Site-specific mutation of Staphylococcus aureus VraS reveals a crucial role for the VraR-VraS sensor in the emergence of glycopeptide resistance

Abstract

An initial response of Staphylococcus aureus to encounter with cell wall-active antibiotics occurs by transmembrane signaling systems that orchestrate changes in gene expression to promote survival. Histidine kinase two-component sensor-response regulators such as VraRS contribute to this response. In this study, we examined VraS membrane sensor phosphotransfer signal transduction and explored the genetic consequences of disrupting signaling by engineering a site-specific vraS chromosomal mutation. We have used in vitro autophosphorylation assay with purified VraS[64-347] lacking its transmembrane anchor region and tested site-specific kinase domain histidine mutants. We identified VraS H156 as the probable site of autophosphorylation and show phosphotransfer in vitro using purified VraR. Genetic studies show that the vraS(H156A) mutation in three strain backgrounds (ISP794, Newman, and COL) fails to generate detectable first-step reduced susceptibility teicoplanin mutants and severely reduces first-step vancomycin mutants. The emergence of low-level glycopeptide resistance in strain ISP794, derived from strain 8325 (ΔrsbU), did not require a functional σ(B), but rsbU restoration could enhance the emergence frequency supporting a role for this alternative sigma factor in promoting glycopeptide resistance. Transcriptional analysis of vraS(H156A) strains revealed a pronounced reduction but not complete abrogation of the vraRS operon after exposure to cell wall-active antibiotics, suggesting that additional factors independent of VraS-driven phosphotransfer, or σ(B), exist for this promoter. Collectively, our results reveal important details of the VraRS signaling system and predict that pharmacologic blockade of the VraS sensor kinase will have profound effects on blocking emergence of cell wall-active antibiotic resistance in S. aureus.

FIG. 1.

Schematic diagram of VraS and VraR highlighting their domain architecture and the location of amino acids mutated in the present study. The predicted transmembrane region (TM), histidine kinase (HisKA), and histidine kinase ATP-binding domain (HATPase) are shown. VraS_cyt indicates the point of truncation used for the purification of the cytoplasmic and soluble portion of the protein used in the present study. The VraR response regulator is depicted and shows the receiver domain and position of phosphorylated aspartate, along with the C-terminal DNA-binding domain. The sequence alignment shows the H-box sequence context of VraS-H156, together with other conserved motifs within the ATP-binding domain. Sequences used were from Swiss-Prot accession: Staphylococcus aureus VraS, Q99SZ7; Bacillus subtilis LiaS, O32198; Escherichia coli UhpB, P09835; and Haemophilus influenzae NarQ, P44604.

FIG. 2.

Purified VraR and VraS and their indicated mutant derivatives used in the present study. Proteins were resolved on 12% polyacrylamide-SDS gels and stained with Coomassie brilliant blue. Molecular mass standards are indicated in kilodaltons for the marker ladder.

FIG. 3.

In vitro autophosphorylation and phosphotransfer assay using purified VraS and VraR. (A) VraS autophosphorylation time course assay in the presence of [γ-³²P]ATP. Reactions were assembled for the indicated times and applied to 12% polyacrylamide-SDS gels, dried, and autoradiographed. Wild-type VraS lacking the N-terminal transmembrane domain VraS_cyt and two purified histidine mutants, H156A and H238A, are shown. (B) VraS-VraR phosphotransfer assay. Autophosphorylation reactions were performed as described for panel A, and then VraR was added. Aliquots were removed at the indicated times and resolved on SDS-protein gels as described above. Note that a minor phosphorylated degradation product of VraS migrates at a position above phosphorylated VraR. S, VraS; R, VraR.

FIG. 4.

Schematic showing the various genetic steps used for the construction of a chromosomal mutant encoding vraS(H156A) and marked with a nearby kanamycin resistance cassette in the VraR-SA1699 (N315 ordered sequence tag numbering) intergenic region. A second strain harboring only the kanamycin resistance marker, but otherwise wild type for the entire VraSR four-gene operon, was designed in parallel. A thermosensitive shuttle plasmid, pAR747, was introduced into AR756. The correct double-crossover event yielded the desired vraS-H156 mutation by allelic exchange. The vraS(H156A) allele was then backcrossed into strain ISP794 by bacteriophage-mediated transduction using selection for the tightly linked nearby kanamycin marker to give AR828. The entire operon and upstream promoter sequences were completely sequence verified. Arrows indicate the transcription direction. Kpn and Pst denote the positions of the restriction sites used for the construction of pAR747 targeting vector.

FIG. 5.

Transcriptional analysis showing the effects of uncoupling of VraS phosphotransfer. (A) Northern blot analysis showing strong oxacillin-induced induction of vraRS operon transcription in wild-type cells but severe reduction in the vraS(H156A) mutant. Note that the vraRS operon is autoregulated. The 2.7-kb band encodes the entire four-gene operon. Estimated lengths of additional transcripts are indicated that may represent strong pause sites or partial transcript degradation. Ethidium bromide-stained 16S and 23S rRNAs are shown as loading controls. (B) Quantitation of VraR mRNA levels by qRT-PCR analysis showing that oxacillin-stimulated transcriptional induction of the vraRS operon is severely reduced but not completely abolished by vraS(H156A). An asterisk denotes Student two-tailed t test analysis of transcript levels in the presence or absence of oxacillin (P < 0.05) from three independent determinations.