Bacillus anthracis Protease InhA Increases Blood-Brain Barrier Permeability and Contributes to Cerebral Hemorrhages

Dhritiman V Mukherjee,Taissia G Popova,Serguei Popov,Charles Bailey,Jessica H Tonry,Myung-Chul Chung,Nalini Ramarao,Kwang Sik Kim,Ramy Aziz

doi:10.1371/journal.pone.0017921

Abstract

Hemorrhagic meningitis is a fatal complication of anthrax, but its pathogenesis remains poorly understood. The present study examined the role of B. anthracis-secreted metalloprotease InhA on monolayer integrity and permeability of human brain microvasculature endothelial cells (HBMECs) which constitute the blood-brain barrier (BBB). Treatment of HBMECs with purified InhA resulted in a time-dependent decrease in trans-endothelial electrical resistance (TEER) accompanied by zonula occluden-1 (ZO-1) degradation. An InhA-expressing B. subtilis exhibited increased permeability of HBMECs, which did not occur with the isogenic inhA deletion mutant (ΔinhA) of B. anthracis, compared with the corresponding wild-type strain. Mice intravenously administered with purified InhA or nanoparticles-conjugated to InhA demonstrated a time-dependent Evans Blue dye extravasation, leptomeningeal thickening, leukocyte infiltration, and brain parenchymal distribution of InhA indicating BBB leakage and cerebral hemorrhage. Mice challenged with vegetative bacteria of the ΔinhA strain of B. anthracis exhibited a significant decrease in leptomeningeal thickening compared to the wildtype strain. Cumulatively, these findings indicate that InhA contributes to BBB disruption associated with anthrax meningitis through proteolytic attack on the endothelial tight junctional protein zonula occluden (ZO)-1.

Highlights

Bacillus anthracis is a gram-positive, spore-forming, rod-shaped bacterium that causes three distinct clinical forms of anthrax depending on the major routes of infection: inhalational, cutaneous, and gastrointestinal [1]
Using human brain microvasculature endothelial cells (HBMECs) as an in vitro model of bloodbrain barrier (BBB) we demonstrate that increased permeability takes place upon internalization of Inhibitor A (InhA) into HBMECs followed by degradation of zonula occluden-1 (ZO-1)
Incubation of the cells with InhA and B. anthracis deltaSterne culture supernatant significantly increased monolayer permeability, whereas o-phenanthroline (100 mM) an inhibitor of metalloproteases abrogated the permeabilizing activity of InhA and the bacterial culture supernatant

Summary

Introduction

Bacillus anthracis is a gram-positive, spore-forming, rod-shaped bacterium that causes three distinct clinical forms of anthrax depending on the major routes of infection: inhalational, cutaneous, and gastrointestinal [1]. Clinical case reports indicate that bacteria disseminated to the brain can cause hemorrhagic meningitis, a life-threatening pathology manifested as a ‘‘cardinal’s cap’’, due to the characteristic appearance of meninges upon autopsy [1,2,3,4]. During the epidemic of inhalation anthrax in Sverdlovsk, Russia, fatal hemorrhagic meningitis was reported in 50% of the individuals infected with aerosolized B. anthracis spores [3,4]. Histopathological analysis indicates that during anthrax the secreted pathogenic factors of B. anthracis exert devastating effects on the BBB, rendering it leaky and permeable [2,3,4]

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Results

Conclusion