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Activity of SigB Modulates Virulence Gene Expression in a Murine Staphylococcus aureus Infection Model but Does Not Influence the Host Kidney Gene Expression

ABSTRACT: Background. Infections caused by Staphylococcus aureus are associated with significant morbidity and mortality and are an increasing threat not only in hospital settings. The expression of the staphylococcal virulence factor repertoire is known to be affected by the alternative sigma factor B (SigB). However, its impact during infection still is a matter of debate. Methods. Kidney tissue of controls or mice infected with S. aureus HG001 or its isogenic sigB mutant was analyzed by transcriptome profiling to monitor the host response, and additionally expression of selected S. aureus genes was monitored by RT-qPCR. Results. Direct transcript analysis by RT-qPCR revealed significant SigB activity in all mice infected with the wild type strain (WT), but not in its isogenic sigB mutant (p<0.0001). Despite a clear cut difference in the SigB-dependent transcription pattern of virulence genes (clfA, aur, and hla), the host reaction to infection (either WT or sigB mutant) was almost identical. Conclusions. Despite its significant activity in vivo, loss of SigB did not have an effect on the outcome of infection as well as on murine kidney gene expression pattern. Thus, these data support the role of SigB as virulence modulator rather than being a virulence determinant by itself.

ORGANISM(S): Mus musculus

PROVIDER: GSE28540 | GEO | 2011/10/24

SECONDARY ACCESSION(S): PRJNA139051

REPOSITORIES: GEO

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Project description:Listeria monocytogenes is well known to have the ability to survive and grow under a variety of stress conditions. The ability to survive and grow under osmotic stress conditions in particular appears to be important for both growth in certain foods and food associated environments as well as for host infection. To characterize the contributions of transcriptional regulators important for stress response and virulence (i.e., Sigma B - M-OM-^CB and PrfA), we initially analyzed three L. monocytogenes parent strains and isogenic mutants ( delta sigB, delta prfA, and delta sigB delta prfA), representing different serotypes and lineages, for their ability to grow in BHI with 11% NaCl (1.9M) at 25M-BM-0C. No significant differences were observed in terms of growth between the parent strains and their respective mutants lacking prfA (i.e. delta prfA, and delta sigB delta prfA mutant strains). While for all strains, the delta sigB mutant showed a prolonged lag phase as compared to the parent strains, maximum growth rates were only reduced for the delta sigB mutant of lineage I and IV strains. Interestingly, for the serotype 1/2b strain, the delta sigB mutant reached a higher maximum cell density than the parent strain or the delta prfA mutant. Caco-2 intestinal epithelial cells invasion assays and hemolytic activity assays showed a significant role for M-OM-^CB in the former and for PrfA in the latter. To initially explore the mechanism that may contribute to the extended lag phase in the delta sigB mutant, microarray was performed to compare transcript levels between the lineage I, serotype 1/2b, parent strain and its isogenic delta sigB mutant in lag phase at 25M-BM-0C in the presence of 11% NaCl. Microarray data showed significant lower and higher transcript levels for 135 and 173 genes, respectively, in the parent strain as compared to the delta sigB strains. Overall, 51 of the 173 M-OM-^CB up-regulated genes had previously been found among the 249 M-OM-^CB-dependent genes identified in a microarray study conducted in stationary phase cells, indicating that up to 122 genes may be transcribed in a M-OM-^CB-dependent manner during lag phase under salt stress. Notable genes that showed higher transcript levels in the parent strain include inlD (encoding an internalin protein that may be involved in virulence), sigH (encoding an alternative M-OM-^C factor), glpK (encoding a glycerol kinase) and resD (encoding a two-component response regulator ResD); while, genes that showed lower transcript levels in the parent strain include dnaK (encoding a dihydroxyacetone kinase), groES (encoding a class I heat-shock protein GroES), grpE (encoding a co-chaperone GrpE) and fri (encoding a non-heme iron-binding ferritin). These data showed that although PrfA does not contribute to growth under osmotic stress at 25M-BM-0C, M-OM-^CB does contribute to survival of L. monocytogenes under high salt conditions. Moreover, the M-OM-^CB-dependent transcriptome of L. monocytogenes lag phase cells under salt stress was characterized and includes previously identified as well as novel M-OM-^CB-dependent genes, including a number of stress response and virulence-associated genes. Independent RNA isolations were performed for one wildtype (lineage I) and M-NM-^TsigB strains from cells grown to lag phase. Three biological replicates were used in competitive whole-genome microarray experiments. For each set of hybridizations, RNA from a L. monocytogenes wildtype strain was hybridized with RNA from its isogenic M-NM-^TsigB null mutant.

Project description:Staphylococcus aureus is a human pathogen that can cause a wide range of diseases. Although formerly regarded as extracellular pathogen, it has been shown that S. aureus can also be internalized by host cells and persist within these cells. In the present study, we comparatively analyzed survival and physiological adaptation of S. aureus HG001 after internalization by two human lung epithelial cell lines (S9 and A549), and human embryonic kidney cells (HEK 293). Combining enrichment of bacteria from host-pathogen assays by cell sorting and quantitation of the pathogen's proteome by mass spectrometry we characterized S. aureus adaptation during the initial phase between 2.5 h and 6.5 h post-infection. Starting with about 2 × 10^6 bacteria, roughly 1450 S. aureus proteins, including virulence factors and metabolic enzymes were identified by spectral comparison and classical database searches. Most of the bacterial adaptation reactions, such as decreased levels of ribosomal proteins and metabolic enzymes or increased amounts of proteins involved in arginine and lysine biosynthesis, enzymes coding for terminal oxidases and stress responsive proteins or activation of the sigma factor SigB were observed after internalization into any of the three cell lines studied. However, differences were noted in central carbon metabolism including regulation of fermentation and threonine degradation. Since these differences coincided with different intracellular growth behavior, complementary profiling of the metabolome of the different non-infected host cell types was performed. This revealed similar levels of intracellular glucose but host cell specific differences in the amounts of amino acids such as glycine, threonine or glutamate. With this comparative study we provide an impression of the common and specific features of the adaptation of S. aureus HG001 to specific host cell environments as a starting point for follow-up studies with different strain isolates and regulatory mutants. Metabolome analysis, by nuclear magnetic resonance spectroscopy, was performed for the infection medium (extracellular medium, mock infection of 1 h).

Project description:Listeria monocytogenes is well known to have the ability to survive and grow under a variety of stress conditions. The ability to survive and grow under osmotic stress conditions in particular appears to be important for both growth in certain foods and food associated environments as well as for host infection. To characterize the contributions of transcriptional regulators important for stress response and virulence (i.e., Sigma B - σB and PrfA), we initially analyzed three L. monocytogenes parent strains and isogenic mutants ( delta sigB, delta prfA, and delta sigB delta prfA), representing different serotypes and lineages, for their ability to grow in BHI with 11% NaCl (1.9M) at 25°C. No significant differences were observed in terms of growth between the parent strains and their respective mutants lacking prfA (i.e. delta prfA, and delta sigB delta prfA mutant strains). While for all strains, the delta sigB mutant showed a prolonged lag phase as compared to the parent strains, maximum growth rates were only reduced for the delta sigB mutant of lineage I and IV strains. Interestingly, for the serotype 1/2b strain, the delta sigB mutant reached a higher maximum cell density than the parent strain or the delta prfA mutant. Caco-2 intestinal epithelial cells invasion assays and hemolytic activity assays showed a significant role for σB in the former and for PrfA in the latter. To initially explore the mechanism that may contribute to the extended lag phase in the delta sigB mutant, microarray was performed to compare transcript levels between the lineage I, serotype 1/2b, parent strain and its isogenic delta sigB mutant in lag phase at 25°C in the presence of 11% NaCl. Microarray data showed significant lower and higher transcript levels for 135 and 173 genes, respectively, in the parent strain as compared to the delta sigB strains. Overall, 51 of the 173 σB up-regulated genes had previously been found among the 249 σB-dependent genes identified in a microarray study conducted in stationary phase cells, indicating that up to 122 genes may be transcribed in a σB-dependent manner during lag phase under salt stress. Notable genes that showed higher transcript levels in the parent strain include inlD (encoding an internalin protein that may be involved in virulence), sigH (encoding an alternative σ factor), glpK (encoding a glycerol kinase) and resD (encoding a two-component response regulator ResD); while, genes that showed lower transcript levels in the parent strain include dnaK (encoding a dihydroxyacetone kinase), groES (encoding a class I heat-shock protein GroES), grpE (encoding a co-chaperone GrpE) and fri (encoding a non-heme iron-binding ferritin). These data showed that although PrfA does not contribute to growth under osmotic stress at 25°C, σB does contribute to survival of L. monocytogenes under high salt conditions. Moreover, the σB-dependent transcriptome of L. monocytogenes lag phase cells under salt stress was characterized and includes previously identified as well as novel σB-dependent genes, including a number of stress response and virulence-associated genes.

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Activity of SigB Modulates Virulence Gene Expression in a Murine Staphylococcus aureus Infection Model but Does Not Influence the Host Kidney Gene Expression

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