Induction of fibronectin adhesins in quinolone-resistant Staphylococcus aureus by subinhibitory levels of ciprofloxacin or by sigma B transcription factor activity is mediated by two separate pathways

Abstract

We recently reported on the involvement of a RecA-LexA-dependent pathway in the ciprofloxacin-triggered upregulation of fibronectin-binding proteins (FnBPs) by fluoroquinolone-resistant Staphylococcus aureus. The potential additional contribution of the transcription factor sigma B (SigB) to the ciprofloxacin-triggered upregulation of FnBPs was studied in isogenic mutants of fluoroquinolone-resistant strain RA1 (a topoisomerase IV gyrase double mutant of S. aureus NCTC strain 8325), which exhibited widely different levels of SigB activity, as assessed by quantitative reverse transcription-PCR of their respective sigB and SigB-dependent asp23 transcript levels. These mutants were Tn551 insertion sigB strain TE1 and rsbU(+) complemented strain TE2, which exhibited a wild-type SigB operon. Levels of FnBP surface display and fibronectin-mediated adhesion were lower in sigB mutant TE1 or higher in the rsbU(+)-restored strain TE2 compared to their sigB(+) but rsbU parent, strain RA1, exhibiting low levels of SigB activity. Steady-state fnbA and fnbB transcripts levels were similar in strains TE1 and RA1 but increased by 4- and 12-fold, respectively, in strain TE2 compared to those in strain RA1. In contrast, fibronectin-mediated adhesion of strains TE1, RA1, and TE2 was similarly enhanced by growth in the presence of one-eighth the MIC of ciprofloxacin, which led to a significantly higher increase in their fnbB transcript levels compared to the increase in their fnbA transcript levels. Increased SigB levels led to a significant reduction in agr RNAIII; in contrast, it led to a slight increase in sarA transcript levels. In conclusion, upregulation of FnBPs by increased SigB levels and ciprofloxacin exposure in fluoroquinolone-resistant S. aureus occurs via independent pathways whose concerted actions may significantly promote bacterial adhesion and colonization.

FIG. 1.

Adhesion to fibronectin-coated coverslips (A) or binding of soluble FITC-labeled fibronectin (B) of sigB null strain TE1 and rsbU⁺-restored strain TE2 grown in either the absence or the presence of 4 μg of ciprofloxacin (CFX) per ml (one-eighth the MIC) expressed as the percentages of adhesion (A) or fibronectin binding (B) for strain RA1 in ciprofloxacin-free medium. Values represent means + SEMs (error bars) of individual adhesion data accumulated over the three fibronectin coating concentrations in three experiments. *, results significantly different (P < 0.01) for each strain grown in ciprofloxacin-containing MHB from those for strains grown in ciprofloxacin-free MHB; **, results significantly different (P < 0.01) from those for strain RA1 grown in ciprofloxacin-free medium.

FIG. 2.

Steady-state levels of the fnbA (left) and fnbB (right) transcripts of strains TE1 and TE2 grown in the absence or the presence of 4 μg of ciprofloxacin (CFX) per ml (one-eighth the MIC) expressed as the percentages of those of strain RA1 grown in ciprofloxacin-free medium. mRNA levels were determined by real-time RT-PCR and were normalized on the basis of their 16S rRNA levels. Values represent the means + SEMs of three experiments performed in triplicate. *, results significantly different (P < 0.01) for each strain grown in ciprofloxacin-containing MHB compared to those for the strain grown in ciprofloxacin-free MHB; **, results significantly different (P < 0.01) from those for strain RA1 grown in ciprofloxacin-free medium.

FIG. 3.

Steady-state mRNA levels of the sigB (left) and asp23 (right) genes of strain TE2 grown in the absence or the presence of 4 μg of ciprofloxacin (CFX) per ml (one-eighth the MIC) expressed as the percentages of those of strain RA1 grown in ciprofloxacin-free medium. mRNA levels were determined by real-time RT-PCR and were normalized on the basis of their 16S rRNA levels. Values are the means + SEMs of three experiments performed in triplicate. *, results significantly different (P < 0.05) for each strain grown in ciprofloxacin-containing MHB compared to those for the strain grown in ciprofloxacin-free MHB; **, results significantly different (P < 0.01) from those for strain RA1 grown in ciprofloxacin-free medium. The asp23 transcripts levels of strain TE1 were too small to be visualized (see text).

FIG. 4.

(A) Steady-state levels of RNAII and RNAIII of strains TE1 and TE2 grown in the absence or the presence of 4 μg of ciprofloxacin (CFX) per ml (one-eighth the MIC) expressed as the percentages of those of strain RA1 grown in ciprofloxacin-free medium. mRNA levels were determined by real-time RT-PCR and were normalized on the basis of their 16S rRNA levels. Values represent the means + SEMs of three experiments performed in triplicate. *, results significantly different (P < 0.05) for each strain grown in ciprofloxacin-containing MHB compared to those for the strain grown in ciprofloxacin-free MHB; **, results significantly different (P < 0.05) from those for strain RA1 grown in ciprofloxacin-free medium. (B) Northern blot analysis of RNAIII in strains RA1, TE1, and TE2.

FIG. 5.

Steady-state levels of the sarA transcripts of strains TE1 and TE2 grown in the absence or the presence of 4 μg of ciprofloxacin (CFX) per ml (one-eighth the MIC) expressed as the percentages of those of strain RA1 grown in ciprofloxacin-free medium. mRNA levels were determined by real-time RT-PCR and were normalized on the basis of their 16S rRNA levels. Values represent the means + SEMs of three experiments performed in triplicate. *, results significantly different (P < 0.05) for each strain grown in ciprofloxacin-containing MHB compared to those for the strain grown in ciprofloxacin-free MHB; **, results significantly different (P < 0.05) from those for strain RA1 grown in ciprofloxacin-free medium.

FIG. 6.

Steady-state levels of the hla and spa transcripts of strains TE1 and TE2 grown in the absence or the presence of 4 μg of ciprofloxacin (CFX) per ml (one-eighth the MIC) expressed as the percentages of those of strain RA1 grown in ciprofloxacin-free medium. mRNA levels were determined by real-time RT-PCR and were normalized on the basis of their 16S rRNA levels. Values represent the means + SEMs of three experiments performed in triplicate. *, results significantly different (P < 0.01) for each strain grown in ciprofloxacin-containing MHB compared to those for the strain grown in ciprofloxacin-free MHB; **, results significantly different (P < 0.05) from those for strain RA1 grown in ciprofloxacin-free medium.