The Staphylococcus aureus Thiol/Oxidative Stress Global Regulator Spx Controls trfA , a Gene Implicated in Cell Wall Antibiotic Resistance

Abstract

S. aureus combats cell wall antibiotic stress by altered gene expression mediated by various environmental signal sensors. In this study, we examined the transcriptional regulation of trfA, a gene related to mecA of Bacillus subtilis encoding an adaptor protein implicated in multiple roles, notably, proteolysis and genetic competence. Despite strong sequence similarity to B. subtilis mecA, the function of S. aureus trfA remains largely unexplored; however, its deletion leads to almost complete loss of resistance to oxacillin and glycopeptide antibiotics in glycopeptide-intermediate S. aureus (GISA) derivatives of methicillin-susceptible or methicillin-resistant S. aureus (MRSA) clinical or laboratory isolates. Northern blot analysis and 5= rapid amplification of cDNA ends (RACE) mapping revealed that trfA was expressed monocistronically by three promoters. Cell wall-active antibiotic exposure led to both increased trfA transcription and enhanced steady-state TrfA levels. trfA promoter regulation was not dependent upon the cell wall stress sentinel VraSR and other sensory stress systems, such as GraRS, WalkRK, Stk1/Stp1, and SigB. Notably, we discovered that the global oxidative-stress regulator Spx controlled trfA transcription. This finding was also confirmed using a strain with enhanced Spx levels resulting from a defect in yjbH, encoding a Spx-interacting protein governing Spx proteolytic degradation. A cohort of clinical GISA strains revealed significant steady-state upregulation of trfA compared to corresponding susceptible parental strains, further supporting a role for trfA in antibiotic resistance. These data provide strong evidence for a link between cell wall antibiotic stress and evoked responses mediated by an oxidative-stress sensor.

Role of Adaptor TrfA and ClpPC in Controlling Levels of SsrA-Tagged Proteins and Antitoxins in Staphylococcus aureus

“…trfA is transcribed from three promoters, generating transcripts of closely related sizes of 751, 818, and 898 bases, which can be seen as a single transcript in a 0.7% agarose gel (28). The changes to trfA transcription during various growth phases remain unclear, however.…”

“…Previous studies of spx transcription by RT-PCR have shown that the gene product of spx likely activates one or more of these transcripts of trfA (28). Spx is a thiol-sensitive transcriptional regulator that is itself proteolytically regulated and is responsible for controlling the adaptation and survival of S. aureus in environments with oxidative stress.…”

“…trfA has also been shown to be responsive to certain stresses (28). Reverse transcription-PCR (RT-PCR) analysis showed that one or more of the trfA transcripts were elevated following antibiotic or thiol stress.…”

“…On the basis of homology to adaptors identified in Bacillus subtilis, there are at least three adaptors in S. aureus, namely, McsB (SA0482), YjbH (SA0860), and TrfA (SA0857). Studies have indicated the involvement of each in various stress responses (27)(28)(29), but many gaps remain in our understanding of the various roles played by these adaptors in S. aureus. Details of the protein targets of these S. aureus adaptors are limited, and to date, only two target proteins have been described to be regulated by S. aureus adaptors.…”

Stepwise Decrease in Daptomycin Susceptibility in Clinical Staphylococcus aureus Isolates Associated with an Initial Mutation in rpoB and a Compensatory Inactivation of the clpX Gene

“…Spx controls gene expression by interacting directly with the ␣ subunit of the RNA polymerase (45). The Spx-controlled trfA gene was recently shown to be required for the ability of subinhibitory concentrations of ␤-lactam antibiotics to induce the hVISA phenotype, raising the intriguing possibility that the elevated Spx level may contribute to the decreased susceptibility to daptomycin, vancomycin, and ␤-lactams (46,47). Unfortunately, attempts to examine this hypothesis by introducing an spx mutation into single mutants were unsuccessful.…”