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Adaptation of bacterial pathogens to an intracellular environment requires resetting of the expression levels of a wide range of both virulence and housekeeping genes. We investigated the possibility that changes in DNA supercoiling could modulate the expression of genes known to be important in the intracellular growth of the pathogen Salmonella enterica serovar Typhimurium. Our data show that DNA becomes relaxed when Salmonella grows in murine macrophage but not in epithelial cells, indicating that DNA supercoiling plays a role in discrimination between two types of intracellular environment. The ssrA regulatory gene within the SPI-2 pathogenicity island that is required for survival in macrophage was found to be upregulated by DNA relaxation. This enhancement of expression also required the Fis nucleoid-associated protein. Manipulating the level of the Fis protein modulated both the level of DNA supercoiling and ssrA transcription. We discuss a model of bacterial intracellular adaptation in which Fis and DNA supercoiling collaborate to fine-tune virulence gene expression.

Staphylococcus aureus is a Gram‐positive commensal that can also cause a variety of infections in humans. S. aureus virulence factor gene expression is under tight control by a complex regulatory network, which includes, sigma factors, sRNAs, and two‐component systems (TCS). Previous work in our laboratory demonstrated that overexpression of the sRNA tsr37 leads to an increase in bacterial aggregation. Here, we demonstrate that the clumping phenotype is dependent on a previously unannotated 88 amino acid protein encoded within the tsr37 sRNA transcript (which we named ScrA for S. aureusclumping regulator A). To investigate the mechanism of action of ScrA we performed proteomics and transcriptomics in a ScrA overexpressing strain and show that a number of surface adhesins are upregulated, while secreted proteases are downregulated. Results also showed upregulation of the SaeRS TCS, suggesting that ScrA is influencing SaeRS activity. Overexpression of ScrA in a saeR mutant abrogates the clumping phenotype confirming that ScrA functions via the Sae system. Finally, we identified the ArlRS TCS as a positive regulator of scrA expression. Collectively, our results show that ScrA is an activator of the SaeRS system and suggests that ScrA may act as an intermediary between the ArlRS and SaeRS systems. [ABSTRACT FROM AUTHOR]

Background: Staphylococcus aureus is a widely distributed human pathogen capable of infecting almost every ecological niche of the host. As a result, it is responsible for causing many different diseases. S. aureus has a vast array of virulence determinants whose expression is modulated by an intricate regulatory network, where transcriptional factors (TFs) are the primary elements. In this work, using diverse sequence analysis, we evaluated the repertoire of TFs and sigma factors in the community-associated methicillin resistant S. aureus (CA-MRSA) strain USA300-FPR3757. Results: A total of 135 TFs and sigma factors were identified and classified into 36 regulatory families. From these around 43% have been experimentally characterized to date, which demonstrates the significant work still at hand to unravel the regulatory network in place for this important pathogen. A comparison of the TF repertoire of S. aureus against 1209 sequenced bacterial genomes was carried out allowing us to identify a core set of orthologous TFs for the Staphylococacceae, and also allowing us to assign potential functions to previously uncharacterized TFs. Finally, the USA300 TFs were compared to those in eleven other S. aureus strains including: Newman, COL, JH1, JH9, MW2, Mu3, Mu50, N315, RF122, MRSA252 and MSSA476. We identify conserved TFs among these strains and suggest possible regulatory interactions. Conclusions: The analysis presented herein highlights the complexity of regulatory networks in S. aureus strains, identifies key conserved TFs among the Staphylococacceae, and offers unique insights into several as yet uncharacterized TFs. [ABSTRACT FROM AUTHOR]

Abstract: Staphylococcus aureus is a highly virulent bacterial pathogen capable of causing a variety of ailments throughout the human body. It is a major public health concern due to the continued emergence of highly pathogenic methicillin resistant strains (MRSA) both within hospitals and in the community. Virulence in S. aureus is mediated by an array of secreted and cell wall associated virulence factors, including toxins, hemolysins and proteases. In this work we identify a leucine aminopeptidase (LAP, pepZ) that strongly impacts the pathogenic abilities of S. aureus. Disruption of the pepZ gene in either Newman or USA300 resulted in a dramatic attenuation of virulence in both localized and systemic models of infection. LAP is required for survival inside human macrophages and gene expression analysis shows that pepZ expression is highest in the intracellular environment. We examine the cellular location of LAP and demonstrate that it is localized to the bacterial cytosol. These results identify for the first time an intracellular leucine aminopeptidase that influences disease causation in a Gram-positive bacterium. [Copyright &y& Elsevier]

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 ppiB tig 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 aureus. [ABSTRACT FROM AUTHOR]