Defining the interaction of the protease CpaA with its type II secretion-chaperone CpaB and its contribution to virulence in Acinetobacter species.

Acinetobacter baumannii, A. nosocomialis, and A. pittii are a frequent cause of multi-drug resistant, healthcare-associated infections. Our previous work demonstrated that A. nosocomialis M2 possesses a functional Type II secretion system (T2SS) that is required for full virulence. Further, we identified the metallo-endopeptidase CpaA, which had been previously shown to cleave human factor V and deregulate blood coagulation, as the most abundant Type II-secreted effector protein. We also demonstrated that its secretion is dependent on CpaB, a membrane-bound chaperone. In this study, we show that CpaA expression and secretion is conserved across several medically-relevant Acinetobacter species. Additionally, we demonstrate that deletion of cpaA results in attenuation of A. nosocomialis M2 virulence in moth and mouse models. The virulence defects resulting from the deletion of cpaA were comparable to those observed upon abrogation of T2SS activity. The virulence defects resulting from the deletion of cpaA are comparable to those observed upon abrogation of T2SS activity. We also show that CpaA and CpaB strongly interact, forming a complex in a 1 to 1 ratio. Interestingly, deletion of the N-terminal transmembrane domain of CpaB results in robust secretion of CpaA and CpaB, indicating that the transmembrane domain is dispensable for CpaA secretion and likely functions to retain CpaB inside the cell. Limited proteolysis of spheroplasts revealed that the C-terminal domain of CpaB is exposed to the periplasm, suggesting that this is the site where CpaA and CpaB interact in vivo. Lastly, we show that CpaB does not abolish the proteolytic activity of CpaA against human Factor V. We conclude that CpaA is, to the best of our knowledge, the first characterized, bona-fide virulence factor secreted by Acinetobacter species.