The Vancomycin Resistance-Associated Regulatory System VraSR Modulates Biofilm Formation of Staphylococcus epidermidis in an <i>ica</i> -Dependent Manner

Abstract

The two-component system VraSR responds to the cell wall-active antibiotic stress in Staphylococcus epidermidis. To study its regulatory function in biofilm formation, a <i>vraSR</i> deletion mutant (Δ<i>vraSR</i>) was constructed using S. epidermidis strain 1457 (SE1457) as the parent strain. Compared to SE1457, the Δ<i>vraSR</i> mutant showed impaired biofilm formation both <i>in vitro</i> and <i>in vivo</i> with a higher ratio of dead cells within the biofilm. Consistently, the Δ<i>vraSR</i> mutant produced much less polysaccharide intercellular adhesin (PIA). The Δ<i>vraSR</i> mutant also showed increased susceptibility to the cell wall inhibitor and SDS, and its cell wall observed under a transmission electron microscope (TEM) appeared to be thinner and interrupted, which is in accordance with higher susceptibility to the stress. Complementation of <i>vraSR</i> in the Δ<i>vraSR</i> mutant restored the biofilm formation and the cell wall thickness to wild-type levels. Transcriptome sequencing (RNA-Seq) showed that the <i>vraSR</i> deletion affected the transcription levels of 73 genes, including genes involved in biofilm formation, bacterial programmed cell death (CidA-LrgAB system), glycolysis/gluconeogenesis, the pentose phosphate pathway (PPP), and the tricarboxylic acid (TCA) cycle, etc. The results of RNA-Seq were confirmed by quantitative real-time reverse transcription-PCR (qRT-PCR). In the Δ<i>vraSR</i> mutant, the expression of <i>icaA</i> and <i>lrgAB</i> was downregulated and the expression of <i>icaR</i> and <i>cidA</i> was upregulated, in comparison to that of SE1457. The transcriptional levels of antibiotic-resistant genes (<i>pbp2</i>, <i>serp1412</i>, <i>murAA</i>, etc.) had no significant changes. An electrophoretic mobility shift assay further revealed that phosphorylated VraR bound to the promoter regions of the <i>ica</i> operon, as well as its own promoter region. This study demonstrates that in S. epidermidis, VraSR is an autoregulator and directly regulates biofilm formation in an <i>ica</i>-dependent manner. Upon cell wall stress, it indirectly regulates cell death and drug resistance in association with alterations to multiple metabolism pathways. <b>IMPORTANCE</b> S. epidermidis is a leading cause of hospital-acquired catheter-related infections, and its pathogenicity depends mostly on its ability to form biofilms on implants. The biofilm formation is a complex procedure that involves multiple regulating factors. Here, we show that a vancomycin resistance-associated two-component regulatory system, VraSR, plays an important role in modulating S. epidermidis biofilm formation and tolerance to stress. We demonstrate that S. epidermidis VraSR is an autoregulated system that selectively responds to stress targeting cell wall synthesis. Besides, phosphorylated VraR can bind to the promoter region of the <i>ica</i> operon and directly regulates polysaccharide intercellular adhesin production and biofilm formation in S. epidermidis. Furthermore, VraSR may indirectly modulate bacterial cell death and extracellular DNA (eDNA) release in biofilms through the CidA-LrgAB system. This work provides a new molecular insight into the mechanisms of VraSR-mediated modulation of the biofilm formation and cell death of S. epidermidis.