Ehrlichia ruminantium uses its transmembrane protein Ape to adhere to host bovine aortic endothelial cells

Ehrlichia ruminantium is an obligate intracellular bacterium, transmitted by ticks of the genus Amblyomma and responsible for heartwater, a disease of domestic and wild ruminants. High genetic diversity of E. ruminantium strains hampers the development of an effective vaccine against all strains present in the field. In order to develop strategies for the control of heartwater through both vaccine and alternative therapeutic approaches, it is important to first gain a better understanding of the early interaction of E. ruminantium and its host cell. Particularly, the mechanisms associated with bacterial adhesion remain to elucidate. Herein, we studied the role of E. ruminantium membrane protein ERGA_CDS_01230 (UniProt Q5FFA9), a probable iron transporter, in the adhesion process to host bovine aortic endothelial cells (BAEC). The recombinant version of the protein ERGA_CDS_01230, successfully produced in the Leishmania tarentolae system, is O-glycosylated. Following in vitro culture of E. ruminantium in BAEC, the expression of CDS ERGA_CDS_01230 peaks at the extracellular infectious elementary body stages. This result suggest the likely involvement of ERGA_CDS_01230, named hereafter Ape for Adhesion protein of Ehrlichia, in the early interaction of E. ruminantium with its host cells. We showed using flow cytometry and scanning electron microscopy that beads coated with recombinant ERGA_CDS_01230 (rApe) adheres to BAEC. In addition, we also abserved that rApe interacts with proteins of the cell lysate, membrane and organelle fractions. Additionally, enzymatic treatment degrading dermatan and chondroitin sulfates on the surface of BAEC is associated with a 50% reduction in the number of bacteria in the host cell after a development cycle, indicating that glycosaminoglycans seem to play a role in the adhesion of E. ruminantium to the host cell. Finally, Ape induces a humoral response in vaccinated animals. Globally, our work identifying the role of Ape in E. ruminantium adhesion to host cells makes it a gold vaccine candidate and represents a first step toward the understanding of the mechanisms of cell invasion by E. ruminantium.

Autocrine Bradykinin Release Promotes Ischemic Preconditioning-Induced Cytoprotection in Bovine Aortic Endothelial Cells

The aims of this study were to assess whether ischemic preconditioning (PC) induces bradykinin (Bk) synthesis in bovine aortic endothelial cells (bAECs) and, if so, to explore the molecular mechanisms by which this peptide provides cytoprotection against hypoxia. PC was induced by exposing bAECs to three cycles of 15 min of hypoxia followed by 15 min of reoxygenation. Bk synthesis peaked in correspondence to the early and late phases of PC (10−12 M and 10−11 M, respectively) and was abolished by a selective tissue kallikrein inhibitor, aprotinin. Stimulation with exogenous Bk at concentrations of 10−12 M and 10−11 M reduced the cell death induced by 12 h of hypoxia by 50%. Pretreatment with HOE−140, a Bk receptor 2 (BKR2) inhibitor, in bAECs exposed to 12 h of hypoxia, abrogated the cytoprotective effect of early and late PC, whereas des-Arg-HOE-140, a Bk receptor 1 (BKR1) inhibitor, affected only the late PC. In addition, we found that PC evoked endocytosis and the recycling of BKR2 during both the early and late phases, and that inhibition of these pathways affected PC-mediated cytoprotection. Finally, we evaluated the activation of PKA and Akt in the presence or absence of BKR2 inhibitor. HOE-140 abrogated PKA and Akt activation during both early and late PC. Consistently, BKR2 inhibition abolished cross-talk between PKA and Akt in PC. In bAECs, Bk-synthesis evoked by PC mediates the protection against both apoptotic and necrotic hypoxia-induced cell death in an autocrine manner, by both BKR2- and BKR1-dependent mechanisms.

Tensional homeostasis at different length scales

Traction field temporal fluctuations of bovine aortic endothelial cells; each color corresponds to a single cell (left), and a representative traction field of a single cell (right) (adapted from ref. 18 with permission from Elsevier).