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The chaperone ClpX stimulates expression of Staphylococcus aureus protein A by Rot dependent and independent pathways.

ABSTRACT: The Clp ATPases (Hsp100) constitute a family of closely related proteins that have protein reactivating and remodelling activities typical of molecular chaperones. In Staphylococcus aureus the ClpX chaperone is essential for virulence and for transcription of spa encoding Protein A. The present study was undertaken to elucidate the mechanism by which ClpX stimulates expression of Protein A. For this purpose, we prepared antibodies directed against Rot, an activator of spa transcription, and demonstrated that cells devoid of ClpX contain three-fold less Rot than wild-type cells. By varying Rot expression from an inducible promoter we showed that expression of Protein A requires a threshold level of Rot. In the absence of ClpX the Rot content is reduced below this threshold level, hence, explaining the substantially reduced Protein A expression in the clpX mutant. Experiments addressed at pinpointing the role of ClpX in Rot synthesis revealed that ClpX is required for translation of Rot. Interestingly, translation of the spa mRNA was, like the rot mRNA, enhanced by ClpX. These data demonstrate that ClpX performs dual roles in regulating Protein A expression, as ClpX stimulates transcription of spa by enhancing translation of Rot, and that ClpX additionally is required for full translation of the spa mRNA. The current findings emphasize that ClpX has a central role in fine-tuning virulence regulation in S. aureus.

SUBMITTER: Jelsbak L

PROVIDER: S-EPMC2939077 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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The chaperone ClpX stimulates expression of Staphylococcus aureus protein A by Rot dependent and independent pathways.

Jelsbak Lotte L Ingmer Hanne H Valihrach Lukás L Cohn Marianne Thorup MT Christiansen Mie H G MH Kallipolitis Birgitte H BH Frees Dorte D

PloS one 20100914 9

The Clp ATPases (Hsp100) constitute a family of closely related proteins that have protein reactivating and remodelling activities typical of molecular chaperones. In Staphylococcus aureus the ClpX chaperone is essential for virulence and for transcription of spa encoding Protein A. The present study was undertaken to elucidate the mechanism by which ClpX stimulates expression of Protein A. For this purpose, we prepared antibodies directed against Rot, an activator of spa transcription, and demo ...[more]

PMID: 20856878

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Project description:UNLABELLED:Lipoteichoic acid (LTA) is an important cell wall component of Gram-positive bacteria and a promising target for the development of vaccines and antimicrobial compounds against Staphylococcus aureus Here we demonstrate that mutations in the conditionally essential ltaS (LTA synthase) gene arise spontaneously in an S. aureus mutant lacking the ClpX chaperone. A wide variety of ltaS mutations were selected, and among these, a substantial portion resulted in premature stop codons and other changes predicted to abolish LtaS synthesis. Consistent with this assumption, the clpX ltaS double mutants did not produce LTA, and genetic analyses confirmed that LTA becomes nonessential in the absence of the ClpX chaperone. In fact, inactivation of ltaS alleviated the severe growth defect conferred by the clpX deletion. Microscopic analyses showed that the absence of ClpX partly alleviates the septum placement defects of an LTA-depleted strain, while other phenotypes typical of LTA-negative S. aureus mutants, including increased cell size and decreased autolytic activity, are retained. In conclusion, our results indicate that LTA has an essential role in septum placement that can be bypassed by inactivating the ClpX chaperone. IMPORTANCE:Lipoteichoic acid is an essential component of the Staphylococcus aureus cell envelope and an attractive target for the development of vaccines and antimicrobials directed against antibiotic-resistant Gram-positive bacteria such as methicillin-resistant S. aureus and vancomycin-resistant enterococci. In this study, we showed that the lipoteichoic acid polymer is essential for growth of S. aureus only as long as the ClpX chaperone is present in the cell. Our results indicate that lipoteichoic acid and ClpX play opposite roles in a pathway that controls two key cell division processes in S. aureus, namely, septum formation and autolytic activity. The discovery of a novel functional connection in the genetic network that controls cell division in S. aureus may expand the repertoire of possible strategies to identify compounds or compound combinations that kill antibiotic-resistant S. aureus.

Project description:In Staphylococcus aureus, the global transcriptional regulator CodY modulates the expression of hundreds of genes in response to the availability of GTP and the branched-chain amino acids isoleucine, leucine, and valine (ILV). CodY DNA-binding activity is high when GTP and ILV are abundant. When GTP and ILV are limited, CodY's affinity for DNA drops, altering expression of CodY-regulated targets. In this work, we investigated the impact of guanine nucleotides (GNs) on S. aureus physiology and CodY activity by constructing a guaA null mutant (ΔguaA strain). De novo biosynthesis of guanine monophosphate is abolished due to the guaA mutation; thus, the mutant cells require exogenous guanosine for growth. We also found that CodY activity was reduced when we knocked out guaA, activating the Agr two-component system and increasing secreted protease activity. Notably, in a rich, complex medium, we detected an increase in alternative sigma factor B activity in the ΔguaA mutant, which results in a 5-fold increase in production of the antioxidant pigment staphyloxanthin. Under biologically relevant flow conditions, ΔguaA cells failed to form robust biofilms when limited for guanine or guanosine. Transcriptome sequencing (RNA-Seq) analysis of the S. aureus transcriptome during growth in guanosine-limited chemostats revealed substantial CodY-dependent and -independent alterations of gene expression profiles. Importantly, these changes increase production of proteases and δ-toxin, suggesting that S. aureus exhibits a more invasive lifestyle when limited for guanosine. Further, gene products upregulated under GN limitation, including those necessary for lipoic acid biosynthesis and sugar transport, may prove to be useful drug targets for treating Gram-positive infections.IMPORTANCEStaphylococcus aureus infections impose a serious economic burden on health care facilities and patients because of the emergence of strains resistant to last-line antibiotics. Understanding the physiological processes governing fitness and virulence of S. aureus in response to environmental cues is critical for developing efficient diagnostics and treatments. De novo purine biosynthesis is essential for both fitness and virulence in S. aureus since inhibiting production cripples S. aureus's ability to cause infection. Here, we corroborate these findings and show that blocking guanine nucleotide synthesis severely affects S. aureus fitness by altering metabolic and virulence gene expression. Characterizing pathways and gene products upregulated in response to guanine limitation can aid in the development of novel adjuvant strategies to combat S. aureus infections.

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The chaperone ClpX stimulates expression of Staphylococcus aureus protein A by Rot dependent and independent pathways.

Publications

The chaperone ClpX stimulates expression of Staphylococcus aureus protein A by Rot dependent and independent pathways.

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