A new two-component regulatory system involved in adhesion, autolysis, and extracellular proteolytic activity of Staphylococcus aureus.

Staphylococcus aureus is a major pathogen in human infectious diseases. It appears to differ considerably from the coagulase-negative staphylococci (CoNS), such as Staphylococcus epidermidis and Staphylococcus saprophyticus, with respect to its pathogenicity in that it elaborates a large number of extracellular virulence factors and other proteins, while the CoNS, which are usually more indolent pathogens, generally produce few if any of these factors. CoNS cause infections associated with implanted foreign bodies such as intravascular catheters. Previous reports of adherence by staphylococci have concentrated primarily on the CoNS (8, 24, 33). However, an essential step in the establishment of any staphylococcal infection is attachment to the host tissues themselves or to foreign bodies within host tissues. Environmental stimuli have been shown to play an important role in expression of virulence genes during pathogenesis. Because S. aureus has the ability to invade different tissues, it responds to environmental stimuli in order to survive and express virulence genes during the infection. To survive, the cells must monitor external conditions and adjust their structure, physiology, and behavior accordingly. Thus, bacteria have developed sophisticated signaling systems for eliciting adaptive responses to their environment. The molecular mechanisms responsible for stimulus-response coupling often involve two types of components encoded by an operon, a transmembrane sensor (histidine protein kinase) and its associated response regulator (50). In general, the transmembrane protein binds a specific ligand, the signal, and binding triggers autophosphorylation at a conserved histidine residue. The phosphorylated sensor then transfers the phosphate to an aspartic residue in the response regulator (39). The response regulator can in turn enhance or repress transcription of target genes, as occurs with PhoB in the phosphate regulon (28) and OmpR in the porin regulon (27, 55) of Escherichia coli. A two-component regulatory system, AgrC-AgrA, involved in the production of virulence factors was previously described for S. aureus (21, 37, 43). This locus has also been shown to affect autolysin expression (16). Peptidoglycan hydrolases, enzymes that hydrolyze either the glycan or the peptide moieties of peptidoglycan, participate in a number of important biological processes occurring during cell growth and division, including cell wall synthesis, daughter cell separation, and peptidoglycan turnover and recycling (1). As these hydrolases are also involved in lysis of bacteria, some have been called autolysins. Thus, the same two-component regulatory system can be involved in pathogenesis as well as in cellular physiology. In order to find loci involved in the regulation of the multidrug efflux pump NorA of S. aureus, we used a library of Tn917LTV1 insertions in the chromosome of strain MT23142 with selection for higher and lower levels of resistance to tetraphenylphosphonium bromide (TPP), a substrate of NorA, than are found in MT23142. Overproduction of norA leads to resistance to drugs such as quinolones, ethidium bromide, and TPP (14, 35). MT23142 carries the flqB mutation. flqB, a cis-acting mutant of norA, is localized downstream of the initiation site of norA and overexpresses norA (35). One transposition mutant, BF15, obtained from the library showed a slight increase of resistance to TPP and modifies norA expression (14). In addition to effects on norA expression, the mutant unexpectedly showed alterations in autolysis. In order to characterize this mutant, we analyzed the sequence of chromosomal DNA flanking the insertional site of Tn917LTV1. We found a new two-component regulatory system, ArlS-ArlR. Insertion of Tn917 in the arlS gene resulted in changes in adhesive properties, the rate of autolysis, and extracellular proteolytic activity. All these properties were complemented by the cloned arlR and arlS genes, indicating that the disruption of arlS was responsible for the mutant phenotype.