<i>Acinetobacter Baumannii</i> Secreted Protease CpaA Inhibits Factor XII-Mediated Bradykinin Generation and Neutrophil Activation.

Abstract

FXII (coagulation factor XII) is best known for its roles in the contact and kallikrein-kinin pathways. FXII is converted to FXIIa (activated factor XII) by PKa (plasma kallikrein) or its unique ability to autoactivate on bacterial or other biologic surfaces. In vivo, FXIIa initiates the intrinsic coagulation pathway and promotes inflammation by reciprocal activation of prekallikrein, which cleaves HK (high-molecular-weight kininogen) to liberate bradykinin. CpaA (coagulation targeting metallo-endopeptidase of <i>A baumannii</i>) is a secreted metalloprotease identified in a human clinical isolate of <i>Acinetobacter baumannii</i> that cleaves FXII at O-linked glycosylated sites, inhibiting contact activation. While CpaA facilitates a modest in vivo fitness advantage in mice, the role of CpaA in human infection remains unclear. As such, the objectives of this study were to characterize the structural details of the interaction between CpaA, FXII, and the KKS (kallikrein-kinin system) and to determine the downstream consequences on thromboinflammatory responses. The effect of purified CpaA on the coagulant activity of FXII and the generation of bradykinin was characterized. Neutrophil signaling, flow cytometry, and functional assays were performed to define how CpaA-mediated cleavage of FXII affects innate immune functions. Bacterial killing by human neutrophils was performed with wild-type and mutant <i>A baumannii</i> strains lacking CpaA. We found that CpaA cleaves both FXII zymogen and FXIIa but not beta Factor XII. However, cleavage of FXIIa by CpaA does not significantly inhibit its clotting activity, demonstrating that CpaA does not inactivate FXIIa, but rather prevents activation of zymogen FXII. CpaA also cleaves HK, resulting in reduced kallikrein activation and bradykinin generation. We previously identified that zymogen FXII interacts with the urokinase receptor on neutrophils and upregulates neutrophil activation. Here, we demonstrate that CpaA cleaves neutrophil FXII, resulting in reduced Akt2 phosphorylation, chemotaxis, oxidative burst, and neutrophil extracellular trap formation. Importantly, CpaA decreases the human neutrophil killing efficiency of <i>A baumannii</i> in culture. These data identify a role for FXII in responding to bacterial infection and suggest that by inhibiting the contact and kallikrein-kinin pathways and impairing neutrophil activation, CpaA may blunt the innate immune response and help prevent the elimination of <i>A baumannii</i> from the human host.