Global regulatory impact of ClpP protease of Staphylococcus aureus on regulons involved in virulence, oxidative stress response, autolysis, and DNA repair

Abstract

Staphylococcus aureus is an important pathogen, causing a wide range of infections including sepsis, wound infections, pneumonia, and catheter-related infections. In several pathogens ClpP proteases were identified by in vivo expression technologies to be important for virulence. Clp proteolytic complexes are responsible for adaptation to multiple stresses by degrading accumulated and misfolded proteins. In this report clpP, encoding the proteolytic subunit of the ATP-dependent Clp protease, was deleted, and gene expression of DeltaclpP was determined by global transcriptional analysis using DNA-microarray technology. The transcriptional profile reveals a strong regulatory impact of ClpP on the expression of genes encoding proteins that are involved in the pathogenicity of S. aureus and adaptation of the pathogen to several stresses. Expression of the agr system and agr-dependent extracellular virulence factors was diminished. Moreover, the loss of clpP leads to a complete transcriptional derepression of genes of the CtsR- and HrcA-controlled heat shock regulon and a partial derepression of genes involved in oxidative stress response, metal homeostasis, and SOS DNA repair controlled by PerR, Fur, MntR, and LexA. The levels of transcription of genes encoding proteins involved in adaptation to anaerobic conditions potentially regulated by an Fnr-like regulator were decreased. Furthermore, the expression of genes whose products are involved in autolysis was deregulated, leading to enhanced autolysis in the mutant. Our results indicate a strong impact of ClpP proteolytic activity on virulence, stress response, and physiology in S. aureus.

FIG. 1.

(A) Growth kinetics of S. aureus 8325 wild-type (♦), 8325ΔclpP (▪), and 8325ΔclpP⁺ (▴) strains grown at 37°C, 30°C, 20°C, 42°C, and 45°C. The results are representative of three independent experiments. (B) Northern blot analysis of clpP transcription in S. aureus 8325 at various temperatures (left) and at various time points during the growth phase (at indicated OD₆₀₀ values) at 37°C.

FIG. 2.

Scanning electron microscopy of strain 8325 wild-type (A and B) and the isogenic ΔclpP mutant (C and D). Cells of the ΔclpP strain show a rougher and more irregular surface and decreased cell size than the wild-type strain. Preparation of samples was performed as described in Materials and Methods.

FIG. 3.

Transcriptional analysis of selected genes in 8325 wild-type (lane 1), ΔclpP (lane 2), and ΔclpP⁺ (lane 3) strains. RNA was isolated from exponentially growing cells (OD₆₀₀ of 1.0). (A) Northern blot analysis of RNAIII expression by hybridization with an RNAIII-specific probe. (B) Semiquantitative RT-PCR for transcriptional analysis of hla, fnbA, fnbB, and clfA. As a control, expression of 16S rRNA and clpP was determined.

FIG. 4.

Internalization of ΔclpP mutant cells was increased in 293T cells. Relative internalization of different isogenic mutants of strain 8325 (ΔagrA, ΔagrC, ΔclpP, and complemented ΔclpP⁺ strains) is compared to internalization of 8325 wild type (Wt). Means ± standard deviations of four experiments are given.

FIG. 5.

Autolysis of whole cells of S. aureus 8325 wild-type (♦), 8325ΔclpP (▪), and complemented mutant ΔclpP⁺ (▴) strains by Triton X-100. The results are expressed as lysis percentages as described in Materials and Methods. The average of two independent experiments is shown.

FIG. 6.

Arginine deiminase (A) and urease (B) activity of 8325 wild-type (Wt), ΔclpP, and complemented mutant ΔclpP⁺ strains after 4 h of incubation (urease) or after 16 h of incubation (arginine deiminase). API Staph test was performed according to the manufacturer's instructions (BioMérieux). +++, ++, and − indicate very high, high, and no enzymatic activity, respectively.