The Small RNA RgsA Can Enhance The Oxidative Stress Resistance Of Pseudomonas Aeruginosa

ObjectivePseudomonas aeruginosa (P.aeruginosa), is a conditional pathogen in clinicalmedicine and usually causes serious infections in patients with metabolic diseases,blood diseases and cancers. It is also a common cause of postoperative infection insurgery patients. It is recently been considered as an important bacterium due to high-level resistance to multiple drugs. The regulation of gene expression by P. aeruginosain response to environmental stresses is the focus of research in the field.Bacterial sRNAs are a class of small regulatory RNAs with40-500nt in size. Most ofthem play important roles in survival and pathogenicity of bacteria, and may evenexceed protein regulators in number and diversity. The sRNA RgsA which exists in P.aeruginosa and Pseudomonas fluorescens (P. fluorescens), has been identified inbioinformtic screen by Nicolas González in2008. Previous experiments showed thatit might be involved in protecting the cell from oxidative stress. RgsA has higherexpression level in P. aeruginosa biofilm than plankton. Biofilms may contribute tothe persistent infections of bacteria, and at least65%of human infections areassociated with biofilms. Infectious processes in which biofilms have been implicatedinclude common problems such as dental plaque, cystic fibrosis, middle-ear infections,chronic osteomyelitis, chronic nasosinusitis and chronic wounds infections. In thisresearch, we further study the basic structure, functional mechanism and generegulation of RgsA, and to investigate its effect in P. aeruginosa.Methods(1) Total RNA of P. aeruginosa PAO1strain was extracted by Trizol. Then5′RACEwas used to determine the potential transcriptional start sites of rgsA gene.(2) PCR amplifie each arm from wild-type genomic DNA of P. aeruginosa PAO1using primers that add the necessary restriction sites at either end. Gene targetingvectors was constructed by inserting homology arms and accC1gene into thesuicide vector pEX18-Ap, and then P. aeruginosa PAO1ΔRgsA mutant wasestablished by hybridization method.(3) The open reading frame of rgsA gene of P. aeruginosa PAO1was amplified byPCR from genomic DNA, then introduce into downstream of arabinose-induciblepromoter pBAD of the expression vector， pJN105-SH to construct the RgsA prokaryotic expression vector. P. aeruginosa PAO1ΔRgsA mutant harboring theexpression vector pJN105-SH was established by biparental mating.(4) The growth ability of wild strain and ΔRgsA mutant strain were calculated by timeas X-axis and changes of OD600during the observation as Y-axis.(5) P. aeruginosa PAO1cells were treated with the different concentrations ofhydrogen peroxide or organic peroxide. Total RNA was extracted with Trizolreagent. The expression of RgsA was analyzed by real-time PCR,(6) The survival ability of wild-type, deficient and over-expressing strains weredetected by MTT and plate assays under simulated circumstance of oxidativestress.(7) Three-day-old biofilms of wild-type, deficient and over-expressing strains weretreated with the different concentrations of hydrogen peroxide, and then werestained by using a BacLight LIVE/DEAD kit and examined by fluorescencemicroscope.Results(1) Two transcripts, sized90bp and300bp, were found in P. aeruginosa PAO1.(2) The deficient and over-expressing strains of P. aeruginosa PAO1identified byPCR and sequencing were constructed successfully.(3) The ΔrgsA mutant strain had a slower growth rate than wild strain.(4) Expression of RgsA was upregulated after treated with different concentrations ofhydrogen peroxide.(5) The survival ability of ΔrgsA mutant was significantly affected by the oxidativestress compared with the wild strain. The strain harboring the expression vectorpJN105-SH has similar survival ability as that of wild strain, when induced with1%L-arabinose.(6) Large scale dead cells were only observed in biofilm of over-expressing strainstreated with30mM hydrogen peroxide. And dead cells were stained to yellow bySYTO9.ConclusionsWe detected two transcripts of rgsA in P. aeruginosa PAO1. RgsA plays an importantrole in protecting P. aeruginosa PAO1cells from oxidative stress.