Studies in Blood-Brain Barrier Disruption in Anthrax Meningitis

STUDIES ON BLOOD-BRAIN BARRIER DISRUPTION IN ANTHRAX MENINGITIS Dhritiman V. Mukherjee, Ph.D. George Mason University, 2009 Dissertation Director: Dr. Serguei G. Popov B. anthracis causes hemorrhagic meningitis. The pathogenesis and molecular mechanisms associated with blood-brain barrier (BBB) dysfunction in anthrax meningitis remain poorly understood. We reported previously that anthrax-secreted metalloprotease Immune-Inhibitor A (InhA) degrades various substrates, including extracellular matrix proteins, endogenous inhibitors, and coagulation proteins. To understand the possible role and mechanism(s) of this potential virulence factor in disruption of BBB, we investigated its effects on the integrity and permeability of human brain microvasculature endothelial cells (HBMEC), as a model of blood-endothelial barriers. A time-dependent decrease of trans-epithelial and endothelial resistance by InhA suggests that HBMEC are highly sensitive to InhA. One of the major tight junction (TJ) proteins in vascular endothelial cells, ZO-1, was found to be a molecular target of InhA treatment. ZO-1 existing in cell lysates, intact cells, and recombinant forms was effectively degraded by InhA. Inhibition of phagocytosis by cytochalasin D and monodansylcadaverine disrupted the ZO-1 degradation, and immunostaining of InhA showed its colocalization with ZO-1 in cell periphery, strongly suggesting that InhA is delivered to target ZO-1 by phagocytosis. In addition, InhA-transformed B. subtilis efficiently invaded, compared to its wild type, and anti-InhA antibodies inhibited this invasion of B. anthracis spores into HBMEC, suggesting thereby InhA-mediated spore entry. Finally, mice challenged with purified InhA underwent the BBB leakage in a time-dependent manner. Injection of Quantum dotconjugated InhA into mice provided further evidence that InhA is required at the BBB leakage of anthrax meningitis. These findings present a previously unknown role and mechanism of the bacterial (B. anthracis) protease, InhA, in CNS vascular endothelial disruption, involving a mechanism facilitated by degradation of ZO-1 by InhA. Persuasive evidence is presented of the selective targeting of the major scaffolding protein, zonulin, in brain endothelial tight junction by the anthrax-secreted metalloprotease, InhA, and its close association with increased BBB permeability and in vivo extravasation (hemorrhage) in brain. These observations suggest that B. anthracis meningitis may primarily result from disintegration of the structural organization of the junctional complex of endothelial cells by bacterial product (InhA). However, it remains uncertain whether or not direct passage of the bacterial has a role in barrier disruption. Nonetheless, the mechanism proposed adds a new dimension to our understanding of anthrax meningitis.