VraCP regulates cell wall metabolism and antibiotic resistance in vancomycin-intermediate Staphylococcus aureus strain Mu50

Abstract

Objectives: Vancomycin-intermediate Staphylococcus aureus (VISA) is increasingly being reported. Previous studies have shown that vraC and vraP may be involved in vancomycin resistance, although the molecular mechanism remains elusive.

Methods: The vraC (SAV0577), vraP (SAV0578) and vraCP mutants were constructed in Mu50 by allelic replacement. Some common VISA phenotypes were assessed in mutants, such as, susceptibility to the cell wall-associated antibiotics, cell wall thickness, autolysis activity and growth rate. RT-qPCR was performed to reveal the differential genes associated with these phenotypes. The binding abilities of VraC and VraCP to the promoters of target genes were determined by electrophoretic mobility shift assay (EMSA).

Results: VraP forms a stable complex with VraC to preserve their own stability. The vraC, vraP and vraCP mutants exhibited increased susceptibility to the cell wall-associated antibiotics and thinner cell walls compared with the WT strain. Consistent with these phenotypes, RT-qPCR revealed downregulated transcription of glyS, sgtB, ddl and alr2, which are involved in cell wall biosynthesis. Moreover, the transcription of cell wall hydrolysis genes, including sceD, lytM and isaA, was significantly downregulated, supporting the finding that mutants exhibited reduced autolysis rates. EMSA confirmed that both VraC and VraCP can directly bind to the sceD, lytM and isaA promoter regions containing the consensus sequence (5'-TTGTAAN2AN3TGTAA-3'), which is crucial for the binding of VraCP with target genes. GFP-reporter assays further revealed VraC and VraCP can enhance promoter activity of sceD to positively regulate its expression.

Conclusions: vraCP plays a significant role in cell wall metabolism and antibiotic resistance in Mu50.

Figure 1.

vraC and vraP are co-transcribed and induced by vancomycin treatment. (a) Schematic of the vraCP locus in S. aureus Mu50 and the positions of primers used in the PCR assay. The black arrows indicate the genes we studied, and the grey arrows indicate neighbouring genes. (b) Gel electrophoresis analysis of PCR products amplified with primers spanning fragments of vraP and vraCP. Genomic DNA, reverse-transcription cDNA and the same amount of total RNA from strain Mu50 were used as templates. G, Genomic DNA; C, reverse-transcription cDNA; R, RNA. (c) Transcription of vraC and vraP is growth-phase dependent. (d) Transcription of vraC and vraP was induced by vancomycin treatment. (e) Protein electrophoresis of VraC, VraC and VraCP by SDS-PAGE.

Figure 2.

Growth curves of S. aureus Mu50 and its derivative strains in different concentrations of vancomycin. Growth of the WT strain (a), the vraCP mutant (b), the vraC mutant (c), the vraP mutant (d) and the vraCP complemented strain (e) in MH broth at 37°C containing 0, 1, 2 and 3 mg/L of vancomycin.

Figure 3.

Population analysis profiles of S. aureus Mu50 and its derivative strains. The vancomycin (a), daptomycin (b), teicoplanin (c) and oxacillin (d) resistance levels were evaluated by population analysis profiles. The colonies were counted after incubation at 37°C for 48 h.

Figure 4.

Transmission electron micrographs of cell walls of S. aureus Mu50 and its derivative strains. TEM of the WT strain (a), the vraCP mutant (b), the vraC mutant (c), the vraP mutant (d) and the vraCP complemented strain (e) at the early exponential phase. (f) Comparison of cell wall thickness between Mu50 and its derivative strains. Data are expressed as the mean ± SD for 15 cells of each strain. (g) Transcriptional analysis of cell wall biosynthesis-related genes. The transcriptional levels of glys, sgtB, ddl and alr2 significantly decreased in the vraC, vraP and vraCP mutants compared with that of Mu50.

Figure 5.

The vraC, vraP and vraCP mutants displayed decreased autolysis rate. (a) 0.2% Triton X-100-induced autolysis assay showed that the vraC, vraP and vraCP mutants exhibited reduced autolysis rates than the WT strain. (b) Genes involved in cell wall degradation, such as sceD, lytM and isaA, have decreased expression in the vraC, vraP and vraCP mutants compared with that of Mu50. (c) SDS-PAGE analysis of purified SceD, LytM and IsaA proteins. (d) Zymographic analyses were performed to confirm cell wall hydrolytic activity of SceD, LytM and IsaA proteins. Purified SceD, LytM and IsaA proteins were separated by 15% SDS-PAGE with 2 mg/L S. aureus purified peptidoglycan. (e) Promoter PsceD shows dramatically weaker activity in the vraC, vraP and vraCP mutants compared with that of WT strain. Promoter activities of strains harbouring a transcriptional fusion of PsceD with GFP (pPsceD-GFP) were assessed at different growth phases by measuring fluorescence as well as the OD₆₀₀. Depicted is GFP emission normalized to the OD₆₀₀ (GFP/OD₆₀₀).

Figure 6.

EMSA of VraCP or VraC with the biotin-labelled promoters PsceD, PlytM and PisaA. The promoter regions of sceD, lytM and isaA were amplified by PCR, and incubated with purified VraCP (a–c) and VraC (d–f), respectively. The unlabelled probes were used as the specific competitors, and the unlabelled partial fragment of hu ORF region was used as the non-specific competitor.

Figure 7.

The consensus sequence (5′-TTGTAAN₂AN₃TGTAA-3′) plays an important role in the binding of VraCP with target genes. (a)The consensus sequence (5′-TTGTAAN₂AN₃TGTAA-3′) in promoter regions of sceD, lytM and isaA was identified by sequences alignment. The locations of PsceD, PlytM and PisaA truncated probes with different lengths are marked by black arrows. The partial relative dispositions of sequence are illustrated in the lower panel. The consensus sequences (5′-TTGTAAN₂AN₃TGTAA-3′) are marked in red, the two pentanucleotide direct repeats (TGTAA) are highlighted in yellow (b–d). EMSA analysis of VraCP with PsceD, PlytM and PisaA truncated probes (e–g).

Figure 8.

VraCP is involved in cell wall metabolism and antibiotic resistance in vancomycin-intermediate S. aureus strain Mu50. VraSR can induce the expression of vraCP to response to vancomycin treatment. VraCP promotes the expression of both cell wall synthesis genes (glyS, sgtB, ddl and alr2) and hydrolysis genes (sceD, lytM and isaA), and thus influences cell wall thickness, antibiotic resistance and autolysis rate.