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Inactivation of alternative sigma factor 54 (RpoN) leads to increased acid resistance, and alters locus of enterocyte effacement (LEE) expression in Escherichia coli O157 : H7.

ABSTRACT: Alternative sigma factor 54 (RpoN) is an important regulator of stress resistance and virulence genes in many bacterial species. In this study, we report on the gene expression alterations that follow rpoN inactivation in Escherichia coli O157 : H7 strain Sakai (Sakai rpoN : : kan), and the influence of RpoN on the acid resistance phenotype. Microarray gene expression profiling revealed the differential expression of 103 genes in SakairpoN : : kan relative to Sakai. This included the growth-phase-dependent upregulation of genes required for glutamate-dependent acid resistance (GDAR) ( gadA, gadB, gadC and gadE), and the downregulation of locus of enterocyte effacement (LEE) genes, which encode a type III secretion system. Upregulation of gad genes in SakairpoN : : kan during exponential growth correlated with increased GDAR and survival in a model stomach system. Complementation of SakairpoN : : kan with a cloned version of rpoN restored acid susceptibility. Genes involved in GDAR regulation, including rpoS (sigma factor 38) and gadE (acid-responsive regulator), were shown to be required for the survival of SakairpoN : : kan by the GDAR mechanism. This study describes the contribution of rpoN to acid resistance and GDAR gene regulation, and reveals RpoN to be an important regulator of stress resistance and virulence genes in E. coli O157 : H7.

SUBMITTER: Riordan JT

PROVIDER: S-EPMC2889430 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Inactivation of alternative sigma factor 54 (RpoN) leads to increased acid resistance, and alters locus of enterocyte effacement (LEE) expression in Escherichia coli O157 : H7.

Riordan James T JT Tietjen Jillian A JA Walsh Coilin W CW Gustafson John E JE Whittam Thomas S TS

Microbiology (Reading, England) 20091126 Pt 3

Alternative sigma factor 54 (RpoN) is an important regulator of stress resistance and virulence genes in many bacterial species. In this study, we report on the gene expression alterations that follow rpoN inactivation in Escherichia coli O157 : H7 strain Sakai (Sakai rpoN : : kan), and the influence of RpoN on the acid resistance phenotype. Microarray gene expression profiling revealed the differential expression of 103 genes in SakairpoN : : kan relative to Sakai. This included the growth-phas ...[more]

PMID: 19942657

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Project description:In this study, we present evidence that proteins encoded by the Locus of Enterocyte Effacement (LEE), considered critical for Escherichia coli O157 (O157) adherence to follicle-associated epithelial (FAE) cells at the bovine recto-anal junction (RAJ), do not appear to contribute to O157 adherence to squamous epithelial (RSE) cells also constituting this primary site of O157 colonization in cattle.Antisera targeting intimin-?, the primary O157 adhesin, and other essential LEE proteins failed to block O157 adherence to RSE cells, when this pathogen was grown in DMEM, a culture medium that enhances expression of LEE proteins. In addition, RSE adherence of a DMEM-grown-O157 mutant lacking the intimin protein was comparable to that seen with its wild-type parent O157 strain grown in the same media. These adherence patterns were in complete contrast to that observed with HEp-2 cells (the adherence to which is mediated by intimin-?), assayed under same conditions. This suggested that proteins other than intimin-? that contribute to adherence to RSE cells are expressed by this pathogen during growth in DMEM. To identify such proteins, we defined the proteome of DMEM-grown-O157 (DMEM-proteome). GeLC-MS/MS revealed that the O157 DMEM-proteome comprised 684 proteins including several components of the cattle and human O157 immunome, orthologs of adhesins, hypothetical secreted and outer membrane proteins, in addition to the known virulence and LEE proteins. Bioinformatics-based analysis of the components of the O157 DMEM proteome revealed several new O157-specific proteins with adhesin potential.Proteins other than LEE and intimin-? proteins are involved in O157 adherence to RSE cells at the bovine RAJ. Such proteins, with adhesin potential, are expressed by this human pathogen during growth in DMEM. Ongoing experiments to evaluate their role in RSE adherence should provide both valuable insights into the O157-RSE interactions and new targets for more efficacious anti-adhesion O157 vaccines.

Project description:There is increasing evidence to support a role for sigma factor 54 (RpoN) in the regulation of stress resistance factors and protein secretion systems important to bacterial transmission and pathogenesis. In enterohemorrhagic E. coli O157:H7, acid resistance and type III secretion are essential determinants of gastric passage and colonization. This study thus described the transcriptome of an rpoN null strain of E. coli O157:H7 (EcJR-8) to determine the influence of RpoN on virulence and stress resistance gene regulation, and further explored its contribution to glutamate-dependent acid resistance (GDAR). Inactivation of rpoN resulted in the growth phase-dependent, differential expression of 104 genes. This included type III secretion structural and regulatory genes encoded on the locus of enterocyte effacement (LEE), as well as GDAR genes gadA, gadBC and gadE. Upregulation of gad transcript levels in EcJR-8 during logarithmic growth correlated with increased GDAR and survival in a model stomach. Acid susceptibility was reconstituted in EcJR-8 complemented in trans with wild-type rpoN. Acid resistance in EcJR-8 was dependent on exogenous glutamate, gadE and rpoS, but was independent of hns. Results also suggest that GDAR may be controlled by RpoN at multiple regulatory levels. This study supports the hypothesis that RpoN is an important regulator of virulence and stress resistance factors in E. coli O157:H7, and is the first to examine the mechanism by which it represses GDAR. Hybridizations measured transcriptional differences between an rpoN null and wild-type (WT) strain of E. coli O157:H7 Sakai at logarithmic and transition phase. Image files (TIFF) of hybridized microarray slides were generated using an Axon 4000B scanner (Molecular Devices), and analyzed using GenePix Pro software (Molecular Devices, ver. 6.0). The resulting microarray intensity data was log2-transformed, and normalized using the LOWESS algorithm in MAANOVA ver. 0.98-8 (R ver. 2.2.1).

Project description:BackgroundEdwardsiella tarda is an enterobacterium which causes edwardsiellosis, a fatal disease of cultured fishes such as red sea bream, eel, and flounder. Preventing the occurrence of E. tarda infection has thus been an important issue in aquaculture. E. tarda has been isolated from other animals and from many environments; however, the relationship between the genotype and evolutionary process of this pathogen is not fully understood. To clarify this relationship, we sequenced and compared the genomes of pathogenic and non-pathogenic E. tarda strains isolated from fish, human, and eel pond using next-generation sequencing technology.ResultsEight strains of E. tarda were sequenced with high accuracy (>99.9%) with coverages from 50- to 400-fold. The obtained reads were mapped to a public reference genome. By comparing single nucleotide and insertion/deletion polymorphisms, we found that an attenuated strain of E. tarda had a loss-of-function mutation in a gene related to the type III secretion system (T3SS), suggesting that this gene is involved in the virulence of E. tarda. A comprehensive gene comparison indicated that fish pathogenic strains possessed a type VI secretion system (T6SS) and pilus assembly genes in addition to the T3SS. Moreover, we found that an E. tarda strain isolated from red sea bream harbored two pathogenicity islands of T3SS and T6SS, which were absent in other strains. In particular, this T3SS was homologous to the locus of enterocyte effacement (LEE) in enteropathogenic and enterohemorrhagic Escherichia coli. Evolutionary analysis suggested that this locus, here named Et-LEE (E. tarda LEE), was introgressed into the E. tarda genome through horizontal transfer.ConclusionsWe found significant differences in the presence/absence of virulence-related genes among E. tarda strains, reflecting their evolutionary relationship. In particular, a single genotype previously proposed for fish-pathogenic strains may be further divided into two subgroups. Furthermore, the current study demonstrated, for the first time, that a fish pathogenic bacterium carried a LEE-like pathogenicity island which was previously reported only in zoonotic pathogenic enterobacteria. These findings will contribute to the exploration of strain-specific drug targets against E. tarda in aquafarms, while also shedding light on the evolution of pathogenesis in enterobacteria.

Dataset Information

Inactivation of alternative sigma factor 54 (RpoN) leads to increased acid resistance, and alters locus of enterocyte effacement (LEE) expression in Escherichia coli O157 : H7.

Publications

Inactivation of alternative sigma factor 54 (RpoN) leads to increased acid resistance, and alters locus of enterocyte effacement (LEE) expression in Escherichia coli O157 : H7.

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