The role of ArlRS in regulating oxacillin susceptibility in methicillin-resistant Staphylococcus aureus indicates it is a potential target for antimicrobial resistance breakers

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA), also known as oxacillin-resistant S. aureus, is a leading cause of community and hospital associated infections globally. In this work, we found that deletion of the arlRS two-component system genes in the USA300 and USA500 strains resulted in increased susceptibilities to oxacillin (8–16-fold decrease in minimal inhibitory concentrations). In USA300Δ arlRS, transcriptional levels of mecA or blaZ showed no obvious change, while mRNA levels of spx showed a 4-fold decrease at 4 h and a 6.3-fold decrease at 10 h. Overexpression of spx in Δ arlRS restored oxacillin resistance to a similar level in USA300. In addition, gel shift assay showed that the recombinant ArlR bound to spx promoter region. Furthermore, silencing of spx led to a significant increase of oxacillin susceptibility in multiple MRSA isolates. Our results indicate that ArlRS plays a strong role in regulating oxacillin resistance in MRSA strains, which involves direct modulation of spx expression. Moreover, oritavancin showed inhibition to ATPase activity of the recombinant histidine kinase ArlS (IC 50 = 5.47 μM). Oritavancin had synergy effect on oxacillin activity against the MRSA strains in both planktonic and biofilm state. Our data suggest that ArlRS is an attractive target for breaking antimicrobial resistance of MRSA.

The Staphylococcus aureus ArlRS two‐component system regulates virulence factor expression through MgrA

“…It was recently shown that ArlRS activates expression of the transcriptional regulator spx (Bai et al , ). Spx interacts directly with RNA polymerase and is involved in the disulfide stress response in Firmicutes (Hillion and Antelmann, ).…”

“…). The ArlRS TCS was also recently shown to directly regulate spx expression (Bai et al , ). We show that this regulation occurs at the spx P2 promoter, which contains a similar ArlR‐binding site (Fig.…”

“…The Spx protein is only stabilized when a CXXC motif near its N‐terminus is oxidized, and in this oxidized state it binds to RNA polymerase to affect gene expression (Hillion and Antelmann, ). Spx is a key stress response regulator that controls expression of thioredoxin reductase ( trxB ), the ica locus for poly‐N‐acetylglucosamine synthesis and TrfA, a Clp proteasome adaptor involved in resistance to cell wall‐active antibiotics (Pamp et al , ; Jousselin et al , ; Bai et al , ). Under the conditions of our RNA‐seq experiment, we did not see regulation of trxB , icaADBC or trfA .…”

“…EMSA experiments were modified from the protocol described by Bai et al (). DNA probes 5ʹ end‐labeled with IRDye700 were generated by annealing 50 nt oligos HC656 and HC657 (probe 2), and HC789 and HC790 (probe 3).…”

“…It was also reported that ArlRS is required for leukocidin expression, perhaps through regulation of agr (Harper et al , ). Recent work showed that ArlRS also activates expression of Spx, a transcriptional regulator involved in the response to oxidative stress and antibiotic resistance (Bai et al , ). ArlR was shown to directly bind to the spx promoter region, although the exact binding site was not determined (Bai et al , ).…”