore-forming toxin. STY1499 encodes a stably expressed protein of unknown function transcribed in the same operon as HlyE. CdtB is a cytolethal distending toxin associated with DNA damage, cell cycle arrest, and cellular distension. Gene expression studies confirmed up-regulation of mRNA of HlyE, STY1499, and CdtB in S. Typhi in PhoP-inducing conditions. CONCLUSIONS/SIGNIFICANCE:This study is the first protein expression study of the PhoP virulence associated regulon using strains of Salmonella mutant in PhoP, has identified three Typhi-unique proteins (CdtB, HlyE and STY1499) that are not present in the genome of the wide host-range Typhimurium, and includes the first protein expression profiling of a live attenuated bacterial vaccine studied in humans (Ty800).
Project description:Salmonella enterica subsp. enterica contains more than 2,600 serovars of which four are of major medical relevance for humans. While the typhoidal serovars (Typhi and Paratyphi A) are human-restricted and cause enteric fever, non-typhoidal Salmonella serovars (Typhimurium and Enteritidis) have a broad host range and predominantly cause gastroenteritis. In this study, we compared the core proteomes of Salmonella Typhi, Paratyphi A, Typhimurium and Enteritidis using contemporary proteomics. Five isolates, covering different geographical origins, and one reference strain per serovar were grown in vitro to the exponential phase. Protein levels of orthologous proteins between serovars were compared and subjected to gene ontology term enrichment and inferred regulatory interactions. Differential expression of the core proteomes of the typhoidal serovars appears mainly related to cell surface components and, for the non-typhoidal serovars, to pathogenicity. Our findings may guide future development of novel diagnostics and vaccines, and understanding of disease progression.
Project description:PhoP is considered a regulator of virulence despite being conserved in both pathogenic and non-pathogenic Enterobacteriaceae. While Escherichia coli strains represent both non-pathogenic commensal isolates and numerous virulent pathotypes, the PhoP virulence regulator has only been studied in commensal E. coli. To better understand how conserved transcription factors contribute to virulence, we characterized PhoP in pathogenic E. coli. Loss of phoP significantly attenuated E. coli during extraintestinal infection. This was not surprising since we demonstrated that PhoP differentially regulated the transcription of >600 genes. In addition to survival at acidic pH and resistance to polymyxin B, PhoP was required for repression of motility and oxygen-independent changes in the expression of primary dehydrogenase and terminal reductase respiratory chain components. All phenotypes have in common a reliance on an energized membrane. Thus, we hypothesized that PhoP mediated these effects by regulating genes that generate a proton motive force. Indeed, bacteria lacking PhoP exhibited a hyper-polarized membrane, and dissipation of the transmembrane electrochemical gradient increased the susceptibility of the phoP mutant to acidic pH, while inhibiting respiratory generation of the proton gradient restored resistance to antimicrobial peptides independent of lipopolysaccharide modification. These findings demonstrate a connection between PhoP, virulence, and the energized state of the membrane.
Project description:Bacterial pathogens causing systemic disease commonly evolve from organisms associated with localized infections but differ from their close relatives in their ability to overcome mucosal barriers by mechanisms that remain incompletely understood. Here we investigated whether acquisition of a regulatory gene, tviA, contributed to the ability of Salmonella enterica serotype Typhi to disseminate from the intestine to systemic sites of infection during typhoid fever. To study the consequences of acquiring a new regulator by horizontal gene transfer, tviA was introduced into the chromosome of S. enterica serotype Typhimurium, a closely related pathogen causing a localized gastrointestinal infection in immunocompetent individuals. Modulation of gene expression by TviA in serotype Typhi and Typhimurium was determined by profiling and found to be very comparable. Expression of flagellin, a pathogen associated molecular pattern (PAMP), was repressed by TviA when bacteria were grown at osmotic conditions encountered in tissue, but not at higher osmolarity present in the intestinal lumen. TviA-mediated flagellin repression enabled bacteria to evade sentinel functions of human model epithelia and resulted in increased bacterial dissemination to the spleen in a chicken model.
Project description:In this study, we have defined the NsrR regulon in Salmonella enterica sv. Typhimurium 14028s using a transcriptional microarray. Wild-type and nsrR mutant S. Typhimurium were grown aerobically to early log-phase (OD600~0.5) at 37C in LB medium. Total RNA was isolated from three independent cultures of both strains and interrogated on a PCR product array representing almost all ORFs.