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, the rcsA expression was dominant under HOCl stress and highly increased under HOCl-related stress conditions. Functional analysis of RcsA showed that the distinguishing core amino acid resides Cys60, Cys63, and His67 were required for the degradation of sodium hypochlorite (NaOCl), suggesting an extended motif in the AhpD family. After producing allelic exchange mutagenesis in the P. aeruginosa rcsA, the P. aeruginosa rcsA-deletion mutant showed a significantly decreased HOCl resistance. Ectopic expression of P. aeruginosa rcsA 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. Finally, the Cys60, Cys63, and His67 variants of RcsA were unsuccessful to complement to 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 involve the catalytic Cys60, Cys63, and His67 residues. Importance Pseudomonas 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. aeruginosa rcsA 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. aeruginosa rcsA plays an important role in bleach tolerance with expression of P. aeruginosa rcsA 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.

Chlorine redox chemistry is not rare in biology

“…Among these proteins were proteins with functions already known to be connected to Cld, such as (per)chlorate reductases, oxidative stress response, genetic mobility, and signaling 50-52 . Additionally, since this work began, an alkylhydroperoxidase AhpD-like protein (subfamily 84), identified here as having a low clustering coefficient (Table 1), was found to be the enzyme RcsA involved in hypochlorous acid degradation in Pseudomonas aeruginosa 53 . Therefore, this method identified true genetic associations between Cld and other protein subfamilies.…”

Chlorine redox chemistry is widespread in microbiology

“…tRNA as an insertion site) [ 47 – 49 ]. Additionally, since this work began, an alkylhydroperoxidase AhpD-like protein (subfamily 84), identified here as having a low clustering coefficient, was found to be the enzyme RcsA involved in hypochlorous acid degradation in Pseudomonas aeruginosa [ 50 ]. Therefore, this method identified true genetic associations with Cld.…”

Monochloramine Induces Release of DNA and RNA from Bacterial Cells: Quantification, Sequencing Analyses, and Implications

“…For instance, transcripts of hypothetical proteins (PA3284, PA3283, PA0565, and PA0566) were detected in higher abundance in emRNA from MCA-treated biofilms. One of these proteins PA0565 (now annotated as rcsA ) was recently shown to be highly upregulated in response to reactive chlorine species …”