SummaryClp proteolytic complexes are essential for virulence and for survival under stress conditions in several pathogenic bacteria. Recently, a study using signature-tagged mutagenesis identified the ClpX ATPase as also being required for virulence in Staphylococcus aureus . Presently, we have constructed deletion mutants removing either ClpX or the proteolytic subunit, ClpP, in S. aureus 8325-4 in order to examine a putative link between stress tolerance and virulence. When exposed to stress, we found that, although clpP mutant cells were sensitive to conditions generating misfolded proteins, the absence of ClpX improved survival. In the presence of oxidative stress or at low temperature, both ClpP and ClpX were important for growth. Virulence was examined in a murine skin abscess model and was found to be severely attenuated for both mutants. S. aureus pathogenicity is largely dependent on a set of extracellular and cell wall-associated proteins. In the mutant cells, the amount of a a a a -haemolysin ( hla ) and several other extracellular proteins was greatly decreased, and analysis of hla expression revealed that the reduction occurred at the transcriptional level. Essential for transcriptional regulation of hla is the quorum-sensing agr locus. Interestingly, the absence of ClpX or ClpP reduced both transcription of the agr effector molecule, RNA III, and the activity of the autoinducing peptide (AIP). In addition, ClpX was required independently of ClpP for transcription of spa encoding Protein A. Thus, our results indicate that ClpX and ClpP contribute to virulence by controlling the activity of major virulence factors rather than by promoting stress tolerance.

SummaryThe Hsp100/Clp ATPases constitute a family of closely related proteins of which some members function solely as chaperones whereas others additionally can associate with the unrelated ClpP peptidase forming a Clp proteolytic complex. We have investigated the role of four Clp ATPases in the versatile pathogen, Staphylococcus aureus . Previously, we showed that ClpX is required for expression of major virulence factors and for virulence of S. aureus , but not for survival during heat shock. In the present study, we have inactivated clpC , clpB and clpL and, while none of these mutations affected toxin production, both ClpC and ClpB and to a minor extent ClpL were required for intracellular multiplication within bovine mammary epithelial cells. These defects were paralleled by an inability of the clpC mutant to grow at high temperature and of the clpB mutant to induce thermotolerance indicating that the protective functions of these proteins are required both at high temperature and during infection. By primer extension analysis and footprint studies, we show that expression of clpC and clpB is controlled by the negative heatshock regulator, CtsR, and that ClpC is required for its repressor activity. Thus, ClpC is a likely sensor of stress encountered during both environmental stress and infection. In addition to virulence factor production the ability to form biofilms is of importance to S. aureus as a nosocomial pathogen. Interestingly, biofilm formation was reduced in the absence of ClpX or ClpC whereas it was enhanced in the absence of ClpP. Thus, our data show that Clp proteolytic complexes and the Clp ATPases control several key processes of importance to the success of S. aureus as a pathogen.

Staphylococcus aureus is a versatile pathogen capable of causing life-threatening infections. Many of its cell wall and exoproduct virulence determinants are controlled via the accessory gene regulator (agr). Although considered primarily as an extracellular pathogen, it is now recognized that S. aureus can be internalized by epithelial and endothelial cells. Traditional experimental approaches to investigate bacterial internalization are extremely time-consuming and notoriously irreproducible. We present here a new reporter gene method to assess intracellular growth of S. aureus in MAC-T cells that utilizes a gfp-luxABCDE reporter operon under the control of the Bacillus megaterium xylA promoter, which in S. aureus is expressed in a growth-dependent manner. This facilitates assessment of the growth of internalized bacteria in a nondestructive assay. The dual gfplux reporter cassette was also evaluated as a reporter of agr expression and used to monitor the temporal induction of agr during the MAC-T internalization process. The data obtained suggest that agr induction occurs prior to endosomal lysis and that agr-regulated exoproteins appear to be required prior to the release and replication of S. aureus within the infected MAC-T cells.Staphylococcus aureus is the etiologic agent of numerous infections in humans and domesticated animals and has been implicated in a multitude of diseases, ranging from minor wound infections to more serious diseases, including endocarditis, osteomyelitis, and septic shock (reviewed by Projan and Novick [34]). The expression of many S. aureus virulence factors is under the control of the accessory gene regulator (agr) which, on entering post-exponential phase, downregulates the production of cell surface-associated proteins and upregulates the expression of secreted toxins and extracellular enzymes (28,33,38). The role of the agr regulon is supported by in vivo studies, which show that agr mutants are greatly attenuated in several animal models, including intramammary infections (13), arthritis in mice (1), and endocarditis in rabbits (7). The agr locus is a quorum-sensing-regulated system activated by autoinducing peptide pheromone (AIP) (21, 25). The agr locus consists of two divergent transcriptional units, RNAII and RNAIII, which are under the control of the P2 and P3 promoters, respectively (reviewed by Novick and Muir [30]). RNAII is a polycistronic mRNA that encodes the agrB and agrD genes required for the synthesis of the AIP and also the two component signal transduction proteins, AgrA and AgrC, which are responsible for sensing and responding to the AIP. RNAIII is the effector molecule in the agr regulon acting primarily at the level of gene transcription. Different S. aureus strains produce AIPs with distinct structures, and strains can be grouped on this basis since they will activate the agr response of strains within the same group and inhibit the agr response of strains from different groups by competitive inhibition (21,30). This inhibitory action of AIPs has identi...