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Aro mutations in Salmonella enterica cause defects in cell wall and outer membrane integrity.

ABSTRACT: In this study we characterized aro mutants of Salmonella enterica serovars Enteritidis and Typhimurium, which are frequently used as live oral vaccines. We found that the aroA, aroD, and aroC mutants were sensitive to blood serum, albumen, EDTA, and ovotransferrin, and this defect could be complemented by an appropriate aro gene cloned in a plasmid. Subsequent microarray analysis of gene expression in the aroD mutant in serovar Typhimurium indicated that the reason for this sensitivity might be the upregulation of murA. To confirm this, we artificially overexpressed murA from a multicopy plasmid, and this overexpression caused sensitivity of the strain to albumen and EDTA but not to serum and ovotransferrin. We concluded that attenuation of aro mutants is caused not only by their inability to synthesize aromatic metabolites but also by their defect in cell wall and outer membrane functions associated with decreased resistance to components of innate immune response.

SUBMITTER: Sebkova A

PROVIDER: S-EPMC2347392 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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aro mutations in Salmonella enterica cause defects in cell wall and outer membrane integrity.

Sebkova Alena A Karasova Daniela D Crhanova Magdalena M Budinska Eva E Rychlik Ivan I

Journal of bacteriology 20080229 9

In this study we characterized aro mutants of Salmonella enterica serovars Enteritidis and Typhimurium, which are frequently used as live oral vaccines. We found that the aroA, aroD, and aroC mutants were sensitive to blood serum, albumen, EDTA, and ovotransferrin, and this defect could be complemented by an appropriate aro gene cloned in a plasmid. Subsequent microarray analysis of gene expression in the aroD mutant in serovar Typhimurium indicated that the reason for this sensitivity might be ...[more]

PMID: 18310348

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Project description:Typhoid caused by Salmonella enterica serovar Typhi remains a major health concern worldwide. The emergence of multidrug-resistant strains of Salmonella with increased virulence, communicability, and survivability leading to increased morbidity and mortality has further complicated its management. Currently available vaccines for typhoid have less-than-desired efficacy and certain unacceptable side effects, making it pertinent to search for new immunogens suitable for vaccine formulation. The outer membrane proteins (OMPs) of Salmonella have been considered possible candidates for conferring protection against typhoid. OMPs interface the cell with the environment, thus representing important virulence factors with a significant role in the pathobiology of gram-negative bacteria and bacterial adaptation. An OMP of Salmonella enterica serovar Typhimurium with an apparent molecular mass of 49 kDa that is highly immunogenic, evokes humoral and cell-mediated immune responses, and confers 100% protection to immunized rats against challenge with very high doses (up to 100 times the 50% lethal dose) of Salmonella enterica serovar Typhimurium has been identified. Further, very efficient clearance of bacteria from the reticuloendothelial systems of immunized animals was seen. This protein is recognized by the antibodies present in serum of typhoid patients. When sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel-eluted protein was further analyzed by high-performance liquid chromatography (HPLC) and two-dimensional electrophoresis, two polypeptides with the same molecular weight were resolved. These have different isoelectric points and gave two peaks with different retention times in reverse-phase HPLC. However, only one of the two bands interacted with patient serum. The immunogenicity studies (enzyme-linked immunosorbent assay and delayed-type hypersensitivity [DTH]) indicated that the immunoreactive protein evoked a strong immune response in rats. The N-terminal sequencing and analysis of the homology of this protein with sequences in the protein database of Salmonella resulted in a match with the N-terminal sequences of a protein in Salmonella enterica serovar Typhi (CT18 and Ty2 strains). The homology search further revealed it to be a hypothetical protein, whose gene had unidentified open reading frames in Salmonella serovar Typhi encoding 447 amino acid residues, corresponding to a molecular mass of 49 kDa. The nucleotide sequence of the encoding gene was deduced, and the gene was amplified by PCR using appropriate primers. An amplified 1.3-kb band was purified and sequenced to confirm its identity. These OMPs provide promising targets for the development of a candidate vaccine against typhoid.

Project description:Bacterial outer membrane vesicles (OMVs), as well as OMV-associated small RNAs, have been demonstrated to play a role in host-pathogen interactions. The presence of larger RNA transcripts in OMVs has been less studied and their potential role in host-pathogen interactions remains largely unknown. Here we analyze RNA from OMVs secreted by Salmonella enterica serovar Typhimurium (S. Typhimurium) cultured under different conditions, which mimic host-pathogen interactions. S. Typhimurium was grown to exponential and stationary growth phases in minimal growth control medium (phosphate-carbon-nitrogen, PCN), as well as in acidic and phosphate-depleted PCN, comparable to the macrophage environment and inducing therefore the expression of Salmonella pathogenicity island 2 (SPI-2) genes. Moreover, Salmonella pathogenicity island 1 (SPI-1), which is required for virulence during the intestinal phase of infection, was induced by culturing S. Typhimurium to the stationary phase in Lysogeny Broth (LB). For each condition, we identified OMV-associated RNAs that are enriched in the extracellular environment relative to the intracellular space. All RNA classes could be observed, but a vast majority of rRNA was exported in all conditions in variable proportions with a notable decrease in LB SPI-1 inducing media. Several mRNAs and ncRNAs were specifically enriched in/on OMVs dependent on the growth conditions. Important to note is that some RNAs showed identical read coverage profiles intracellularly and extracellularly, whereas distinct coverage patterns were observed for other transcripts, suggesting a specific processing or degradation. Moreover, PCR experiments confirmed that distinct RNAs were present in or on OMVs as full-length transcripts (IsrB-1/2; IsrA; ffs; SsrS; CsrC; pSLT035; 10Sa; rnpB; STM0277; sseB; STM0972; STM2606), whereas others seemed to be rather present in a processed or degraded form. Finally, we show by a digestion protection assay that OMVs are able to prevent enzymatic degradation of given full-length transcripts (SsrS, CsrC, 10Sa, and rnpB). In summary, we show that OMV-associated RNA is clearly different in distinct culture conditions and that at least a fraction of the extracellular RNA is associated as a full-length transcripts with OMVs, indicating that some RNAs are protected by OMVs and thereby leaving open the possibility that those might be functionally active.

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Aro mutations in Salmonella enterica cause defects in cell wall and outer membrane integrity.

Publications

aro mutations in Salmonella enterica cause defects in cell wall and outer membrane integrity.

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