The carboxyl-terminal processing proteases Prc and CtpA modulate cell-surface signalling activity and Pseudomonas aeruginosa virulence

Cell-surface signalling (CSS) is a signal transfer system of Gram-negative bacteria used to detect extracellular signals and modulate gene transcription in response. These three-protein systems are formed by an outer membrane receptor, a cytoplasmic membrane-embedded anti-{sigma} factor and a cytosolic extracytoplasmic function {sigma} factor ({sigma}ECF). In absence of an inducing signal, the anti-{sigma} factor binds to and keeps the {sigma}ECF factor sequestered, thus preventing its interaction with the RNA polymerase and the transcription of signal response genes. Presence of the signal triggers a signalling cascade that extends from the outer membrane to the cytosol and results in {sigma}ECF factor activation. Recently, we and others have reported that CSS {sigma}ECF factor activation requires the regulated and sequential proteolysis of the cognate anti-{sigma} factor, and the function of the Prc and RseP proteases. However, many features of this proteolytic cascade are still unclear. In this work, we have identified another protease that modulates CSS activity, namely the periplasmic carboxyl-terminal processing protease CtpA. We show that both CtpA and the previously identified protease Prc control CSS activation by modulating the levels of the anti-{sigma} factor. CtpA functions upstream of Prc in the proteolytic cascade and seems to prevent the Prc-mediated proteolysis of the CSS anti-{sigma} factor. Importantly, using zebrafish embryos and the A549 cell line as hosts, we show that mutants in the rseP and ctpA proteases of the human pathogen Pseudomonas aeruginosa are considerably attenuated in virulence while the prc mutation increases virulence likely by enhancing the production of outer membrane vesicles. Because proteases are druggable proteins, the identification of regulatory proteases involved in P. aeruginosa virulence holds promise for the development of novel strategies to fight this clinically relevant pathogen.