Staphylococcus aureus–induced endothelial permeability and inflammation are mediated by microtubule destabilization

Pratap Karki,Yunbo Ke,Yufeng Tian,Tomomi Ohmura,Albert Sitikov,Nicolene Sarich,Christopher P Montgomery,Anna A Birukova

doi:10.1074/jbc.ra118.004030

Abstract

Staphylococcus aureus is a major etiological agent of sepsis and induces endothelial cell (EC) barrier dysfunction and inflammation, two major hallmarks of acute lung injury. However, the molecular mechanisms of bacterial pathogen-induced EC barrier disruption are incompletely understood. Here, we investigated the role of microtubules (MT) in the mechanisms of EC barrier compromise caused by heat-killed S. aureus (HKSA). Using a customized monolayer permeability assay in human pulmonary EC and MT fractionation, we observed that HKSA-induced barrier disruption is accompanied by MT destabilization and increased histone deacetylase-6 (HDAC6) activity resulting from elevated reactive oxygen species (ROS) production. Molecular or pharmacological HDAC6 inhibition rescued barrier function in HKSA-challenged vascular endothelium. The HKSA-induced EC permeability was associated with impaired MT-mediated delivery of cytoplasmic linker-associated protein 2 (CLASP2) to the cell periphery, limiting its interaction with adherens junction proteins. HKSA-induced EC barrier dysfunction was also associated with increased Rho GTPase activity via activation of MT-bound Rho-specific guanine nucleotide exchange factor-H1 (GEF-H1) and was abolished by HDAC6 down-regulation. HKSA activated the NF-κB proinflammatory pathway and increased the expression of intercellular and vascular cell adhesion molecules in EC, an effect that was also HDAC6-dependent and mediated, at least in part, by a GEF-H1/Rho-dependent mechanism. Of note, HDAC6 knockout mice or HDAC6 inhibitor-treated WT mice were partially protected from vascular leakage and inflammation caused by both HKSA or methicillin-resistant S. aureus (MRSA). Our results indicate that S. aureus-induced, ROS-dependent up-regulation of HDAC6 activity destabilizes MT and thereby activates the GEF-H1/Rho pathway, increasing both EC permeability and inflammation.

Highlights

Staphylococcus aureus is a major etiological agent of sepsis and induces endothelial cell (EC) barrier dysfunction and inflammation, two major hallmarks of acute lung injury
We further investigated the MT-dependent mechanism of EC permeability and inflammation caused by heat-killed S. aureus (HKSA)
This study shows that HKSA-induced pulmonary EC permeability and inflammation is associated with destabilization of the MT cytoskeleton caused by HKSA-induced activation of histone deacetylase-6 (HDAC6) enzymatic activity, which has not been shown previously

Summary

To whom correspondence should be addressed

Staphylococcus aureus (SA) infections are the predominant cause of sepsis, which is still the twelfth leading cause of death in the U. The small GTPases RhoA, Rac, and Rap play an active role in vascular endothelial cytoskeletal remodeling and the regulation of endothelial barrier integrity [10] These GTPases act as a molecular switch by cycling between GTP-bound active and. CLASP2 interaction with adherens junction protein p120 catenin observed in keratinocytes [36, 37] suggests its additional role in linking MT with cell junction complexes These features suggest that CLASP2 is a potentially important regulator of MT–actin cytoskeleton cross-talk and EC barrier regulation. By employing molecular and pharmacological inhibitors, we elucidated the mechanisms of HKSA-induced activation of HDAC6 that cause MT destabilization and MT-dependent signaling, leading to increased endothelial permeability and vascular inflammation. We evaluated the protective effects of HDAC6 inhibition as a potential therapeutic for HKSA or MRSA-induced vascular leak and inflammation in vivo

Results

Discussion

Experimental procedures

Evaluation of lung injury parameters