Staphylococcus aureus ClpC is required for stress resistance, aconitase activity, growth recovery, and death

Abstract

The ability of Staphylococcus aureus to adapt to various conditions of stress is the result of a complex regulatory response. Previously, it has been demonstrated that Clp homologues are important for a variety of stress conditions, and our laboratory has shown that a clpC homologue was highly expressed in the S. aureus strain DSM20231 during biofilm formation relative to expression in planktonic cells. Persistence and long-term survival are a hallmark of biofilm-associated staphylococcal infections, as cure frequently fails even in the presence of bactericidal antimicrobials. To determine the role of clpC in this context, we performed metabolic, gene expression, and long-term growth and survival analyses of DSM20231 as well as an isogenic clpC allelic-replacement mutant, a sigB mutant, and a clpC sigB double mutant. As expected, the clpC mutant showed increased sensitivity to oxidative and heat stresses. Unanticipated, however, was the reduced expression of the tricarboxylic acid (TCA) cycle gene citB (encoding aconitase), resulting in the loss of aconitase activity and preventing the catabolization of acetate during the stationary phase. clpC inactivation abolished post-stationary-phase recovery but also resulted in significantly enhanced stationary-phase survival compared to that of the wild-type strain. These data demonstrate the critical role of the ClpC ATPase in regulating the TCA cycle and implicate ClpC as being important for recovery from the stationary phase and also for entering the death phase. Understanding the stationary- and post-stationary-phase recovery in S. aureus may have important clinical implications, as little is known about the mechanisms of long-term persistence of chronic S. aureus infections associated with formation of biofilms.

FIG. 1.

A. Growth analysis of the WT and the clpC and sigB mutants. Growth curves (OD₆₀₀) of WT S. aureus DSM20231(▪), the clpC mutant (PBM001; •), the sigB mutant (MB290; ▾), and the clpC sigB double mutant (MB288; ♦) were determined in BHI medium. For analysis of the clpC-complemented strain in PBM001 (MGM001; ▴), medium was supplemented with xylose (1%). Data are means ± standard errors of the means of values obtained in three independent experiments *, P < 0.001 compared to WT (t test). B. Inactivation of clpC enhances stationary-phase survival. Single colonies of WT S. aureus DSM20231(□), the clpC mutant (PBM001; ○), the sigB mutant (MB290; ▿), and the clpC sigB double mutant (MB288; ⋄) were inoculated into BHI medium, grown at 37°C, and aerated by being shaken at 230 rpm for up to 1 week. At different intervals, aliquots were removed and CFU per milliliter were determined in triplicate. For analysis of the clpC-complemented strain in PBM001 (MGM001; ▵), medium was supplemented with xylose (1%). Data are means ± standard deviations of values obtained in two independent experiments *, P < 0.05 compared to WT (t test).

FIG. 2.

Sensitivity of clpC or sigB mutants to oxidative stress. (A and B) Cells of the WT (upper left plates), PBM001 (clpC mutant; upper right plates), MB290 (sigB mutant; lower left plates), and MB288 (clpC sigB double mutant; lower right plates) grown for 24 h (in TSB) were plated on TSA. A disk containing 10 μl of hydrogen peroxide at a concentration of either 15% (A), or 30% (B) was placed, and then plates were incubated (37°C, 18 h). (C) Fifty-milliliter cultures of the wild type and the clpC mutant were grown to an OD₆₀₀ of 0.1. The cultures were split, 7.5 mM of hydrogen peroxide was added to one half, and then both halves were incubated (37°C). Shown are OD₆₀₀ values obtained at the indicated time points (▪, WT without H₂O₂; ▴, WT with H₂O₂; •, PBM001 without H₂O₂; ▾, PBM001 with H₂O₂). Shown are representative results of at least two independent experiments.

FIG. 3.

Growth and clp gene expression after heat shock. (A to D) Cultures of the WT (1), PBM001 (2), MB290 (3), or MB288 (4) were grown to the exponential phase in TSB at 37°C. At an OD₆₀₀ of 0.25 to 0.30, cultures were diluted 10⁻¹, 10⁻², 10⁻³, and 10⁻⁴-fold, and 10 μl of each dilution (top to bottom, respectively) was spotted on TSA plates. The plates were then incubated at 37°C (A), 44°C (B), 46°C (C), and 47°C (D) for 24 h. (E) Real-time RT-PCR quantification of clp gene expression after heat shock at 55°C for 15 min. clpC, clpP, and clpB mRNA concentrations in the WT (DSM 20231) and the clpC mutant (PBM001) after heat shock at 0 (□), 5 (▨), and 30 (▪) min were determined as described in Materials and Methods. Shown are transcript quantities relative to the control (gyrB) transcript, expressed as fold increase. Data are means ± standard deviations of triplicate determinations.

FIG. 4.

A. Determination of acetate in the culture supernatant. After the indicated time intervals, supernatants of the WT (▪), PBM001 (•), MGM001 (▴), MB290 (▾), and MB288 (♦), cultivated in BHI medium, were analyzed for acetate concentration as described in Materials and Methods. Shown are representative results of at least two independent experiments. B. Expression of acetyl-CoA synthetase (acsA) in the WT, PBM001, and MB290. Gene expression of acsA was determined in the WT, PBM001 (clpC mutant), and MB290 (sigB mutant) by real-time RT-PCR at different time intervals as described in the legend to Fig. 3.

FIG. 5.

Expression of citB, asp23, and clpC. Gene expression in the WT (A) and the clpC mutant PBM001 (B) was determined by real-time RT-PCR as described in the legend to Fig. 3. Expression of clpC in PBM001 was not determined.

FIG. 6.

Aconitase activity of the WT and clpC or sigB mutants. The WT (▪), PBM001 (•), MGM001(▴), MB290 (▾), and MB288 (♦) were grown as described in the legend to Fig. 1 for 24 h, 48 h, 72 h, and 96 h. Aconitase activities were determined as described in Materials and Methods in triplicate analysis. Results are means ± standard errors from two independent experiments.

FIG. 7.

Hypothetical model of the role of ClpC in oxidative metabolism of S. aureus. During exponential growth in rich medium, glucose is rapidly catabolized and finally accumulates as acetate in the medium. During late stationary phase (such as in biofilm populations), the bacterial cell yields free ATP primarily by oxidative metabolism provided by TCA cycle activity. The function of the key TCA cycle enzyme aconitase requires ClpC, since functional deletion of clpC results in a marked reduction in citB (aconitase) transcription along with loss of aconitase activity and persistent acetate accumulation. Under oxidative stress conditions, ClpC might be restoring/activating a yet-unknown activator(s) of citB, which in turn encodes active aconitase, and/or activating/restoring the activity of aconitase protein. This confers post-stationary-phase growth followed by entry into death phase. Free σ^B also seems to cooperate in activating/restoring the aconitase activity, through yet-unknown pathways.