Characterization of Acanthamoeba adherence and benzimidazole susceptibility

Vicki R Gordon, Purdue University

Abstract

Acanthamoeba is a genus of amoebae which contains species which include opportunistic pathogens. These species can cause granulomatous amoebic encephalitis, a chronic fatal infection, mostly among debilitated and/or immunocompromised patients. These amoebae can also cause Acanthamoeba keratitis in mostly healthy individuals, a severe corneal infection which is difficult to treat. The thesis research summarized in this dissertation addresses adhesion, the postulated second step in the pathogenesis of these diseases, by studying the interaction of Acanthamoeba with the extracellular matrix proteins, collagen type I and fibronectin, and the basal lamina proteins collagen type II and laminin. The absence of optimal drug therapies was addressed by the screening of several drugs for inhibition of Acanthamoeba growth. An in vitro binding assay was developed in which culture clusters coated with matrix proteins were seeded with $\sp{35}$S methionine-labeled amoebae in the presence or absence of putative inhibitors of adhesion. These potential inhibitors included $\alpha$-methylmannopyranoside (AMM), fucose, Acanthamoeba-specific antisera, AIIB$\sb2$ (a monoclonal antibody against $\sb1$-integrins), and the RGDS peptide (inhibits the binding of some integrins). The amoebae adherence to human corneal fibroblasts and endothelial cells (with or without these inhibitors) was also monitored. The effect these inhibitors had on binding was determined by scintillation counting of the remaining bound amoebae after they were solubilized with 1% sodium dodecyl sulfate. Fucose and AMM had similar effects on Acanthamoeba binding to the matrix proteins and cornea cells. Fucose had no effect on binding while AMM decreased binding. Affinity chromatography using an AMM matrix column isolated a 20 kilodalton (kD) protein. Surface iodination and biotinylation both suggested that all or part of the 20 kD molecule is exposed at the amoeba surface. Neither the AIIB$\sb2$ antibody or the RGDS peptide affected binding and suggests that the major mediator of binding to the proteins was not an integrin. Acanthamoeba appeared to bind more strongly to corneal fibroblast cells than to corneal endothelial cells. The amoebae were incubated with varying concentrations of several drugs to determine their ability to inhibit Acanthamoeba growth. Itraconazole, a triazole compound presently used to treat Acanthamoeba keratitis, was a positive control. Two drugs tested, benomyl and carbendazim, were more effective than itraconazole at inhibiting the growth of the amoeba.

Degree

Ph.D.

Advisors

McLaughlin, Purdue University.

Subject Area

Pathology|Microbiology|Veterinary services

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