Besides its central role in regulation of virulence, the agr locus has also been connected to β-lactam resistance possibly via regulation of autolysis (Piriz et al., 1996; Fujimoto & Bayles, 1998). The agr system consists of two adjacent transcriptional units, RNAII and RNAIII, which are transcribed in opposite directions from two divergent promoters, P2 and P3, respectively. RNAII encodes the precursor and the transporter of the autoinducing peptide, as well as a membrane-bound sensor that responds to the autoinducer check details by activating the transcriptional
regulator AgrA. AgrA, which is also encoded by RNAII, in turn activates several promoters including P2 and P3 at the agr locus (Queck et al., 2008). RNAIII is the major effector of the agr system and regulates the expression of a large number of genes by base pairing with target mRNAs. This directly affects the expression of some virulence genes; for example, reduced translation of the spa mRNA encoding the cell surface-associated Protein A (Huntzinger et
al., 2005) and induced translation of the hla mRNA encoding the α-hemolysin (Novick et al., 1993; Morfeldt et al., 1995). However, the most widespread effect of RNAIII is probably caused by its inhibition of rot translation (Geisinger et al., 2006). Rot is a repressor of many genes encoding extracellular virulence factors like proteases and hemolysins, but also functions as a positive regulator of transcription (Said-Salim et al., 2003). Our previous model of reversal of methicillin resistance in MRSA by thioridazine only included the effect of the combinatorial treatment
on mecA, blaZ, SAHA HDAC mw and their regulators. However, we expect that treatment with thioridazine causes profound changes in gene expression as recently demonstrated in a clinical isolate of multidrug-resistant Mycobacterium tuberculosis (Dutta et al., 2010). Based on this, we find it likely that thioridazine in combination with oxacillin affects the expression or activity of additional proteins involved in the resistance mechanism besides PBP2a. In the present study, we sought to explore the possibility that additional genes besides mecA and blaZ are involved in the mechanism by which thioridazine resensitizes MRSA to oxacillin. We analyzed the expression levels of selected genes involved in Chlormezanone β-lactam resistance and peptidoglycan biosynthesis in response to the combinatorial treatment. Furthermore, to assess the suitability of the treatment, we also tested the effect on selected toxicity and virulence/pathogenesis genes. MRSA ATCC strain 33591 was routinely grown at 37 °C with shaking in brain heart infusion medium (Difco) and Mueller–Hinton medium and agar (BBL) for subculture and maintenance. Minimal inhibitory concentrations for oxacillin and thioridazine are >256 and 16 mg L−1, respectively. MRSA cultures were grown to an OD600 nm of 0.