Roles of RcsA, an AhpD Family Protein, in Reactive Chlorine Stress Resistance and Virulence in Pseudomonas aeruginosa

Abstract

Reactive chlorine species (RCS), particularly hypochlorous acid (HOCl), are powerful antimicrobial oxidants generated by biological pathways and chemical syntheses. Pseudomonas aeruginosa is an important opportunistic pathogen that has adapted mechanisms for protection and survival in harsh environments, including RCS exposure. Based on previous transcriptomic studies of HOCl exposure in P. aeruginosa, we found that the expression of PA0565, or rcsA, which encodes an alkyl hydroperoxidase D-like protein, exhibited the highest induction among the RCS-induced genes. In this study, rcsA expression was dominant under HOCl stress and greatly increased under HOCl-related stress conditions. Functional analysis of RcsA showed that the distinguishing core amino acid residues Cys60, Cys63, and His67 were required for the degradation of sodium hypochlorite (NaOCl), suggesting an extended motif in the AhpD family. After allelic exchange mutagenesis in the P. aeruginosarcsA, the P. aeruginosarcsA deletion mutant showed significantly decreased HOCl resistance. Ectopic expression of P. aeruginosarcsA led to significantly increased NaOCl resistance in Escherichia coli Moreover, the pathogenicity of the rcsA mutant decreased dramatically in both Caenorhabditis elegans and Drosophila melanogaster host model systems compared to the wild type (WT). Finally, the Cys60, Cys63, and His67 variants of RcsA were unsuccessful at complementing phenotypes of the rcsA mutant. Overall, our data indicate the importance of P. aeruginosa RcsA in defense against HOCl stress under disinfections and during infections of hosts, which involves the catalytic Cys60, Cys63, and His67 residues.IMPORTANCEPseudomonas aeruginosa is a common pathogen that is a major cause of serious infections in many hosts. Hypochlorous acid (HOCl) is a potent antimicrobial agent found in household bleach and is a widely used disinfectant. P. aeruginosa has evolved adaptive mechanisms for protection and survival during HOCl exposure. We identified P. aeruginosarcsA as a HOCl-responsive gene encoding an antioxidant protein that may be involved in HOCl degradation. RcsA has a distinguishing core motif containing functional Cys60, Cys63, and His67 residues. P. aeruginosarcsA plays an important role in bleach tolerance, with expression of P. aeruginosarcsA in Escherichia coli also conferring HOCl resistance. Interestingly, RcsA is required for full virulence in worm and fruit fly infection models, indicating a correlation between mechanisms of bleach toxicity and host immunity during infection. This provides new insights into the mechanisms used by P. aeruginosa to persist in harsh environments such as hospitals.

Keywords: AhpD family protein; Pseudomonas aeruginosa; RcsA; bleach; reactive chlorine species; virulence.