OC 25.3 - The Acinetobacter baumannii Secreted Protease CpaA Inhibits Factor XII-mediated Neutrophil Activation

Sunday, June 23, 2024

15:15 – 15:30 ICT

Room: 110 A-C

Presenting Author(s)

Dillon Bohinc

PhD. Student
Case Western Reserve University
Cleveland, Ohio, United States

Co-Author(s)

KB

Kara Bane

Laboratory Manager
Case Western Reserve University
Cleveland, Ohio, United States
KB

Kris Blair

Research Development Specialist
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium
Seattle, Washington, United States
EG

Eduarda Grinsztejn

Research Coordinator
Medicine Service, Section of Hematology-Oncology, Louis Stokes Veterans Administration Medical Center
Cleveland, Ohio, United States
SM

Steven Marshall

Research Technician
5Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center
Cleveland, Ohio, United States
RB

Robert Bonomo

Professor
Department of Medicine, Division of Infectious Diseases, Case Western Reserve University School of Medicine,
Cleveland, Ohio, United States
Daniel Lawrence

Professor
Department of Internal Medicine, Division of Cardiovascular Medicine, University of Michigan Medical School
Ann Arbor, Michigan, United States
Maria Sandkvist

Professor
1Department of Microbiology and Immunology, University of Michigan Medical School
Ann Arbor, Michigan, United States
Evi X. Stavrou, MD

Associate Professor
Department of Medicine, Hematology and Oncology Division, Case Western Reserve University (CWRU) School of Medicine
Cleveland, Ohio, United States

Background: Coagulation factor XII (FXII) is best known for its role in the contact pathway. Autoactivation of zymogen FXII initiates intrinsic coagulation and FXIIa activation of plasma kallikrein leads to cleavage of high-molecular-weight kininogen (HK) and generation of the proinflammatory peptide bradykinin. We have previously shown that zymogen FXII binds to uPAR on the surface of neutrophils, upregulating pro-inflammatory neutrophil functions. CpaA is a metalloprotease secreted by the bacterial pathogen Acinetobacter baumannii that targets the proline-rich domain of FXII, cleaving between prolines and O-linked glycosylated sites.

Aims: To examine the effects of CpaA on enzymatic and zymogen functions of FXII.

Methods: We utilized protein and cell-based assays to examine the effects of CpaA on the contact pathway and the FXII/uPAR/neutrophil signaling axis.

Results: CpaA cleaves FXII before O-glycosylated residues Thr280 and Thr309. CpaA cleaves both zymogen FXII and FXIIa but not βFXIIa. CpaA significantly increased clotting time in normal human plasma (NHP), which was partially restored by adding exogenous FXIIa in a concentration-dependent manner. CpaA-cleaved FXIIa mostly restored clotting time in FXII-deficient plasma, suggesting CpaA-cleaved FXIIa retains some enzymatic activity.
CpaA was able to cleave neutrophil-derived autocrine FXII and inhibit FXII-mediated neutrophil signaling through pAkt2S474 and phospho-p47phox. Neutrophils treated with CpaA-cleaved FXII showed a significant decrease in reactive oxygen species (ROS) generation and NET formation compared to neutrophils treated with intact FXII. A. baumannii bacteria secreting CpaA (wild type; WT) presented a significant survival advantage when incubated with healthy human neutrophils ex vivo compared to a mutant A. baumannii strain lacking the cpaA gene (ΔcpaA).

Conclusion(s): Here, we show that CpaA may serve as a compensatory bacterial rheostat that disrupts contact pathway initiation and FXII-driven proinflammatory neutrophil functions to promote bacterial survival.