Abstract
Rickettsial infections continue to cause serious morbidity and mortality in severe human cases around the world. Host cell adhesion and invasion is an essential requisite for intracellular growth, replication, and subsequent dissemination of pathogenic rickettsiae. Heparan sulfate proteoglycans [HSPGs] facilitate the interactions between fibroblast growth factor(s) and their tyrosine kinase receptors resulting in receptor dimerization/activation and have been implicated in bacterial adhesion to target host cells. In the present study, we have investigated the contributions of fibroblast growth factor receptors [FGFRs] in rickettsial entry into the host cells. Inhibition of HSPGs by heparinase and FGFRs by AZD4547 (a selective small-molecule inhibitor) results in significant reduction in rickettsial internalization into cultured human microvascular endothelial cells (ECs), which represent the primary targets of pathogenic rickettsiae during human infections. Administration of AZD4547 during R. conorii infection in a murine model of endothelial-target spotted fever rickettsiosis also diminishes pulmonary rickettsial burden in comparison to mock-treated controls. Silencing of FGFR1 expression using a small interfering RNA also leads to similar inhibition of R. rickettsii invasion into ECs. Consistent with these findings, R. rickettsii infection of ECs also results in phosphorylation of tyrosine 653/654, suggesting activation of FGFR1. Using isobaric tag for relative and absolute quantitation [iTRAQ]-based proteomics approach, we further demonstrate association of β-peptide of rickettsial outer membrane protein OmpA with FGFR1. Mechanistically, FGFR1 binds to caveolin-1 and mediates bacterial entry via caveolin-1 dependent endocytosis. Together, these results identify host cell FGFR1 and rickettsial OmpA as another novel receptor-ligand pair contributing to the internalization of pathogenic rickettsiae into host endothelial cells and the potential application of FGFR-inhibitor drugs as adjunct therapeutics against spotted fever rickettsioses.
Highlights
Employing a coalescence of biochemical, molecular, and pharmacological approaches, we have identified that pathogenic Rickettsia species belonging to the spotted fever group avail the host FGFR1/heparan sulfate proteoglycans (HSPGs) complex to gain entry into target endothelial cells (ECs)
These findings represent the first report on the contributions of outer membrane protein A (OmpA)-FGFR1 as yet another ligand-receptor system and associated downstream signaling mechanisms in facilitating rickettsial internalization into host endothelium
FGFR1, FGFR2, FGFR3, and FGFR4 constitute the FGF receptors (FGFRs) family of receptor tyrosine kinases with three immunoglobulin G-like domains in the extracellular region [25, 26]
Summary
Pathogenic spotted fever group (SFG) rickettsiae predominantly infect microvascular endothelium lining of blood vessels as their preferred primary targets in mammalian hosts, resulting in endothelial dysfunction characterized by induction of acute phase responses, infiltration of inflammatory cells, and compromised barrier function [1]. Resultant activation of FGFR1 leads to the recruitment and subsequent tyrosine phosphorylation of the docking protein FGFR substrate (FRS)-2α linking the receptor to intracellular signaling pathways [10]. Using a proteomics-based approach, we have identified β-peptide of rickettsial OmpA as an interacting partner of host FGFR1. These results demonstrate the importance of rickettsial interactions with FGFR1 in facilitating host cell invasion and substantiate the concept of exploitation of redundant entry mechanisms by pathogenic rickettsiae. A small-molecule FGFR inhibitor AZD4547 exhibits a significant inhibitory effect on rickettsial invasion of microvascular endothelium and pulmonary rickettsial burden in a mouse model of infection, suggesting inhibition of FGFR1-mediated pathogen entry into host cells as a useful adjunct strategy to combat rickettsial infections
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