All biosafety level (BSL) 3 or ABSL3 experiments were performed in CDC-certified facilities in the Galveston National Laboratory at UTMB, Galveston, TX, using established procedures and precautions

All biosafety level (BSL) 3 or ABSL3 experiments were performed in CDC-certified facilities in the Galveston National Laboratory at UTMB, Galveston, TX, using established procedures and precautions. bacterial infections, which is needed for developing therapeutics that could limit infection at this initial stage. Obligately intracellular bacteria in the genus ([22, 23], [24], and [25, 26]. Typically, rickettsiae are transmitted through the bite of infected ticks, and ECs are the primary vertebrate host target cells [11, 27]. The initial step in establishing a productive intracellular infection is for the bacterium to recognize and establish an adhesive interaction with specific cellular receptor(s) to firmly anchor itself on the host EC luminal surface, thus overcoming detachment by shear stress from blood flow prior to invasion into the EC [11, 28]. Therefore, rickettsial infection is a suitable model to employ for studying endovascular bacterial adhesion. Remarkable insights into the rickettsial components involved in this initial interaction have come from identification of rickettsial adhesins [29C35], although comparatively little is known about host surface receptor(s) and the mechanism for establishing the connection between the host cell surface and rickettsiae. Host proteins Ku70 [36], 21 integrin [29], clathrin [37], caveolin 2 [37], and exchange protein activated by cAMP (EPAC) [38] have been identified as being involved in rickettsial invasion into nonphagocytic host cells via endocytic mechanisms [36]. Yet, as analyzed by an immunofluorescence (IF)-based assay, only the 21 integrin heterodimer [29] and EPAC1 [38] were shown to be involved in rickettsial adhesion to the host cell surface. 21 integrin mainly serves as a endothelial receptor for extracellular matrix molecules [39]. EPAC is an intracellular cAMP receptor [40] and is speculated to play a regulatory role, rather than as a direct receptor for rickettsial adherence on the host cell surface. Using a functional antibody specific to KU70, it was shown that rickettsial invasion into Vero cells was effectively blocked, but there was no effect on rickettsial adhesion to Vero cell surfaces [36]. ECs express abundant plasminogen (Plg), and Plg activator binding sites on their vascular luminal surfaces serve plasmin-based fibrinolytic functions [41], among which the annexin A2 (ANXA2) is the best recognized and is emerging as the focus of research on a growing spectrum of biologic and pathologic processes [42, 43]. ANXA2 is a Ca2+-regulated and phospholipid-binding protein that associates with cell membrane lipid rafts and the actin cytoskeleton [42, 44, 45]. It is detected on endothelial surfaces in the form of a complex with S100A10, (ANXA2-S100A10)2. Of note, there is in vitro IITZ-01 evidence that ANXA2 participates in efficient invasion of [46], [47, 48], [49], [50], and [51] in epithelial IITZ-01 CDC42EP1 cells via regulation of cytoskelton remodeling in the vicinity of lipid rafts. Neutralization of the ligands on by incubation of the bacteria with recombinant, soluble ANXA2 prevents bacterial entry into human epithelial cells, suggesting ANXA2 may be a receptor for bacterial adherence and/or invasion [49]. Although ANXA2 has been identified as a binding partner of adhesin clumping factor A in a proteinCprotein IITZ-01 binding assay [46], direct in vitro or in vivo evidence are completely lacking in the field of endovascular infections after adherence to ECs. In the present study, we employ a novel, anatomically based, in vivo quantitative bacterial-adhesion-to-vascular-EC analysis system, combined with atomic force microscopy (AFM), to examine the role of endothelial luminal surface ANXA2 during rickettsial adherence to ECs. We identified endothelial surface ANXA2 as a receptor for SFG rickettsial adhesion in vivo using AFM.