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Global Screening of Salmonella enterica Serovar Typhimurium Genes for Desiccation Survival.

ABSTRACT: Salmonella spp., one of the most common foodborne bacterial pathogens, has the ability to survive under desiccation conditions in foods and food processing facilities for years. This raises the concerns of Salmonella infection in humans associated with low water activity foods. Salmonella responds to desiccation stress via complex pathways involving immediate physiological actions as well as coordinated genetic responses. However, the exact mechanisms of Salmonella to resist desiccation stress remain to be fully elucidated. In this study, we screened a genome-saturating transposon (Tn5) library of Salmonella Typhimurium (S. Typhimurium) 14028s under the in vitro desiccation stress using transposon sequencing (Tn-seq). We identified 61 genes and 6 intergenic regions required to overcome desiccation stress. Salmonella desiccation resistance genes were mostly related to energy production and conversion; cell wall/membrane/envelope biogenesis; inorganic ion transport and metabolism; regulation of biological process; DNA metabolic process; ABC transporters; and two component system. More than 20% of the Salmonella desiccation resistance genes encode either putative or hypothetical proteins. Phenotypic evaluation of 12 single gene knockout mutants showed 3 mutants (atpH, atpG, and corA) had significantly (p < 0.02) reduced survival as compared to the wild type during desiccation survival. Thus, our study provided new insights into the molecular mechanisms utilized by Salmonella for survival against desiccation stress. The findings might be further exploited to develop effective control strategies against Salmonella contamination in low water activity foods and food processing facilities.

SUBMITTER: Mandal RK

PROVIDER: S-EPMC5596212 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Global Screening of Salmonella enterica Serovar Typhimurium Genes for Desiccation Survival.

Mandal Rabindra K RK Kwon Young M YM

Frontiers in microbiology 20170908

Salmonella spp., one of the most common foodborne bacterial pathogens, has the ability to survive under desiccation conditions in foods and food processing facilities for years. This raises the concerns of Salmonella infection in humans associated with low water activity foods. Salmonella responds to desiccation stress via complex pathways involving immediate physiological actions as well as coordinated genetic responses. However, the exact mechanisms of Salmonella to ...[more]

PMID: 28943871

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Project description:In this study, the changes in the global proteome of Salmonella in response to desiccation and thermal treatment were investigated by using an iTRAQ multiplex technique. A Salmonella enterica serovar Typhimurium strain was dried, equilibrated at high (1.0) and low (0.11) water activity (aw), and thermally treated at 75°C. The proteomes were characterized after every treatment. The proteomes of the different treatments differed in the expression of 175 proteins. On the basis of their proteomic expression profiles, the samples were clustered into two major groups, namely, "dry" samples and "moist" samples. The groups had different levels of proteins involved in DNA synthesis and transcription and in metabolic reactions, indicating that cells under either of the aw conditions need to strictly control energy metabolism, the rate of replication, and protein synthesis. The proteins with higher expression levels in moist samples were flagellar proteins (FlgEFGH), membrane proteins, and export systems (SecF, SecD, the Bam complex), as well as stress response proteins, suggesting that rehydration can trigger stress responses in moist cells. Dry samples had higher levels of ribosomal proteins, indicating that ribosomal proteins might be important for additional regulation of the cellular response, even when the synthesis of proteins is slowed down. At both aws, no differences in protein expression were observed between the thermally treated samples and the nonheated cells. In conclusion, our study indicates that the preadaptation to a dry condition was linked to increased thermal tolerance, while reversion from a dry state to a moist state induced a significant change in protein expression, possibly linked to the observed loss of thermal tolerance.IMPORTANCESalmonella enterica is able to survive in dry environments for very long periods. While it is well known that the initial exposure to desiccation is fundamental to trigger thermal tolerance in this organism, the specific physiological and molecular processes involved in this cross-protection phenomenon have not been fully characterized. Several studies have focused on the low-aw transcriptome of this pathogen when inoculated in different food matrices or on abiotic surfaces, but proteomic analyses have not been reported in the literature. Our study investigated the changes in proteomic expression in Salmonella enterica serovar Typhimurium during desiccation, exposure to low aw, and thermal treatment. A better knowledge of the systems involved in the response to desiccation and thermal tolerance, as well as a better understanding of their interplay, is fundamental to identify the most effective combination of interventions to prevent Salmonella's contamination of foods.

Project description:Penetration of intestinal epithelial cells by Salmonella enterica serovar Typhimurium requires the expression of invasion genes, found in Salmonella pathogenicity island 1 (SPI1), that encode components of a type III secretion apparatus. These genes are controlled in a complex manner by regulators within SPI1, including HilA and InvF, and those outside SPI1, such as the two-component regulators PhoP/PhoQ and BarA/SirA. We report here that epithelial cell invasion requires the serovar Typhimurium homologue of Escherichia coli csrA, which encodes a regulator that alters the stability of specific mRNA targets. A deletion mutant of csrA was unable to efficiently invade cultured epithelial cells and showed reduced expression of four tested SPI1 genes, hilA, invF, sipC, and prgH. Overexpression of csrA from an induced araBAD promoter also negatively affected the expression of these genes, indicating that CsrA can act as both a positive and a negative regulator of SPI1 genes and suggesting that the bacterium must tightly control the level or activity of CsrA to achieve maximal invasion. We found that CsrA affected hilA, a regulator of the other three genes we tested, probably by controlling one or more genetic elements that regulate hilA. We also found that both the loss and the overexpression of csrA reduced the expression of two regulators of hilA, hilC and hilD, suggesting that csrA exerts its control of hilA through one or both of these regulators. We further found, however, that CsrA could affect the expression of both invF and sipC independent of its effects on hilA. One additional striking phenotype of the csrA mutant, not observed in a comparable E. coli mutant, was its slow growth. Phenotypic revertants that had normal growth rates, while maintaining the csrA mutation, were common. These suppressed strains, however, did not recover the ability to invade cultured cells, indicating that the csrA-mediated loss of invasion cannot be attributed simply to poor growth and that the growth and invasion deficits of the csrA mutant arise from effects of CsrA on different targets.

Project description:The rpoS gene encodes the alternative sigma factor sigma(S) (RpoS) and is required for survival of bacteria under starvation and stress conditions. It is also essential for Salmonella virulence in mice. Most work on the RpoS regulon has been in the closely related enterobacterial species Escherichia coli. To characterize the RpoS regulon in Salmonella, we isolated 38 unique RpoS-activated lacZ gene fusions from a bank of Salmonella enterica serovar Typhimurium mutants harboring random Tn5B21 mutations. Dependence on RpoS varied from 3-fold to over 95-fold, and all gene fusions isolated were regulated by growth phase. The identities of 21 RpoS-dependent fusions were determined by DNA sequence analysis. Seven of the fusions mapped to DNA regions in Salmonella serovar Typhimurium that do not match any known E. coli sequence, suggesting that the composition of the RpoS regulon differs markedly in the two species. The other 14 fusions mapped to 13 DNA regions very similar to E. coli sequences. None of the insertion mutations in DNA regions common to both species appeared to affect Salmonella virulence in BALB/c mice. Of these, only three (otsA, katE, and poxB) are located in known members of the RpoS regulon. Ten insertions mapped in nine open reading frames of unknown function (yciF, yehY, yhjY, yncC, yjgB, yahO, ygaU, ycgB, and yeaG) appear to be novel members of the RpoS regulon. One insertion, that in mutant C52::H87, was in the noncoding region upstream from ogt, encoding a O(6)-methylguanine DNA methyltransferase involved in repairing alkylation damage in DNA. The ogt coding sequence is very similar to the E. coli homolog, but the ogt 5' flanking regions were found to be markedly different in the two species, suggesting genetic rearrangements. Using primer extension assays, a specific ogt mRNA start site was detected in RNAs of the Salmonella serovar Typhimurium wild-type strains C52 and SL1344 but not in RNAs of the mutant strains C52K (rpoS), SL1344K (rpoS), and C52::H87. In mutant C52::H87, Tn5B21 is inserted at the ogt mRNA start site, with lacZ presumably transcribed from the identified RpoS-regulated promoter. These results indicate that ogt gene expression in Salmonella is regulated by RpoS in stationary phase of growth in rich medium, a finding that suggests a novel role for RpoS in DNA repair functions.

Dataset Information

Global Screening of Salmonella enterica Serovar Typhimurium Genes for Desiccation Survival.

Publications

Global Screening of <i>Salmonella enterica</i> Serovar Typhimurium Genes for Desiccation Survival.

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