The major role of sarA in limiting Staphylococcus aureus extracellular protease production in vitro is correlated with decreased virulence in diverse clinical isolates in osteomyelitis

Abstract

We previously demonstrated that MgrA, SarA, SarR, SarS, SarZ, and Rot bind at least three of the four promoters associated with genes encoding primary extracellular proteases in Staphylococcus aureus (Aur, ScpA, SspA/SspB, SplA-F). We also showed that mutation of sarA results in a greater increase in protease production, and decrease in biofilm formation, than mutation of the loci encoding any of these other proteins. However, these conclusions were based on in vitro studies. Thus, the goal of the experiments reported here was to determine the relative impact of the regulatory loci encoding these proteins in vivo . To this end, we compared the virulence of mgrA , sarA , sarR , sarS , sarZ , and rot mutants in a murine osteomyelitis model. Mutants were generated in the methicillin-resistant USA300 strain LAC and the methicillin-sensitive USA200 strain UAMS-1, which was isolated directly from the bone of an osteomyelitis patient during surgical debridement. Mutation of mgrA and rot limited virulence to a statistically significant extent in UAMS-1, but not in LAC, while the sarA mutant exhibited reduced virulence in both strains. The reduced virulence of the sarA mutant was correlated with reduced cytotoxicity for osteoblasts and osteoclasts, reduced biofilm formation, and reduced sensitivity to the antimicrobial peptide indolicidin, all of which were directly attributable to increased protease production in both LAC and UAMS-1. These results illustrate the importance of considering diverse clinical isolates when evaluating the impact of regulatory mutations on virulence and demonstrate the significance of SarA in limiting protease production in vivo in S. aureus .

Increased production of aureolysin and staphopain A is a primary determinant of the reduced virulence of Staphylococcus aureus sarA mutants in osteomyelitis

“…Mutation of sarA resulted in a greater increase in protease production than mutation of the loci encoding any of these other regulatory proteins, which suggests that sarA is the major regulator of extracellular proteases and that sarA -mediated repression of protease production occurs via a direct interaction between SarA and its DNA targets ( 6 ). Moreover, we recently extended these studies to demonstrate that mutation of sarA limits the virulence of LAC and UAMS-1 in our osteomyelitis model to a greater extent than mutation of any of the loci encoding these other regulatory proteins ( 3 ).…”

“…Despite their differences, mutation of sarA limits the virulence of both strains in multiple animal models, and this attenuation can be attributed to a significant extent to the increased production of extracellular proteases ( 3 , 5 – 8 , 15 ). This is not to discount the importance of sarA as a transcriptional regulator of other S. aureus genes; indeed, eliminating the ability of sarA mutants to produce extracellular proteases did not entirely restore virulence in our osteomyelitis model, as new bone formation and cortical bone destruction values for mice infected with the protease/ sarA mutants were generally lower than those observed in mice infected with the isogenic parent strain.…”

“…Collectively, these studies demonstrate that mutation of sarA , which encodes the highly conserved transcriptional regulator SarA ( 12 ), increases expression of the genes encoding these proteases, limits the abundance of over 1,000 S . aureus proteins present in conditioned medium (CM) from overnight cultures and, more importantly, limits virulence in murine models of sepsis, implant-associated infection, and osteomyelitis ( 2 – 8 ). This work also confirmed that eliminating the production of these 10 extracellular proteases restores the abundance of most of these S. aureus proteins and increases the virulence of sarA mutants to levels comparable to the parent strain in these same animal models ( Fig.…”

“…Because mutation of sarA results in an increase in the abundance of all 10 extracellular proteases ( 2 , 6 , 7 ), it remains unknown whether specific proteases also play a primary role in the attenuation of sarA mutant osteomyelitis or any of the protease-dependent sarA mutant phenotypes identified ( 3 – 6 , 13 – 15 ). Given the large number of full-length proteins that are present in reduced amounts in CM from sarA mutants due to protease-mediated degradation ( 2 , 7 ), and based on the presumption that this includes S. aureus proteins that contribute to these phenotypes, determining which of the dysregulated proteases is responsible for attenuation could prove invaluable in identifying specific virulence factors of importance for various forms of S. aureus infection including osteomyelitis.…”

Differences in Biofilm Formation by Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Strains

“…In addition, it was shown that sarA mutants decreased PIA production and biofilm formation by downregulating ica operon transcription. Furthermore, sarA mutants showed higher protease activity beyond the downregulation of PIA synthesis [68][69][70]. It was also found in S. aureus clinical isolates amenable to genetic manipulation that biofilm formation was abolished for sarA MRSA mutants in a glucose-containing medium and for sarA MSSA mutants in a NaCl-containing medium [19].…”