ABSTRACT: Investigation of whole genome gene expression level changes in a Salmonella enterica serovar Typhimurium 14028 delta GidA mutant The mutant described in this study is further analyzed in Shippy, D. C., N. M. Eakley, P. N. Bochsler, and A. A. Fadl. 2011. Biological and virulence characteristics of Salmonella enterica serovar Typhimurium following deletion of glucose-inhibited division (gidA) gene. Microb Pathog. A single chip study using three separate cultures of wild-type Salmonella enterica serovar Typhimurium 14028 and three separate cultures of a single mutant, delta GidA Salmonella enterica serovar Typhimurium 14028.
Project description:Investigation of whole genome gene expression level changes in a Salmonella enterica serovar Typhimurium UK1 delta-iacP mutant, compared to the wild-type strain. IacP is resoponsible for the secretion of virulence effector proteins via the type III secretion system, thereby contributing the virulence of S. Typhimurium. The mutants analyzed in this study are further described in Kim et al. 2011. Role of Salmonella Pathogenicity Island 1 Protein IacP in Salmonella enterica Serovar Typhimurium Pathogenesis. Infection and Immunity 79(4):1440-1450 (PMID 21263021). A chip study using total RNA recovered from two separate wild-type cultures of Salmonella enterica serovar Typhimurium UK1 and two separate cultures of a mutant strain, Salmonella enterica serovar Typhimurium UK1 delta-iacP. Each chip measures the expression level of 4,302 genes from Salmonella enterica serovar Typhimurium.
Project description:Genomic subtractive hybridization was performed between Salmonella enterica serovar Typhimurium LT2 and DT104 to search for novel Salmonella serovar Typhimurium DT104-specific sequences. The subtraction resulted mainly in the isolation of DNA fragments with sequence similarity to phages. Two fragments identified were associated with possible virulence factors. One fragment was identical to irsA of Salmonella serovar Typhimurium ATCC 14028, which is suggested to be involved in macrophage survival. The other fragment was homologous to HldD, an Escherichia coli O157:H7 lipopolysaccharide assembly-related protein. Five selected DNA fragments-irsA, the HldD homologue, and three fragments with sequence similarity to prophages-were tested for their presence in 17 Salmonella serovar Typhimurium DT104 isolates and 27 non-DT104 isolates by PCR. All five selected DNA fragments were Salmonella serovar Typhimurium DT104 specific among the serovar Typhimurium isolates tested. These DNA fragments can be useful for better detection and typing of Salmonella serovar Typhimurium DT104.
Project description:Salmonella enterica serovar Typhimurium is one of approximately 2,500 distinct serovars of the genus Salmonella but is exceptional in its wide distribution in the environment, livestock, and wild animals. S Typhimurium causes a large proportion of nontyphoidal Salmonella (NTS) infections, accounting for a quarter of infections, second only to S. enterica serovar Enteritidis in incidence. S Typhimurium was once considered the archetypal broad-host-range Salmonella serovar due to its wide distribution in livestock and wild animals, and much of what we know of the interaction of Salmonella with the host comes from research using a small number of laboratory strains of the serovar (LT2, SL1344, and ATCC 14028). But it has become clear that these strains do not reflect the genotypic or phenotypic diversity of S Typhimurium. Here, we review the epidemiological record of S Typhimurium and studies of the host-pathogen interactions of diverse strains of S Typhimurium. We present the concept of distinct pathovariants of S Typhimurium that exhibit diversity of host range, distribution in the environment, pathogenicity, and risk to food safety. We review recent evidence from whole-genome sequencing that has revealed the extent of genomic diversity of S Typhimurium pathovariants, the genomic basis of differences in the level of risk to human and animal health, and the molecular epidemiology of prominent strains. An improved understanding of the impact of genome variation of bacterial pathogens on pathogen-host and pathogen-environment interactions has the potential to improve quantitative risk assessment and reveal how new pathogens evolve.
Project description:DegQ is a serine protease that is highly homologous to HtrA, an important virulence determinant of Salmonella enterica serovar Typhimurium. We examined if DegQ is involved in serovar Typhimurium pathogenesis. A serovar Typhimurium degQ mutant was as virulent as the wild-type strain in mice. However, a serovar Typhimurium htrA degQ mutant survived less well in murine organs, particularly in the liver, than a serovar Typhimurium htrA mutant. DegQ is not essential for serovar Typhimurium pathogenesis but may play a small role during salmonella growth at systemic sites.
Project description:BACKGROUND: The global ppGpp-mediated stringent response in pathogenic bacteria plays an important role in the pathogenesis of bacterial infections. In Salmonella enterica serovar Typhimurium (S. Typhimurium), several genes, including virulence genes, are regulated by ppGpp when bacteria are under the stringent response. To understand the control of virulence genes by ppGpp in S. Typhimurium, agarose 2-dimensional electrophoresis (2-DE) combined with mass spectrometry was used and a comprehensive 2-DE reference map of amino acid-starved S. Typhimurium strain SH100, a derivative of ATCC 14028, was established. RESULTS: Of the 366 examined spots, 269 proteins were successfully identified. The comparative analysis of the wild-type and ppGpp0 mutant strains revealed 55 proteins, the expression patterns of which were affected by ppGpp. Using a mouse infection model, we further identified a novel virulence-associated factor, STM3169, from the ppGpp-regulated and Salmonella-specific proteins. In addition, Salmonella strains carrying mutations in the gene encoding STM3169 showed growth defects and impaired growth within macrophage-like RAW264.7 cells. Furthermore, we found that expression of stm3169 was controlled by ppGpp and SsrB, a response regulator of the two-component system located on Salmonella pathogenicity island 2. CONCLUSIONS: A proteomic approach using a 2-DE reference map can prove a powerful tool for analyzing virulence factors and the regulatory network involved in Salmonella pathogenesis. Our results also provide evidence of a global response mediated by ppGpp in S. enterica.
Project description:Innovative vaccines against typhoid and other Salmonella diseases that are safe, effective, and inexpensive are urgently needed. In order to address this need, buoyant, self-adjuvating gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1 were bioengineered to display the highly conserved Salmonella enterica antigen SopB, a secreted inosine phosphate effector protein injected by pathogenic bacteria during infection into the host cell. Two highly conserved sopB gene segments near the 3'-coding region, named sopB4 and B5, were each fused to the gvpC gene, and resulting GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and B5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of recombinant GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 ?pmrG-HM-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-?, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Th1 response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5-GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were found to be stable at elevated temperatures for extended periods without refrigeration in Halobacterium cells. The results all together show that bioengineered GVNPs are likely to represent a valuable platform for the development of improved vaccines against Salmonella diseases.
Project description:Virulence and persistence in the BALB/c mouse gut was tested for 32 strains of Salmonella enterica for which genome sequencing is complete or underway, including 17 serovars within subspecies I (enterica), and two representatives of each of the other five subspecies. Only serovar Paratyphi C strain BAA1715 and serovar Typhimurium strain 14028 were fully virulent in mice. Three divergent atypical Enteritidis strains were not virulent in BALB/c, but two efficiently persisted. Most of the other strains in all six subspecies persisted in the mouse intestinal tract for several weeks in multiple repeat experiments although the frequency and level of persistence varied considerably. Strains with heavily degraded genomes persisted very poorly, if at all. None of the strains tested provided immunity to Typhimurium infection. These data greatly expand on the known significant strain-to-strain variation in mouse virulence and highlight the need for comparative genomic and phenotypic studies.
Project description:Loss of the Salmonella MsbB enzyme, which catalyzes the incorporation of myristate destined for lipopolysaccharide in the outer membrane, results in a strong phenotype of sensitivity to salt and chelators such as EGTA and greatly diminished endotoxic activity. MsbB- salmonellae mutate extragenically to EGTA-tolerant derivatives at a frequency of 10(-4) per division. One of these derivatives arose from inactivation of somA, which suppresses sensitivity to salt and EGTA. Here we show that a second mode of MsbB- suppression is a RecA-dependent deletion between two IS200 insertion elements present in Salmonella enterica serovar Typhimurium strain ATCC 14028 but not in two other wild-type strains, LT2 and SL1344, which lack one of the IS200 elements. This deletion occurs spontaneously in wild-type and MsbB- strain 14028 salmonellae and accounts for about one-third of all of the spontaneous suppressors of MsbB- in strain 14028. It spans the region corresponding to 17.7 to 19.9 centisomes, which includes somA, on the sequenced map of Salmonella LT2 (136 ORFs in that strain; ATCC 14028 and other strains showed variability in this region). In addition to conferring EGTA resistance correlated with somA, the deletion confers a MacConkey galactose resistance phenotype on MsbB- Salmonella, indicating that at least one additional gene (distinct from somA) within the deletion is responsible for this phenotype. In the wild type, the deletion mutant grows with normal exponential growth rate in Luria broth but is chlorate resistant and does not grow on citrate agar. The deletion strains have lost hydrogen sulfide production, nitrate reductase activity, and gas production from glucose fermentation.
Project description:We have recently demonstrated that an attenuated strain of Salmonella enterica serovar Typhimurium unable to synthesize the zinc transporter ZnuABC (S. Typhimurium ?znuABC), is able to protect mice against systemic and enteric salmonellosis and is safe in pigs. Here, we have tested the protective effects of S. Typhimurium ?znuABC in pigs. Resistance to challenge with the fully virulent strain S. Typhimurium ATCC 14028 was assessed in animals vaccinated with S. Typhimurium ?znuABC (two dosages tested), in controls vaccinated with a formalin-inactivated virulent strain and in unvaccinated controls. Clinical signs of salmonellosis, faecal shedding and bacterial colonization of organs were used to assess vaccine-induced protection. After the challenge, pigs vaccinated with the attenuated S. Typhimurium ?znuABC strain did not display clinical signs of salmonellosis (fever or diarrhoea). The vaccine also reduced intestinal tract colonization and faecal shedding of the fully virulent Salmonella strain, as compared to control groups. S. Typhimurium ?znuABC represents a promising candidate vaccine against salmonellosis in pigs.
Project description:Static broth culture favors Salmonella enterica subsp. enterica serovar Typhimurium to produce type 1 fimbriae, while solid agar inhibits its expression. A transposon inserted in stbC, which would encode an usher for Stb fimbriae of a non-flagellar Salmonella enterica subsp. enterica serovar Typhimurium LB5010 strain, conferred it to agglutinate yeast cells on both cultures. RT-PCR revealed that the expression of the fimbrial subunit gene fimA, and fimZ, a regulatory gene of fimA, were both increased in the stbC mutant when grown on LB agar; fimW, a repressor gene of fimA, exhibited lower expression. Flagella were observed in the stbC mutant and this phenotype was correlated with the motile phenotype. Microarray data and RT-PCR indicated that the expression of three genes, motA, motB, and cheM, was enhanced in the stbC mutant. The stbC mutant was resistant to several antibiotics, consistent with the finding that expression of yhcQ and ramA was enhanced. A complementation test revealed that transforming a recombinant plasmid possessing the stbC restored the mannose-sensitive agglutination phenotype to the stbC mutant much as that in the parental Salmonella enterica subsp. enterica serovar Typhimurium LB5010 strain, indicating the possibility of an interplay of different fimbrial systems in coordinating their expression.