Abstract
P. aeruginosa is an opportunistic human pathogen and a major cause of death in patients with cystic fibrosis. Recently, an elaborate regulatory network has been discovered that regulates transition between two different virulent modes of the bacterium, one that causes an acute and another that causes chronic infection. In response to unknown environmental signals this network modulates activity of a single protein, RsmA. RsmA is a translational regulator and an RNA binding protein. The objective of this work is to identify direct mRNA targets of RsmA and to elucidate the mechanisms underlying RsmA-mediated regulation. Using a data-independent, label-free LC-MS method, protein expression patterns were monitored in wt, rsmA mutant, and rsmYZ double mutant (analogous to a strain overexpressing RsmA) cultures of P. aeruginosa. Separate analyses were carried out for the cell-associated and secreted proteins. LC-MS data were acquired using a nano-ACQUITY UPLC system and a QTof Premier mass spectrometer operating in alternate scanning mode. In parallel, pull down experiments were carried out in which RNA species that co-purified with His-tagged RsmA were isolated, reverse transcribed, cloned and sequenced. Comparison of the proteomics results and the pull down data revealed that majority of the proteins that were differentially expressed in the proteomics study were also identified among the pulled-down RNA species, including mRNAs encoding major virulence factors. These results suggest that RsmA regulates all of the differentially regulated genes directly by binding their cognate mRNAs. In addition, comparison of the proteomics and the pull down data showed that RsmA binds mRNAs of genes that are regulated either positively or negatively by RsmA, suggesting at least two different mechanisms for RsmA-mediated translational regulation.