Rickettsiae Induce Microvascular Hyperpermeability via Phosphorylation of VE-Cadherins: Evidence from Atomic Force Microscopy and Biochemical Studies

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Abstract
The most prominent pathophysiological effect of spotted fever group (SFG) rickettsial infection of microvascular endothelial cells (ECs) is an enhanced vascular permeability, promoting vasogenic cerebral edema and non-cardiogenic pulmonary edema, which are responsible for most of the morbidity and mortality in severe cases. To date, the cellular and molecular mechanisms by which SFG Rickettsia increase EC permeability are largely unknown. In the present study we used atomic force microscopy (AFM) to study the interactive forces between vascular endothelial (VE)-cadherin and human cerebral microvascular EC infected with R. montanensis, which is genetically similar to R. rickettsii and R. conorii, and displays a similar ability to invade cells, but is non-pathogenic and can be experimentally manipulated under Biosafety Level 2 (BSL2) conditions. We found that infected ECs show a significant decrease in VE-cadherin-EC interactions. In addition, we applied immunofluorescent staining, immunoprecipitation phosphorylation assay, and an in vitro endothelial permeability assay to study the biochemical mechanisms that may participate in the enhanced vascular permeability as an underlying pathologic alteration of SFG rickettsial infection. A major finding is that infection of R. montanensis significantly activated tyrosine phosphorylation of VE-cadherin beginning at 48 hr and reaching a peak at 72 hr p.i. In vitro permeability assay showed an enhanced microvascular permeability at 72 hr p.i. On the other hand, AFM experiments showed a dramatic reduction in VE-cadherin-EC interactive forces at 48 hr p.i. We conclude that upon infection by SFG rickettsiae, phosphorylation of VE-cadherin directly attenuates homophilic protein–protein interactions at the endothelial adherens junctions, and may lead to endothelial paracellular barrier dysfunction causing microvascular hyperpermeability. These new approaches should prove useful in characterizing the antigenically related SFG rickettsiae R. conorii and R. rickettsii in a BSL3 environment. Future studies may lead to the development of new therapeutic strategies to inhibit the VE-cadherin-associated microvascular hyperpermeability in SFG rickettsioses. Rickettsial diseases are serious human infections. Some spotted fever group (SFG) rickettsial pathogens are bioterror agents. A major clinical hallmark of SFG rickettsial disease is the infection of endothelial cells leading to enhanced vascular permeability. Previous studies show that SFG rickettsiae cause dose-dependent hyperpermeability, which was associated with disruption of intercellular adherens junctions (AJs). The underlying molecular mechanism by which the junctional complexes are disrupted, ultimately causing changes in the endothelial paracellular milieu during rickettsial infection, remains largely unclear. The available evidence suggests that inflammatory stimuli can trigger tyrosine phosphorylation of various components of AJs, mainly the vascular endothelial–cadherin (VE-cadherin). This causes gaps at AJs, partially due to phosphorylation-induced destabilization of VE-cadherins at the plasma membrane and increased endocytosis, greatly increasing paracellular leaks. Here, we hypothesize that infection by SFG rickettsiae induces endothelial cells to develop altered VE-cadherin in association with phosphorylation of tyrosine residues. Utilizing nano-mechanical studies with atomic force microscopy and biochemical analysis of the major AJ protein VE-cadherin, we have implicated that phosphorylation of VE-cadherin directly attenuates homophilic interactions between VE-cadherins. The experimental approach advances a new way of studying rickettsial infection. This strategy should prove useful in uncovering novel therapeutic strategies for virulent arthropod-borne rickettsioses.