Mechanisms of Pyocyanin Toxicity and Genetic Determinants of Resistance in Staphylococcus aureus

Abstract

<i>Pseudomonas aeruginosa</i> and <i>Staphylococcus aureus</i> are commonly isolated from polymicrobial infections, such as wound infections and chronic respiratory infections of persons with cystic fibrosis. Despite their coisolation, <i>P. aeruginosa</i> produces substances toxic to <i>S. aureus</i>, including pyocyanin, a blue-pigmented molecule that functions in <i>P. aeruginosa</i> virulence. Pyocyanin inhibits <i>S. aureus</i> respiration, forcing it to derive energy from fermentation and adopt a small-colony variant (SCV) phenotype. The mechanisms by which <i>S. aureus</i> sustains infection in the presence of pyocyanin are not clear. We sought to clarify the mechanisms of pyocyanin toxicity in <i>S. aureus</i> as well as identify the staphylococcal factors involved in its resistance to pyocyanin toxicity. Nonrespiring <i>S. aureus</i> SCVs are inhibited by pyocyanin through pyocyanin-dependent reactive oxygen species (ROS) production, indicating that pyocyanin toxicity is mediated through respiratory inhibition and ROS generation. Selection on pyocyanin yielded a menadione auxotrophic SCV capable of growth on high concentrations of pyocyanin. Genome sequencing of this isolate identified mutations in four genes, including <i>saeS</i>, <i>menD</i>, NWMN_0006, and <i>qsrR</i> QsrR is a quinone-sensing repressor of quinone detoxification genes. Inactivation of <i>qsrR</i> resulted in significant pyocyanin resistance, and additional pyocyanin resistance was achieved through combined inactivation of <i>qsrR</i> and menadione biosynthesis. Pyocyanin-resistant <i>S. aureus</i> has an enhanced capability to inactivate pyocyanin, suggesting QsrR-regulated gene products may degrade pyocyanin to alleviate toxicity. These findings demonstrate pyocyanin-mediated ROS generation as an additional mechanism of pyocyanin toxicity and define QsrR as a key mediator of pyocyanin resistance in <i>S. aureus</i> <b>IMPORTANCE</b> Many bacterial infections occur in the presence of other microbes, where interactions between different microbes and the host impact disease. In patients with cystic fibrosis, chronic lung infection with multiple microbes results in the most severe disease manifestations. <i>Staphylococcus aureus</i> and <i>Pseudomonas aeruginosa</i> are prevalent cystic fibrosis pathogens, and infection with both is associated with worse outcomes. These organisms have evolved mechanisms of competing with one another. For example, <i>P. aeruginosa</i> produces pyocyanin, which inhibits <i>S. aureus</i> growth. Our research has identified how pyocyanin inhibits <i>S. aureus</i> growth and how <i>S. aureus</i> can adapt to survive in the presence of pyocyanin. Understanding how <i>S. aureus</i> sustains infection in the presence of <i>P. aeruginosa</i> may identify means of disrupting these microbial communities.