These results suggest that serralysin-mediated ENaC activation requires active protease and that modulation of these protease activities could potentially be leveraged to effectively reduce the virulence associated with bacterial metalloprotease production and secretion. Proteolytic activation of ENaC has been postulated to play a key role in both normal and disease physiologies in the airway. As such, it is possible that both endogenous and exogenous proteases may play a role in establishing and remodeling the airway environment. Here we demonstrate that multiple members of the serralysin metalloprotease family are capable of activating ENaC. These data suggest that ENaC may serve as a target for the serralysin virulence factors from multiple human pathogens. Further, the Pseudomonas aeruginosa AprI, alkaline protease inhibitor can be effectively used to block the in vitro activities of purified serralysin proteases and reverse their effects in physiological experiments on cultured and primary epithelial cells. Our previous studies showed that ENaC can be activated by the addition of AP at the apical surface of cultured and primary epithelial cells. This activation may contribute to the virulence of Pseudomonas by remodeling the local airway environment to be more Vismodegib favorable for bacterial adhesion and subsequent colonization. The current study demonstrates that this activation is more general to this class of bacterial exoproteases, as serralysin from Serratia marcescens is similarly capable of activating ENaC. This activation is slow when compared to trypsin under maximal stimulating conditions. The slow activation of ENaC by both AP and SmP suggest that the physical basis of activation may also be similar for both proteases. However the kinetics of ENaC activation were slightly accelerated in SmP treated epithelia compared to AP, in line with the biophysical characterization of the protease activities. Binding of the inhibitor to AP and SmP is tight, as measured in vitro using purified proteins, and completely abolishes protease activity, consistent with prior reports of binding between the protease and inhibitor. This tight in vitro binding is observed as a complete loss of protease-induced ENaC current in two different model epithelia. This inhibition provides evidence that the activation of ENaC is mediated through cleavage of a host protein by the bacterial protease. The coincident inhibition of protease activity and loss of ENaC activation suggests that the observed activation is occurring through one or more cleavage events and is not mediated by other non-catalytic binding or protein-protein interactions. The AP and SmP mediated activation is slow when compared to that elicited by trypsin. The kinetics of ENaC activation by AP and SmP are slowed by,3.5 to 20 fold when compared to trypsin in the two cell lines. Though previous studies have demonstrated that cleavage of the c-subunit is required for AP induced ENaC activation, it is not immediately clear why the activation kinetics vary between the trypsin and the bacterial proteases. The relatively slow and submaximal activation may arise from conformational constraints limiting GDC-0879 purchase access to one or more cleavage sites in the ENaC ectodomain. This would be consistent with a model wherein the mechanisms of ENaC cleavage and activation did not co-evolve with the bacterial proteases. Alternatively, this slow activation may be the result of indirect activation via an additional protease-sensitive pathway. Further work to evaluate these differences in activation kinetics is ongoing. Both the AP and SmP proteases have been implicated in bacterial virulence. Previous studies have suggested that AP is associated with exacerbations in CF and complications in treating Pseudomonas.