Characterization of NorR Protein, a Multifunctional Regulator of norA Expression in Staphylococcus aureus

Staphylococcus aureus is a leading cause of human infectious diseases worldwide, ranging from superficial skin lesions to systemic and life-threatening infections, such as osteomyelitis, endocarditis, pneumonia, and septicemia. The virulence of S. aureus has been associated with the production of a large number of extracellular toxins, enzymes, and cell-surface-associated proteins, encoded by diverse genes, the expression of which is controlled by the accessory gene regulator locus (agr) (35, 50). The agr regulatory effector is a 510-base RNA molecule (RNAIII) (42). The agr locus regulates most target genes at the level of transcription, but was also shown to affect translation of some genes (35, 42). sarA is the second known global regulatory locus that was also involved in the regulation of virulence factors in S. aureus (4, 34). SarA activates α-toxin gene transcription but represses transcription of genes for serine protease and protein A (7, 9, 10). SarA acts partly through the agr regulatory pathway by binding to agr promoters and stimulating the transcription of agr (8, 11, 45). Since agr and sarA loci play such important roles in diverse gene regulation, they may also participate in regulation of efflux pump expression, which causes resistance to multiple antibiotics, including fluoroquinolones in S. aureus. Fluoroquinolones are synthetic antimicrobial agents and have been used for treatment of a broad range of bacterial infections (22). Increases in resistance to diverse antibiotics, including fluoroquinolones, have limited the choice of antimicrobial agents in some clinical settings. The genetics and mechanisms of bacterial resistance to fluoroquinolones have been studied extensively. Fluoroquinolones act on DNA gyrase and topoisomerase IV to inhibit bacterial DNA replication (12). Mutations in gyrA and gyrB encoding the subunits of DNA gyrase (21, 23, 26) and grlA and grlB encoding the subunits of DNA topoisomerase IV of S. aureus (13, 38) lead to quinolone resistance in gram-positive and gram-negative bacteria. One of the most intriguing mechanisms underlying resistance to fluoroquinolones as well as a range of other antimicrobial agents involves the extrusion of a variety of structurally unrelated compounds due to active efflux by membrane pumps (19, 51). On the basis of bioenergetic and structural criteria, the multidrug transporters have been divided into five major families (5): the ATP-binding cassette family (ABC), the major facilitator superfamily (MFS), the multidrug and toxic compound extrusion transporters (MATE), the drug/metabolite transporters (DMT), and the resistance/nodulation/division transporters (RND) (31, 44). The S. aureus NorA protein belongs to the MFS group frequently found in bacteria. Overexpression of the NorA multidrug resistance (MDR) efflux pump causes resistance to some quinolones (25, 27, 37, 39, 53). It is characterized by the presence of 12 transmembrane segments (37, 39) and is related to Bmr, an efflux pump of Bacillus subtilis (1, 36, 55). NorA protects the cell from a number of lipophilic and monocationic compounds, such as ethidium bromide, cetrimide, benzalkonium chloride, tetraphenylphosphonium bromide, and acriflavine, as well as hydrophilic quinolones (25, 27). While the physiological function of NorA as a self-sufficient multidrug transporter was demonstrated with cytoplasmic membrane vesicles and reconstituted proteoliposomes (54), the regulation of the expression of the NorA efflux pump is still not well understood. Previous studies demonstrated that the expression of norA is increased by mutations in the promoter region that increase mRNA stability, reported in the case of the chromosomal quinolone resistance locus, flqB, which is linked to fus and the transposon insertion Ω1108 on the SmaI D fragment (17), and by mutation in the arlS gene that alters the two-component regulatory system arlRS, reported in the case of the insertion-disruption of arlS by Tn917LTV1 in the S. aureus chromosome, but it is not known if the effects of arlRS on norA expression are direct or indirect (15, 16). In order to identify direct regulatory elements involved in norA expression, we have purified the 17.6-kDa protein that binds to the norA promoter, identified its gene, and characterized the effects of its disruption and overexpression. Our data show that this putative regulatory protein, which we have named “NorR,” functions as an activator of norA expression but is also multifunctional, affecting cell surface properties and acting as a negative regulator for expression of other effectors of the MDR phenotype that likely represent other as-yet-uncharacterized MDR efflux pumps. (This work was presented in part at the 101st General Meeting of the American Society of Microbiology, Orlando, Fla., 20 to 24 May 2001.)