Whole genome sequencing and complete genetic analysis reveals novel pathways to glycopeptide resistance in Staphylococcus aureus

Abstract

The precise mechanisms leading to the emergence of low-level glycopeptide resistance in Staphylococcus aureus are poorly understood. In this study, we used whole genome deep sequencing to detect differences between two isogenic strains: a parental strain and a stable derivative selected stepwise for survival on 4 µg/ml teicoplanin, but which grows at higher drug concentrations (MIC 8 µg/ml). We uncovered only three single nucleotide changes in the selected strain. Nonsense mutations occurred in stp1, encoding a serine/threonine phosphatase, and in yjbH, encoding a post-transcriptional negative regulator of the redox/thiol stress sensor and global transcriptional regulator, Spx. A missense mutation (G45R) occurred in the histidine kinase sensor of cell wall stress, VraS. Using genetic methods, all single, pairwise combinations, and a fully reconstructed triple mutant were evaluated for their contribution to low-level glycopeptide resistance. We found a synergistic cooperation between dual phospho-signalling systems and a subtle contribution from YjbH, suggesting the activation of oxidative stress defences via Spx. To our knowledge, this is the first genetic demonstration of multiple sensor and stress pathways contributing simultaneously to glycopeptide resistance development. The multifactorial nature of glycopeptide resistance in this strain suggests a complex reprogramming of cell physiology to survive in the face of drug challenge.

Figure 1. Genomic differences detected by whole genome sequencing.

(A) Chromosomal location of detected SNPs and predicted protein changes. (B) Physical map of each locus containing yjbH(K23stop), stp1(Q12stop) and vraS(G45R). For genetic analysis, each mutation was tagged by site-specific insertion of a nearby selectable marker to facilitate strain constructions using bacteriophage-mediated transduction.

Figure 2. Stepwise genetic reconstitution of all SNP permutations and analysis of their contribution to glycopeptide resistance.

(A and B) Teicoplanin spot plating population analysis (Spot PAP) on teicoplanin of ISP794 and each of its strain derivatives. Spot serial dilutions are indicated at the right margin. The first spot 10 µl corresponds to 1×10⁵ colony forming units (CFU). Results for two independent isolates are shown for each single mutant (solid bar). For convenience, genes are marked with an asterisk such that vraS*, stp1* and yjbH* correspond to vraS(G45R), stp1(Q12stop) and yjbH(K23stop) mutations, respectively. (C) Vancomycin spot plating population analysis (Spot PAP) of ISP794 and each of its strain derivatives. Conditions used were as in panels A, B.

Figure 3. Cell wall thickness analysis.

(A) Transmission electron microscopy showing one representative image of each bacterial strain used in this study, after growth in the absence of antibiotic to post-exponential phase on TSB media. Magnification ×37,000. Scale bar automatically inserted by the microscope imaging system is shown. (B) Quantification of cell wall thickness (nm) of each bacterial strain. Reported values correspond to the mean ± SD (n = at least 40) of each bacterial strain. Asterisk (*) represents results significantly different by student's two-tailed t-test (p<0.05) from ISP794.

Figure 4. Western blot showing the effect of yjbH(K23stop) mutation on Spx protein levels.

Total soluble protein extracts (50 µg) from S. aureus strains were loaded per well in an SDS 15% acrylamide gel. Spx protein (15 kDa) was detected using rabbit-polyclonal anti-Spx antibodies. Purified-Spx protein (600 ng) (lane 1), Δspx strain (lane 2), ISP794 containing wild-type yjbH gene (lane 3), yjbH* correspond to yjbH(K23stop) mutant (lane 4), SaΔclpP strain (lane 5) and AR376 (lane 6). The position of protein markers are shown in the right margin.

Figure 5. Effect of spx on msrA1 transcription.

(A) Steady-state levels of msrA1 transcript of wild type strain (spx ⁺) compared to mutant Δspx strain, determined by qRT-PCR. Values represent the mean ± SEM of four independent experiments performed in triplicate. Data are depicted setting spx ⁺ values in the absence of teicoplanin as 100%. Asterisk (*) represents results significantly different by student's two-tailed t-test (p<0.05) from spx ⁺ without teicoplanin. (B) Steady-state levels of msrA1 transcript of ISP794 containing wild-type yjbH gene, AR376 and yjbH* containing both yjbH(K23stop) mutation, determined by qRT-PCR. Values represent the mean ± SEM of three independent experiments performed in triplicate. Data are depicted setting ISP794 values as 100%. Asterisk (*) represents results significantly different by student's two-tailed t-test (p<0.05) from ISP794.