Dataset Information

Trapping and identification of cellular substrates of the Staphylococcus aureus ClpC chaperone.

ABSTRACT: ClpC is an ATP-dependent Hsp100/Clp chaperone involved in protein quality control in low-GC Gram-positive bacteria. Previously, we found that ClpC affected the expression of a large number of genes, including capsule genes in Staphylococcus aureus. Here we constructed a His-tagged ClpC variant (ClpC(trap)) with mutations within the Walker B motifs to identify the direct substrates of ClpC by copurification with ClpC(trap) followed by gel electrophoresis combined with liquid chromatography-tandem mass spectrometry proteomics. We identified a total of 103 proteins that are potential substrates of ClpC in strain Newman. The direct protein-protein interaction of ClpC with a subset of the captured proteins was verified in a bacterial two-hybrid system. The captured proteins could be grouped into various functional categories, but most were related to proteins involved in the stress response. Several known ClpC substrates were captured, including ClpP, TrfA/MecA, ClpB, DnaK, DnaJ, GroL, RecA, and CodY, supporting the validity of our approach. Our results also revealed many new ClpC substrates, including AgrA, CcpA, RsbW, MurG, FtsA, SrtA, Rex, Atl, ClfA, and SbcC. Analysis of capsule production showed that three of the captured proteins, which were not previously known to be transcriptional regulators, did affect capsule production.

SUBMITTER: Graham JW

PROVIDER: S-EPMC3807464 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Trapping and identification of cellular substrates of the Staphylococcus aureus ClpC chaperone.

Graham Justin W JW Lei Mei G MG Lee Chia Y CY

Journal of bacteriology 20130802 19

ClpC is an ATP-dependent Hsp100/Clp chaperone involved in protein quality control in low-GC Gram-positive bacteria. Previously, we found that ClpC affected the expression of a large number of genes, including capsule genes in Staphylococcus aureus. Here we constructed a His-tagged ClpC variant (ClpC(trap)) with mutations within the Walker B motifs to identify the direct substrates of ClpC by copurification with ClpC(trap) followed by gel electrophoresis combined with liquid chromatography-tandem ...[more]

PMID: 23913326

Similar Datasets

Project description:Sunlight influences microbial water quality of surface waters. Previous studies have investigated photoinactivation mechanisms and cellular photostress responses of fecal indicator bacteria (FIB), including Escherichia coli and enterococci, but further work is needed to characterize photostress responses of bacterial pathogens. Here we investigate the photoinactivation of Staphylococcus aureus (strain Newman), a pigmented, waterborne pathogen of emerging concern. We measured photodecay using standard culture-based assays and cellular membrane integrity and investigated photostress response by measuring the relative number of mRNA transcripts of select oxidative stress, DNA repair, and metabolism genes. Photoinactivation experiments were performed in both oxic and anoxic systems to further investigate the role of oxygen-mediated and non-oxygen-mediated photoinactivation mechanisms. S. aureus lost culturability much faster in oxic systems than in anoxic systems, indicating an important role for oxygen in photodecay mechanisms. S. aureus cell membranes were damaged by sunlight exposure in anoxic systems but not in oxic systems, as measured by cell membrane permeability to propidium iodide. After sunlight exposure, S. aureus increased expression of a gene coding for methionine sulfoxide reductase after 12 h of sunlight exposure in the oxic system and after 6 h of sunlight exposure in the anoxic system, suggesting that methionine sulfoxide reductase is an important enzyme for defense against both oxygen-dependent and oxygen-independent photostresses. This research highlights the importance of oxygen in bacterial photoinactivation in environmentally relevant systems and the complexity of the bacterial photostress response with respect to cell structure and transcriptional regulation.IMPORTANCEStaphylococcus aureus is a pathogenic bacterium that causes gastrointestinal, respiratory, and skin infections. In severe cases, S. aureus infection can lead to life-threatening diseases, including pneumonia and sepsis. Cases of community-acquired S. aureus infection have been increasing in recent years, pointing to the importance of considering S. aureus transmission pathways outside the hospital environment. Associations have been observed between recreational water contact and staphylococcal skin infections, suggesting that recreational waters may be an important environmental transmission pathway for S. aureus However, prediction of human health risk in recreational waters is hindered by incomplete knowledge of pathogen sources, fate, and transport in this environment. This study is an in-depth investigation of the inactivation of a representative strain of S. aureus by sunlight exposure, one of the most important factors controlling the fate of microbial contaminants in clear waters, which will improve our ability to predict water quality changes and human health risk in recreational waters.

Project description:Staphylococcus aureus is an important pathogen responsible for nosocomial and community-acquired infections in humans, and methicillin-resistant S. aureus (MRSA) infections have continued to increase despite widespread preventative measures. S. aureus can colonize the female vaginal tract, and reports have suggested an increase in MRSA infections in pregnant and postpartum women as well as outbreaks in newborn nurseries. Currently, little is known about specific factors that promote MRSA vaginal colonization and subsequent infection. To study S. aureus colonization of the female reproductive tract in a mammalian system, we developed a mouse model of S. aureus vaginal carriage and demonstrated that both hospital-associated and community-associated MRSA isolates can colonize the murine vaginal tract. Immunohistochemical analysis revealed an increase in neutrophils in the vaginal lumen during MRSA colonization. Additionally, we observed that a mutant lacking fibrinogen binding adhesins exhibited decreased persistence within the mouse vagina. To further identify novel factors that promote vaginal colonization, we performed RNA sequencing to determine the transcriptome of MRSA growing in vivo during vaginal carriage at 5 h, 1 day, and 3 days postinoculation. Over 25% of the bacterial genes were differentially regulated at all time points during colonization compared to laboratory cultures. The most highly induced genes were those involved in iron acquisition, including the Isd system and siderophore transport systems. Mutants deficient in these pathways did not persist as well during in vivo colonization. These results reveal that fibrinogen binding and the capacity to overcome host nutritional limitation are important determinants of MRSA vaginal colonization.IMPORTANCEStaphylococcus aureus is an opportunistic pathogen able to cause a wide variety of infections in humans. Recent reports have suggested an increasing prevalence of MRSA in pregnant and postpartum women, coinciding with the increased incidence of MRSA infections in neonatal intensive care units (NICUs) and newborn nurseries. Vertical transmission from mothers to infants at delivery is a likely route of MRSA acquisition by the newborn; however, essentially nothing is known about host and bacterial factors that influence MRSA carriage in the vagina. Here, we established a mouse model of vaginal colonization and observed that multiple MRSA strains can persist in the vaginal tract. Additionally, we determined that MRSA interactions with fibrinogen and iron uptake can promote vaginal persistence. This study is the first to identify molecular mechanisms which govern vaginal colonization by MRSA, the critical initial step preceding infection and neonatal transmission.

Project description:Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes that assist in protein folding around proline-peptide bonds, and they often possess chaperone activity. Staphylococcus aureus encodes three PPIases, i.e., PrsA, PpiB, and trigger factor (TF). Previous work by our group demonstrated a role for both PrsA and PpiB in S. aureus; however, TF remains largely unstudied. Here, we identify a role for TF in S. aureus biofilm formation and demonstrate cooperation between TF and the cytoplasmic PPIase PpiB. Mutation of the tig gene (encoding TF) led to reduced biofilm development in vitro but no significant attenuation of virulence in a mouse model of infection. To investigate whether TF possesses chaperone activity, we analyzed the ability of a tig mutant to survive acid and base stress. While there was no significant decrease for a tig mutant, a ppiBtig double mutant exhibited significant decreases in cell viability after acid and base challenges. We then demonstrated that a ppiB tig double mutant had exacerbated phenotypes in vitro and in vivo, compared to either single mutant. Finally, in vivo immunoprecipitation of epitope-tagged PpiB revealed that PpiB interacted with 4 times the number of proteins when TF was absent from the cell, suggesting that it may be compensating for the loss of TF. Interestingly, the only proteins found to interact with TF were TF itself, fibronectin-binding protein B (FnBPB), and the chaperone protein ClpB. Collectively, these results support the first phenotype for S. aureus TF and demonstrate a greater network of cooperation between chaperone proteins in Staphylococcus aureusIMPORTANCES. aureus encodes a large number of virulence factors that aid the bacterium in survival and pathogenesis. These virulence factors have a wide variety of functions; however, they must all be properly secreted in order to be functional. Bacterial chaperone proteins often assist in secretion by trafficking proteins to secretion machinery or assisting in proper protein folding. Here, we report that the S. aureus chaperone TF contributes to biofilm formation and cooperates with the chaperone PpiB to regulate S. aureus virulence processes. These data highlight the first known role for TF in S. aureus and suggest that S. aureus chaperone proteins may be involved in a greater regulatory network in the cell.

Dataset Information

Trapping and identification of cellular substrates of the Staphylococcus aureus ClpC chaperone.

Publications

Trapping and identification of cellular substrates of the Staphylococcus aureus ClpC chaperone.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets