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.

Fig 1

Analysis of trfA mRNA levels in glycopeptide-susceptible and GISA paired clinical strains. Steady-state levels of trfA transcripts were determined by qRT-PCR and normalized to 16S rRNA. All GISA mRNA levels (black bars) were compared to the corresponding glycopeptide-susceptible strains (white bars). The values represent the means and standard errors of the mean (SEM) of three independent experiments. *, results significantly different by Student's two-tailed t test (P < 0.05).

Fig 2

Induction of trfA mRNA levels by cell wall-active antibiotics. Steady-state mRNA levels of trfA and hu (control) were determined by qRT-PCR and normalized to 16S rRNA. The mRNA levels of antibiotic-treated bacteria were compared to that of strain ISP794 in the absence of antibiotic addition. oxa, oxacillin; van, vancomycin; tec, teicoplanin; D-cyclo, d-cycloserine; Cip, ciprofloxacin. The values reported represent the means and SEM of at least three independent experiments. *, results significantly different by Student's two-tailed t test (P < 0.05). Note the absence of trfA induction by the non-cell-wall-targeting drug ciprofloxacin.

Fig 3

Western blot analysis of TrfA. Total soluble protein extracts (75 μg) from E. coli and S. aureus strains were loaded in SDS 15% acrylamide gels. TrfA protein (30 kDa) was detected using rabbit-polyclonal anti-peptide-TrfA antibodies (see Materials and Methods), and a typical Western blot is shown. As reference controls, an aliquot of a whole-cell extract of IPTG-induced S. aureus TrfA produced in E. coli (lane 1 from left) and an extract from an ISP794 control strain lacking trfA (lane 6) were included. Lanes 2 and 3, respectively, compare extracts derived from ISP794 or it isogenic Tei^r derivative. Lanes 4 and 5 show the results for ISP794 and ISP4-2-1, each exposed to 1 μg/ml of oxacillin (lane 4 and 5). The position of the 30-kDa protein marker is shown on the right.

Fig 4

Transcriptional analysis of trfA. (A) Northern blot analysis of trfA and spx in strains ISP794 and ISP4-2-1, using ³²P-radiolabeled RNA trfA- and spx-specific probes. The arrows indicate the trfA (0.7-kb, 0.8-kb, and 0.9-kb) and spx (0.6-kb and 0.5-kb) transcripts. Ethidium bromide-stained rRNA from the agarose gel prior to blot transfer is shown as a loading control. (B) Schematic representation of B. subtilis (B.s.) MecA and S. aureus (S.a.) TrfA/MecA proteins. Throughout the text, we refer to S. aureus TrfA to avoid confusion with S. aureus mecA, a gene unrelated to the MecA adaptor protein of B. subtilis and encoding an alternative penicillin binding protein responsible for the MRSA phenotype in the organism. The N-terminal and C-terminal protein regions are depicted in black and gray and show 57% and 31% protein identity, respectively. The linker region (white) is smaller in B. subtilis MecA. (C) Sequence of the trfA promoter region. The three different nucleotides corresponding to transcriptional start sites detected by 5′RACE are shown in boldface, and the rho-independent transcriptional terminator of spx is underlined. RBS, ribosome binding site. Asterisks mark each 10-nucleotide region. (D) Schematic diagram showing spx and trfA gene transcription organization. The arrows indicate spx or trfA transcripts produced from the corresponding promoters. Predicted rho-independent transcriptional terminators are shown.

Fig 5

Analysis of vraS* (vraSG45R), stp1* (stp1Q12stop), yjbH* (yjbHK23stop), and spx on trfA mRNA levels. Strains harboring each of the three nucleotide changes detected in strain ISP4-2-1 compared to ISP794 (10) were used to determine which mutation(s) conferred enhanced trfA expression in strain ISP4-2-1. Steady-state levels of trfA transcripts were determined by qRT-PCR and normalized to 16S rRNA. Steady-state levels of trfA were also compared between the Δspx mutant and its spx⁺ restored derivative (Δspx-c), as well as between ISP794 treated with the thiol-specific oxidant diamide and untreated ISP794. The values represent the means and SEM of three independent experiments. *, results significantly different by Student's two-tailed t test (P < 0.05).

Fig 6

Effect of oxacillin on spx mRNA levels. (A) Steady-state levels of spx and hu transcripts were determined by qRT-PCR and normalized to 16S rRNA in strain ISP794 compared to oxacillin-treated (oxa) ISP794 cells. The values represent the means and SEM of three independent experiments. (B) Western blot analysis of total soluble protein extracts (75 μg) of S. aureus ISP794 or the same strain treated with oxacillin loaded in SDS-15% acrylamide gels. Spx protein (13 kDa) was detected using rabbit polyclonal anti-S. aureus Spx antibody as previously described (10).

Fig 7

Model of proposed pathways regulating the evoked response to cell wall antibiotic encounter. Stk1/Stp, serine/threonine kinase-phosphatase sensor; RNAP, RNA polymerase. The dashed arrow denotes the presumptive pathway leading to Spx protein stabilization.