Staurosporine as a Potential Treatment for Acanthamoeba Keratitis Using Mouse Cornea as an Ex Vivo Model

Abstract

Acanthamoeba is a ubiquitous genus of amoebae that can trigger a severe and progressive ocular disease known as Acanthamoeba Keratitis (AK). Furthermore, current treatment protocols are based on the combination of different compounds that are not fully effective. Therefore, an urgent need to find new compounds to treat Acanthamoeba infections is clear. In the present study, we evaluated staurosporine as a potential treatment for Acanthamoeba keratitis using mouse cornea as an ex vivo model, and a comparative proteomic analysis was conducted to elucidate a mechanism of action. The obtained results indicate that staurosporine altered the conformation of actin and tubulin in treated trophozoites of A. castellanii. In addition, proteomic analysis of treated trophozoites revealed that this molecule induced overexpression and a downregulation of proteins related to key functions for Acanthamoeba infection pathways. Additionally, the ex vivo assay used validated this model for the study of the pathogenesis and therapies of AK. Finally, staurosporine eliminated the entire amoebic population and prevented the adhesion and infection of amoebae to the epithelium of treated mouse corneas.

Keywords: Acanthamoeba; PCD; ex vivo; mouse cornea; proteomic analysis; staurosporine.

Figure 1

Evaluation of the effect of IC₉₀ of staurosporine on the actin and tubulin cytoskeleton of Acanthamoeba castellanii trophozoites for 24 h. The phalloidin-TRITC dye stains the polymerised actin cytoskeleton showing the normal organization of the networks with an orange fluorescence in the negative control cells (A); scale bar represents 10 µm. Treated cells emitted a lower orange fluorescence, and trophozoites showed disorganized and degraded acanthopodium (B); scale bar represents 10 µm. Tubulin antibodies bind to microtubules and stain them in control cells showing an intense red fluorescence and demonstrating a normal conformation (C); scale bar represents 5 µm. However, trophozoites incubated with staurosporine show disorganization or destruction of the tubulin microtubules (D); scale bar represents 2 µm. Mounting with DAPI solution for DNA staining shows a blue fluorescence (C,D). All images (63×) were obtained using an inverted confocal light microscope Leica DMI 4000 B (Deerfield, IL, USA).

Figure 2

Volcano graph expressing a logarithmic Student’s t-test p-value as a function of staurosporine protein control fold change.

Figure 3

Scanning electron microscopy analysis of the effect of Acanthamoeba castellanii trophozoites and/or staurosporine on mouse corneas during 3 h. The co-incubation of mouse corneas with staurosporine at IC₉₀ shows no evidence of damage or cell disorganization (A); Group 1, scale bar represents 10 µm. Mouse corneas co-cultured with trophozoites of Acanthamoeba castellanii (B); scale bar represents 10 µm; Group 2 presented de-epithelization of the corneal epithelium by the amoebic infection penetrating between the junctions of the epithelial cells (white arrows). Corneas co-incubated with trophozoites of Acanthamoeba castellanii pretreated with staurosporine 30 min prior (C); scale bar represents 10 µm; Group 3 showed that fewer trophozoites adhered to the corneal epithelium (black arrows), which did not migrate toward the inner layers of the corneal epithelium; therefore, the damage was very limited. In the corneas with simultaneous infection and treatment with staurosporine (D)—Group 4, scale bar represents 10 µm—the cellular debris of Acanthamoeba castellanii trophozoites was observed (black arrows), and the corneal epithelium showed no cell damage or signs of amoebic invasion. Corneas co-cultured with trophozoites and treated with staurosporine 30 min after infection (E); scale bar represents 10 µm; Group 5 showed that no-healthy cells adhered to the corneal surface (black arrow), and the corneal epithelium showed signs of early stages of amoebic infection. Images were obtained using a JEOL-JSM 7100F scanning electron microscope (JEOL Ltd., Tokyo, Japan).