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Acanthamoeba is an opportunistic protozoan pathogen that can cause fatal granulomatous encephalitis and a sight-threatening keratitis. However, the pathogenic mechanisms associated with Acanthamoeba infections remain unclear. One of the primary requirement in Acanthamoeba encephalitis is the haematogenous spread, followed by invasion of the blood-brain barrier. Using human brain microvascular endothelial cells (HBMEC) which constitute the blood-brain barrier, here we studied Acanthamoeba interactions with HBMEC. The results revealed that Acanthamoeba binds to HBMEC and adhesion can be blocked using exogenous mannose suggesting a role for mannose-binding protein (MBP). Acanthamoeba mutants expressing reduced levels of MBP showed decreased binding, further suggesting that MBP plays a role in HBMEC interactions. Among 12 isolates belonging to 5 different genotypes, the clinical isolates belonging to T4 genotype exhibited optimum binding. Adhesion most likely leads to secondary processes such as phagocytosis and toxin secretion, which are crucial for the ability ofAcanthamoeba to produce host cell damage. Using heat-killed fluorescein isothiocyanate (FITC)-labelled Escherichia coli, phagocytosis in Acanthamoeba was studied. Genistein as well as cytochalasin D blocked E. coli uptake, suggesting that Acanthamoeba phagocytosis is dependent on protein tyrosine kinasemediated intracellular signalling pathways and involve cytoskeletal rearrangements. In support of this notion, Y27632 (RhoA inhibitor) reduced bacterial uptake. By using L Y294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, we identified PI3K as an important mediator ofAcanthamoeba phagocytosis. Next, we studied the role of proteases in Acanthamoeba traversal of the blood-brain barrier using in vitro permeability assays. Our findings revealed that Acanthamoeba conditioned medium produced approximately 80% increase in HBMEC permeability, by targeting tight junctions. Western blotting assays determined that extracellular proteases of Acanthamoeba degrade zonula-l and occ1udin, key molecules in the formation of tight junctions. Prior treatment with PMSF (a serine protease inhibitor) abolished permeability changes, indicating the role of serine proteases. Zymographic assays revealed thatAcanthamoeba produced two major proteases, one of which was inhibited with PMSF and the second with 1,1O-phenanthroline (metalloprotease inhibitor). We also studied the host defence mechanisms in case of AK. Using tears from healthy individuals and an AK patient, it was demonstrated that both subjects exhibited similar levels of Acanthamoeba-specific IgA as determined by Western blotting and enzymelinked immunosorbent assay. However, normal tears were slightly more potent in reducingAcanthamoeba binding to human corneal epithelial cells, compared with tears from AK patient. Neither normal tears nor AK tears had any protective effects on Acanthamoeba-mediated corneal epithelial cell cytotoxicity. Future studies should continue to identify mechanisms associated with Acanthamoeba pathogenesis, imperative for the rationale development of therapeutic interventions. |
