The post-transcriptional regulator RsmA mediates the protective response against pyocyanin overproduction in Pseudomonas aeruginosa, as revealed by a proteomic approach

Pyocyanin is the primary phenazine produced by Pseudomonas aeruginosa, which possesses redox activity. This compound contributes to its virulence by generating reactive oxygen species and inducing oxidative stress in prokaryotic and eukaryotic cells. P. aeruginosa is able to resist high concentrations of pyocyanin despite its toxicity. In this work, we employed P. aeruginosa ID4365 and PAO1 rsmA mutants, which produce 276.2 μM and 6.9 μM of pyocyanin, respectively, to conduct a proteomic analysis in order to identify proteins involved in the protective response against this phenazine. We found that inactivation of rsmA in ID4365 altered the levels of 474 proteins, including those related to the oxidative stress response, efflux pumps, and chaperones. While in PAO1, rsmA inactivation affected the levels of 177 proteins, and only MexH, which is part of the MexGHI-OpmD efflux pump involved in exporting pyocyanin, was identified as a protein related to the protective response. We further determined whether RsmA controls the pyocyanin protective response in ID4365 and found that expression of genes encoding OxyR, SoxR, and Fur, which are essential for oxidative stress defense and iron homeostasis, were negatively regulated by RsmA. Moreover, we demonstrate that RsmA negatively controls the GroESL chaperone system, suggesting its role in alleviating pyocyanin-induced proteotoxic stress. Our findings indicate that the protective response against pyocyanin is more pronounced in ID4365 compared to the PAO1 strain when rsmA is deleted. Thus, RsmA plays a crucial role in this strain by regulating the response to prevent damage caused by pyocyanin overproduction.