Teicoplanin stress-selected mutations increasing sigma(B) activity in Staphylococcus aureus

Abstract

A natural rsbU mutant of Staphylococcus aureus, unable to activate the alternative transcription factor sigma(B) via the RsbU pathway and therefore forming unpigmented colonies, produced first-step teicoplanin-resistant mutants upon selection for growth in the presence of teicoplanin, of which the majority were of an intense orange color. By using an asp23 promoter-luciferase fusion as an indicator, the pigmented mutants were shown to express increased sigma(B) activity. Increased sigma(B) activity was associated with point mutations in rsbW, releasing sigma(B) from sequestration by the anti-sigma factor RsbW, or to promoter mutations increasing the sigma(B)/RsbW ratio. Genetic manipulations involving the sigB operon suggested that the mutations within the operon were associated with the increase in teicoplanin resistance. The upregulation of sigma(B) suggests that a sigma(B)-controlled gene(s) is directly or indirectly involved in the development of teicoplanin resistance in S. aureus. Carotenoids do not contribute to teicoplanin resistance, since inactivation of the dehydrosqualene synthase gene crtM abolished pigment formation without affecting teicoplanin resistance. The relevant sigma(B)-controlled target genes involved in teicoplanin resistance remain to be identified.

FIG. 1

Proposed model for the regulation of ς^B in S. aureus (adapted from references and 24). Based on the known functions of the RsbUVW homologues from B. subtilis (reviewed in reference 12), it is assumed that the anti-ς^B protein RsbW from S. aureus can form mutually exclusive complexes with either ς^B or its antagonist, RsbV (step 1). RsbV is normally inactive (RsbV-P) due to phosphorylation by RsbW and is thus unable to complex with RsbW, leaving the latter free to interact with ς^B (step 2). When bound to RsbW, ς^B is unable to aggregate with the RNA polymerase core enzyme (E) to form an active holoenzyme (E-ς^B). Upon stress, the RsbV-P-specific phosphatase activity of RsbU, a positive activator of ς^B, becomes activated and thus reactivates RsbV (step 3). Unphosphorylated RsbV interacts and complexes highly specifically with RsbW (step 4), thereby releasing ς^B. RsbW, if complexed with RsbV, is unable to bind to ς^B, leaving the latter free to form an active ς^B-holoenzyme (E-ς^B). Even though the exact mode of activation of RsbU in S. aureus remains unclear, there is evidence that its activation differs substantially from that of the RsbU homologue in B. subtilis.

FIG. 2

Population analysis profiles. Colonies formed from overnight cultures of the parent, MB33 (squares), and its teicoplanin first-step mutant MB128 (circles) were plated on LB agar plates containing increased concentrations of teicoplanin.

FIG. 3

Teicoplanin gradient plate. Suspensions (0.5 McFarland standard) of overnight cultures were swabbed on an LB agar plate along an antibiotic gradient as indicated. Growth was monitored after 24 h of incubation. The asterisks indicate pigmented strains. For a detailed description of the strains, refer to Table 1.

FIG. 4

ς^B activities of different S. aureus strains during growth. The expression profiles of asp23p::luc+ during growth of different S. aureus strains, grown in LB medium at 37°C, are shown. Bacterial growth was measured as the OD₆₀₀ (solid symbols). ς^B transcriptional activity was determined by measuring the luciferase activity of Luc+ (open symbols), the product of the luc+ reporter gene fused to the ς^B-dependent promoters of asp23 (asp23p). Squares, parental strain MB33 (rsbU); triangles, MB49 (MB33 rsbU⁺V⁺W⁺ sigB⁺); circles, MB130 [MB33 rsbW5(Am)]; diamonds, MB137 (MB33 sigB2).

FIG. 5

Western blot analyses of RsbW and ς^B. Cytoplasmic protein fractions (10 μg/lane) of different S. aureus strains, obtained from cells grown to an OD₆₀₀ of 1.5, were separated using sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis and blotted onto nitrocellulose. The blotted proteins were stained with amido black prior to hybridization to ensure equal loading and were subjected to Western blot analyses using either antigen-purified anti-ς^B antibodies (A) or anti-RsbW antibodies (B). The broad-range molecular-weight marker (Gibco-BRL) was used as a size marker. Relevant protein signals are indicated. For a detailed description of the strains, refer to Table 1.