sarA‐mediated repression of protease production plays a key role in the pathogenesis of Staphylococcus aureus USA300 isolates

Mutation of staphylococcal accessory regulator (sarA) results in increased production of extracellular proteases in Staphylococcus aureus, which has been correlated with decreased biofilm formation and decreased accumulation of extracellular toxins. We used murine models of implant‐associated biofilm infection and S. aureus bacteraemia (SAB) to compare virulence of USA300 strain LAC, its isogenic sarA mutant, and derivatives of each of these strains with mutations in all 10 of the genes encoding recognized extracellular proteases. The sarA mutant was attenuated in both models, and this was reversed by eliminating production of extracellular proteases. To examine the mechanistic basis, we identified proteins impacted by sarA in a protease‐dependent manner. We identified 253 proteins where accumulation was reduced in the sarA mutant compared with the parent strain, and was restored in the sarA/protease mutant. Additionally, in SAB, the LAC protease mutant exhibited a hypervirulent phenotype by comparison with the isogenic parent strain, demonstrating that sarA also positively regulates production of virulence factors, some of which are subject to protease‐mediated degradation. We propose a model in which attenuation of sarA mutants is defined by their inability to produce critical factors and simultaneously repress production of extracellular proteases that would otherwise limit accumulation of virulence factors.     

Nuclease modulates biofilm formation in community-associated methicillin-resistant Staphylococcus aureus

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging contributor to biofilm-related infections. We recently reported that strains lacking sigma factor B (sigB) in the USA300 lineage of CA-MRSA are unable to develop a biofilm. Interestingly, when spent media from a USA300 sigB mutant was incubated with other S. aureus strains, biofilm formation was inhibited. Following fractionation and mass spectrometry analysis, the major anti-biofilm factor identified in the spent media was secreted thermonuclease (Nuc). Considering reports that extracellular DNA (eDNA) is an important component of the biofilm matrix, we investigated the regulation and role of Nuc in USA300. The expression of the nuc gene was increased in a sigB mutant, repressed by glucose supplementation, and was unaffected by the agr quorum-sensing system. A FRET assay for Nuc activity was developed and confirmed the regulatory results. A USA300 nuc mutant was constructed and displayed an enhanced biofilm-forming capacity, and the nuc mutant also accumulated more high molecular weight eDNA than the WT and regulatory mutant strains. Inactivation of nuc in the USA300 sigB mutant background partially repaired the sigB biofilm-negative phenotype, suggesting that nuc expression contributes to the inability of the mutant to form biofilm. To test the generality of the nuc mutant biofilm phenotypes, the mutation was introduced into other S. aureus genetic backgrounds and similar increases in biofilm formation were observed. Finally, using multiple S. aureus strains and regulatory mutants, an inverse correlation between Nuc activity and biofilm formation was demonstrated. Altogether, our findings confirm the important role for eDNA in the S. aureus biofilm matrix and indicates Nuc is a regulator of biofilm formation.     

Staphylococcus aureus CodY negatively regulates virulence gene expression

CodY is a global regulatory protein that was first discovered in Bacillus subtilis, where it couples gene expression to changes in the pools of critical metabolites through its activation by GTP and branched-chain amino acids. Homologs of CodY can be found encoded in the genomes of nearly all low-G+C gram-positive bacteria, including Staphylococcus aureus. The introduction of a codY-null mutation into two S. aureus clinical isolates, SA564 and UAMS-1, through allelic replacement, resulted in the overexpression of several virulence genes. The mutant strains had higher levels of hemolytic activity toward rabbit erythrocytes in their culture fluid, produced more polysaccharide intercellular adhesin (PIA), and formed more robust biofilms than did their isogenic parent strains. These phenotypes were associated with derepressed levels of RNA for the hemolytic alpha-toxin (hla), the accessory gene regulator (agr) (RNAII and RNAIII/hld), and the operon responsible for the production of PIA (icaADBC). These data suggest that CodY represses, either directly or indirectly, the synthesis of a number of virulence factors of S. aureus.     

The influence of agr and sigmaB in growth phase dependent regulation of virulence factors in Staphylococcus aureus

The expression of many virulence determinants in Staphylococcus aureus is tightly coordinated generally by global regulatory elements such as accessory gene regulator (agr), staphylococcal accessory regulator and the alternative sigma factor sigmaB. We have compared the two-dimensional (2-D) protein pattern of extracellular protein extracts of wild-type cells with the 2-D patterns of the respective regulatory mutants in order to identify proteins whose amount is influenced by a mutation in agr or sigB. In order to quantify changes in the level of interesting proteins we used the Ettan-fluorescence difference gel electrophoresis technique (Amersham Biosciences). As in most bacteria, the amount of extracellular proteins was strongly regulated and increased mainly in the stationary phase of growth at high cell densities. By comparing the extracellular protein pattern of the RN6390 rsbU strain with that of an isogenic agr mutant RN6911 we show that the level of about 70 protein spots changed in the mutant. To analyze the role of sigmaB in virulence gene expression an RsbU+ (RN6390 RsbU+) derivative was included in this study. The protein pattern of the RsbU+ strain (RN6390 RsbU+) was very similar to that of the Deltaagr/DeltarsbU mutant strain (RN6911) indicating an opposing effect of agr and rsbU on the expression of the same genes.     

Staphylococcus aureus accessory regulators: expression within biofilms and effect on adhesion.

Many of the genes encoding the virulence factors for Staphylococcus aureus are controlled by the accessory gene regulator (agr) and staphylococcal accessory regulator (sar). This regulation may be affected by the environment in which the organisms are grown. In the majority of ecosystems, bacteria grow attached to surfaces and form biofilms. We used S. aureus strains containing mutations inactivating agr and sar to determine whether the presence of these genes influences the attachment of the bacterium to a surface. We also used strains harbouring reporter constructs of the agr and sar operons to determine their expression in biofilms. The attachment study results showed that the sarA mutant strain adhered better to glass than did the agrA mutant or the wild type. There was an increased adherence to fibronectin-coated glass for all three strains compared to glass. Thus, these adhesion studies demonstrate that agr and sar have pleiotrophic effects on the surface expression of molecules responsible for binding to different substrata. In the biofilms higher numbers of bacteria and the greatest expression were observed at the base, but there were no observable differences between the reporter constructs. Expression of the agr and sar reporter fusions was significantly higher in the deepest layers of the biofilms where the greatest numbers of bacteria were also observed, perhaps as one might expect for genes that are regulated in a cell density dependent fashion.     

Functional Analysis of the Ferric Uptake Regulator Gene fur in Xanthomonas vesicatoria

Iron is essential for the growth and survival of many organisms. Intracellular iron homeostasis must be maintained for cell survival and protection against iron toxicity. The ferric uptake regulator protein (Fur) regulates the high-affinity ferric uptake system in many bacteria. To investigate the function of the fur gene in Xanthomonas vesicatoria (Xv), we generated a fur mutant strain, fur-m, by site-directed mutagenesis. Whereas siderophore production increased in the Xv fur mutant, extracellular polysaccharide production, biofilm formation, swimming ability and quorum sensing signals were all significantly decreased. The fur mutant also had significantly reduced virulence in tomato leaves. The above-mentioned phenotypes significantly recovered when the Xv fur mutation allele was complemented with a wild-type fur gene. Thus, Fur either negatively or positively regulates multiple important physiological functions in Xv.