Staurosporine as a potential treatment for Acanthamoeba keratitis using mouse cornea as an ex vivo model

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Rodríguez-Expósito, Ines Sifaoui, Lizbeth Salazar-Villatoro, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3878546/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Currently, six different genera were reported to be pathogenic to humans and animals, which the most common being Acanthamoeba genus. Acanthamoeba is a ubiquitous genus of amoebae that can trigger severe and progressive ocular disease kwon as Acanthamoeba Keratitis (AK). Furthermore, actual treatment protocols are based on the combination of different compounds that are not fully effective in eliminating the parasite in ocular infections. Therefore, this leads to an urgent need to develop new compounds to treat Acanthamoeba infections. In the present study, we have evaluated Staurosporine as a potential treatment for Acanthamoeba keratitis using mouse cornea as an ex vivo model, and to investigate its model of action by comparative proteomic analysis. Staurosporine altered the conformation of actin and tubulin cytoskeleton of treated trophozoites of A. castellanii. In addition, proteomic analysis of the effect of Staurosporine on treated trophozoites revelated that this molecule induced an overexpression and a down-regulation of proteins related to functions vital for Acanthamoeba infections. Additionally, obtained results in this study on the ex vivo assay using mouse corneas validate this animal model for the study of the pathogenesis of AK. Finally, Staurosporine eliminated the entire amoebic population and prevented adhesion and infection of amoebae to the epithelium of treated mouse corneas. Acanthamoeba ex vivo mouse cornea Staurosporine proteomic analysis PCD Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Free-living amoebae are ubiquitous single-celled living organisms isolated from multiple habitats including water and soil. Until present, six different genera were reported to be pathogenic to humans and animals, which the most common being Acanthamoeba genus. This amoeba could cause a threatening eye infection known as Acanthamoeba keratitis: a rare eye infection but commonly associated with contact lens wearers [ 1 , 2 ]. Although, Acanthamoeba keratitis constitutes 2% of the corneal infections, its incidence has been consistently increasing [ 3 ]. Actual treatment protocols are based on the combination of cationic antiseptics such as polyhexamethylene biguanide (0.02%) or chlorhexidine (0.02%) and aromatic diamidines such as propamidine (0.1%) or hexamidine (0.1%) [ 4 – 6 ]. Still those current therapies are not fully effective in eliminating the parasite because of their variable efficacy among different genotypes, the appearance of highly resistant cyst form, or due to their toxicity generated by a prolonged administration. This leads to an urgent need to discover new drugs and/or drug posology. On the one hand, in vitro assays are fundamental for drug discovery against the present infection, because they are simple, reproductible and much more economical than the in vivo assay, but still inefficient to predict the drug’s action inside the host organism. On the other hand, in vivo assays are more clinically relevant and the drug effect and side effect could be much more accurate, yet in vivo assays are much more expensive and harder to control as they include multitude of variables. Ex vivo assays are important preclinical tools between in vitro and in vivo assays. In these ex vivo systems, the cytoarchitecture and intracellular connections and metabolic processes could be conserved, mimicking the in vivo environment [ 7 ]. Rabbits, hamsters and mice were used as models for in vivo assay of Acanthamoeba Keratitis [ 8 , 9 ]. To the best of our knowledge, only the hamster cornea has been used as an ex vivo model for Acanthamoeba keratitis [ 10 – 13 ]. The aim of the present study was to evaluate Staurosporine as potential treatment for Acanthamoeba keratitis using mouse cornea as an ex vivo model and to investigate its model of action by comparative proteomic analysis. Study design/ Material and Methods Acanthamoeba strain Trophozoites of A. castellanii (genotype T4) isolated from a clinical case of AK from “Asociación para evitar la ceguera en México”, Luis Sánchez Bulnes Hospital, Mexico City. The strain was reported to be invasive in the GAE murine model [ 14 – 16 ]. The strain was grown axenically in PYG medium (0.75% (w/v) proteose peptone, 0.75% (w/v) yeast extract and 1.5% (w/v) glucose) containing 40 µg gentamicin ml − 1 (Biochrom AG, Cultek, Granollers, Barcelona, Spain) at 26°C. After 72 hours of incubation (end of growth logarithmic phase), culture was centrifuged (2500 rpm during 5 min) and trophozoites were harvested for the subsequent assays. In vitro assay Fluorescent staining of actin distribution For direct fluorescent staining, trophozoites of A. castellanii were treated first with the IC 90 (2.70 ± 0.015 µM) of the Staurosporine. After 30 min of incubation, cells were fixed with formaldehyde and deposit on pre-coated coverslip. Later, cells were treated with Triton (0.1%) for 30 min followed by Phalloidin–tetramethylrhodamine B isothiocyanate (Phalloidin-TRITC; Sigma-Aldrich, Madrid) for another 30 min at room temperature. Finally, cells were washed with PBS and later examined by Z -stack imaging using an inverted light confocal microscope Leica DMI 4000 B with LAS X software, a 532 nm laser and Leica HCX PL Apo 63x Oil Objective were used. Untreated cells were considered as the negative control. Immunofluorescence staining of tubulin of Acanthamoeba trophozoites. The immunofluorescence staining of tubulin was conducted using the immunofluorescence staining procedure of the manufacture (Sigma-Aldrich) with slight modifications. Briefly, cells were treated first the IC 90 of Staurosporine for 30 min. 50 µL of the cell suspension were placed on a gelatine precoated coverslip for 30 min. Later they were fixed with paraformaldehyde (4%). After 15 min, cells were treated with Triton (0.3%) for 10 min followed by 3 washes with PBS 1X. At this stage, cells were treated with 5% BSA in PBS 1X/150 mM sucrose for 30 min and washed with glycine 100 mM in PBS 1X for 5 min. Later, the trophozoites were incubated with the first anti-tubulin antibody 1:2000 for 2 hours at room temperature (Monoclonal Anti-α-Tubulin antibody produced in mouse, Sigma-Aldrich, Madrid). After 3 washes with PBS 1X cells were incubated with the second antibody labeled with Alexa 594 (1:500) for 1 hour at room temperature in darkness (Goat anti-Mouse IgG (H + L) Highly Cross – Adsorbed Secondary Antibody, Alexa Fluor Plus 594; Thermo Fisher Scientific, Rockford; USA). Finally, cells were washed with PBS 1X and mounted in DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride; Sigma-Aldrich; Madrid) containing mounting solution. 3D and maximum projection imaging of the trophozoites were performed by Z-stack imaging using an inverted light confocal microscope Leica DMI 4000 B with LAS X software, a 405 nm laser and 532 nm laser and Leica HCX PL Apo 63x Oil Objective were used. Proteomic analysis Comparative label-free proteomic analysis was conducted described in our recent study [ 17 ]. 10 6 cells of A. castellanii were treated with the IC 50 of Staurosporine for 24 hours, washed 1 time with PBS and the pellets were subjected to further processing. Untreated cells were prepared as a control group. Both groups were prepared in three biological replicates. Protein identification was made using the data base of https://www.uniprot.org/ . Ex vivo infection Co-incubation and interaction of A. castellanii trophozoites with BALB/c mice cornea. A total of 12 pathogen-free male BALB/c ( Mus musculus ) mice were used, with an average age of 21 to 28 days and an average body weight of 35 g. Experiments were based on protocol previously described in hamster cornea ( Mesocricetus auratus ) [ 11 – 13 ], and were approved by Research Ethics and Animal Welfare Committee of the University of La Laguna, included in the project known as Evaluation of the in vivo amoebicidal activity of eye drops containing active ingredients administered via the ocular route , with the reference number CEIBA2021-3074. The corneas of each mouse were removed and processed. Then, corneas were placed in 96-well polystyrene plates and were washed with 1x PBS twice. Five experimental groups were established in the 96-well plates. The Group 1 included corneas incubated alone with Staurosporine at IC 90 of A. castellanii . Corneas in Group 2 were co-cultured alone with 10 6 Acanthamoeba castellanii trophozoites. Corneas in Group 3 were co-incubated with pretreated A. castellanii with the IC 90 of Staurosporine for 30 min. Group 4 consisted of corneas co-incubated simultaneous with trophozoites and with IC 90 of Staurosporine. Corneas of Group 5 were co-cultured alone with A. castellanii trophozoites and, after 30 min of incubation, amoebas were treated with the IC 90 of Staurosporine. The plates with all groups of mouse corneas were incubated in a humidity chamber at 36 ºC during 3 h. Scanning electron microscopy After co-incubation, all groups of corneas were fixed at room temperature with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, dehydrated in increasing concentrations of ethanol and critical-point dried with liquid CO 2 using a Samdri 780 apparatus (Tousimis, Rockville Maryland, USA). Then, the corneas were coated with a thin layer (30 nm) of gold in a JEOL-JFC I 100 ion-sputtering device. Finally, all groups of corneas were observed using a JEOL-JSM 7100F scanning electron microscope (JEOL Ltd., Tokyo, Japan) [ 12 ]. Results Staurosporine-induced structural damage of the Acanthamoeba cytoskeleton. In eukaryotic cells, the cytoskeleton is mainly composed of proteins like microfilaments, microtubules, and intermediate fibres. Microfilaments, composed essentially of actin filaments, play a crucial role in cellular motility and cell interaction with the extracellular and intracellular environment [ 18 ]. In Acanthamoeba , targeting the actin network could prevent infection by inhibiting adhesion and cyst formation. A staining using the conjugate phalloidin-TRITC revealed the damage induced by Staurosporine on the distribution of actin cytoskeleton: treated cells emitted lower fluorescence and we observed partly degraded and disorganized acanthopodium, reflecting on the lower expression of actin protein (Fig. 1 ). As for the microtubules which are mainly composed of tubulin, they have been involved in motility, intracellular transport, and cell division [ 19 ]. Inhibiting this protein could prevent cells growth and has been suggested as a good target for amoebicidal agents. An indirect immunofluorescence assay for tubulin detection was conducted. After 24 h of incubation with the Staurosporine, we observed a decrease in cell shape with a uniformly distributed tubulin network. The affected cells emit lower fluorescence than the untreated cells (Fig. 2 ). Proteomic analysis. To comprehend the effect of Staurosporine on Acanthamoeba , a mass spectrometry-based proteomic approach was conducted. Two groups of trophozoite stage of Acanthamoeba castellanii cells were prepared: Untreated and treated cells with IC 50 of the present molecule. After 24 hours of treatment total proteins were extracted. Proteomic profiling of both cultures resulted in the identification of 4566 proteins (Supplementary Material, Protein analysis). Compared to untreated cells, the proteomic analysis revealed that a total of 812 proteins were differentially expressed at least twofold, in which, 97 and 715 proteins were downregulated and upregulated, respectively (Fig. 3 ). Selected affected proteins discussed below are listed in Table 1 . Table 1 Changes in the levels of selected proteins upon Staurosporine treatment at IC 50 for 24 hours. Gene ID Product Fold Change Staurosporine/control ACA1_376810 Universal stress domain containing protein N.d. in control ACA1_146430 Catalase 8,87 ACA1_392960 Universal stress domain containing protein 3,44 ACA1_387720 Universal stress domain containing protein 2,92 ACA1_076370 Profilin 2,69 ACA1_265580 Manganese and iron superoxide dismutase 2,67 ACA1_031660 Catalase 2,59 ACA1_062010 Universal stress domain containing protein 2,41 ACA1_236670 Universal stress protein (USP) family protein 2,38 ACA1_045080 Thioredoxin, putative 2,28 ACA1_054650 Glutathione transferase family protein 2,03 ACA1_095580 Serine/threonine kinase -2,16 ACA1_288400 Dual specificity protein kinase shkB, putative -3,73 ACA1_159080 Papain family cysteine protease subfamily protein N.d. in Staurosporine N.d.: not detected. Ex vivo assay in mouse corneas infected with A. castellanii and treated with Staurosporine. Scanning electron microscopy was used to analyse the effect of Staurosporine in the mouse corneas infected with trophozoites of Acanthamoeba castellanii. Mouse corneas incubated alone with Staurosporine at IC 90 for A. castellanii presented no corneal injury. Furthermore, epithelial cells showed a normal conformation and structure (Fig. 4 , A). In the group of mouse corneas cultured alone with trophozoites, amoebas adhere corneal surface and penetrated the junctions of the epithelial cells of the cornea, taking advantage of these junctions to produce infections of the epithelium (Fig. 4 , B). A contact-damage was observed without lysis, corneal epithelium showed de-epithelisation and destabilisation produced by trophozoites, and then, phagocytosis took place. The group co-cultured with trophozoites pretreated with the IC 90 of Staurosporine before incubation showed that epithelium cells were not damaged, and scarce amoebas were observed (Fig. 4 , C). The pre-treatment with Staurosporine affected the adhesion of the amoebae to the epithelial cells, and consequently, prevents the amoebae from invading the corneal epithelium. Mouse corneas co-incubated simultaneously with trophozoites and Staurosporine at IC 90 revealed that corneal epithelium was intact without signs of cellular damage. Moreover, no healthy amoebae were observed on the epithelium. The simultaneous treatment with Staurosporine eliminated all the amoebas, preventing the amoebic invasion of the corneal epithelium (Fig. 4 , D). Mouse corneas co-cultured with trophozoites, and treated with Staurosporine after 30 min of incubation, showed no healthy adherent trophozoites on the corneal epithelium. The amoebae presented morphological alterations after Staurosporine treatment. The corneal epithelium showed cellular damage caused by the early stages of amoebic, but once Staurosporine was administered after 30 min of incubation, it acted directly on the trophozoites preventing a more severe invasion of the corneal epithelium (Fig. 4 , E). Discussion Staurosporine was first isolated by Omura, S. et al. in 1977 from a Streptomyces strain [ 20 ]. Until present, several authors have confirmed its pharmacological activities like hypotensive, antiprotozoal, anticoagulant and antifungal agent [ 21 , 22 ]. In a previous work, we have proved the amoebicidal activity of Staurosporine against various Acanthamoeba strains [ 23 ]. The approach was based on molecule isolation and bio-guided fractionation from a Streptomyces sanyensis extract. In addition, we have confirmed that the present indolocarbazole could induce program cell death in Acanthamoeba castellanii Neff [ 23 , 24 ]. Yet, to confirm the obtained results and to indicate the mechanism of action, we opt to study the effect of the present molecules on the proteomic profile of Acanthamoeba and to confirm its amoebicidal activity using and ex vivo approach. The main objectives of the present work were first to confirm the amoebicidal effect of Staurosporine on this clinical strain of Acanthamoeba castellanii following protein expression in the early stage of treatment. Second to establish a new protocol to study the effect on amoebicidal drug on an amoebic keratitis murine ex vivo model, which turned out to be efficient since data similar to those observed and described in the cornea of hamster ( Mesocricetus auratus ) were obtained. In our previous work, we highlighted the morphological alterations induced by the Staurosporine on Acanthamoeba in the early stage of treatment. To corroborate the effect of the present drug on the cytoskeleton of Acanthamoeba , a specific staining of actin and tubulin was done. After 30 min of incubation with the present drug, we observed a dramatic alteration of the cell cytoskeleton; actin staining revealed the formation of long elongation. Various reports confirmed the present findings, Hedberg et al. , (1990) have reported the alteration of the cytoskeleton of several cell lines, including PTK2 epithelial cells, Swiss 3T3 fibroblasts, and human foreskin fibroblasts, by the Staurosporine [ 25 ]. While Xie et al. , (2017) have observed the formation of filaments in the fungal pathogen Candida albicans [ 26 ]. In fact, they have related the Staurosporine-induced filament to a defect in septin ring formation implicating cell cycles kinases as potential Staurosporine targets [ 26 ]. As for the microtubules network, we observed that the present drug induces alteration in its rearrangement as a disorganisation network with the presence of concentrated points. All those events could be a result of a cell dismantling upon program cell death [ 27 ]. The proteomic analysis of cells treated with the Staurosporine (IC 50 ) for 24 hours revealed that various membrane protein kinases were significantly downregulated, including dual specificity protein kinase shkB and Serine/threonine-protein kinase. Those proteins have been described as regulators of chemotaxis and phagocytosis processes in Acanthamoeba. Inhibiting those proteins could reduce the pathogenicity and growth of Acanthamoeba . Along with this effect, we notice the inhibition of cysteine protease implicated in the tissue invasion as well as in the encystation pathway [ 28 ]. In treated cells, almost 16% of the total identified proteins were overexpressed. Among those proteins, we have observed the upregulation of Profilin. This molecule is implicated in the regulation of actin polymerization and affects the structure of the cytoskeleton. Various reports have confirmed that at lower concentrations this protein enhances the actin polymerization while at high concentrations would prevent it and trigger the autophagy via the mTOR pathway [ 29 , 30 ]. Among the most upregulated proteins, we found proteins involved in cell survival under oxidative stress. Although, we have confirmed in vitro the effect of Staurosporine on Acanthamoeba , these results are still insufficient to scale it up in vivo . For this reason, one of the main objectives of the present study was to establish an ex vivo model to study the effect of drug therapy on AK infection. Omaña-Molina et al. , (2004) established an ex vivo model to study the cytopathic effect of Acanthamoeba castellanii and A. polyphaga on hamster corneas [ 11 ]. In this study, the ex vivo assay revealed that trophozoites of Acanthamoeba castellanii infected mouse corneas, showing that the amoebas invaded corneal epithelium penetrating the junctions of the epithelial cells without lysis process, only phagocyting the detached cells. This type of invasion suggest that Acanthamoeba infections are contact-dependent. In this sense, Omaña-Molina et al. , (2013) demonstrated that A. castellanii and A. polyphaga invasion and disruption of corneal epithelium in the hamster model is performed by the penetration of the amoebae through cell junctions either by the action of proteases and/or a mechanical effect exerted by trophozoites, suggesting that the contact-dependent activity is an important pathogenic mechanism of these strains of Acanthamoeba [ 12 ]. Moreover, in a previous study Omaña-Molina et al. , (2010) co-cultivated A. castellanii trophozoites with human corneas and reported mechanisms of pathogenicity of amoebic infections were very similar to the previous study using a hamster model and our findings using a mouse model, which validate these animal models for the study of the pathogenesis of AK [ 11 , 13 , 31 ]. Currently, the agents normally recommended to treat AK need to be administered for a prolonged period, which often result in severe ocular surface toxicity [ 4 , 32 – 35 ]. In this study, mouse corneas cultured alone with Staurosporine revelated that this compound causes no adverse effect on corneal epithelium compared to other drugs, maintaining the corneal tissues intact. Importantly, it has been shown that Staurosporine with only 30 min pre-treatment affects the adhesion of the amoebae to the corneal epithelium and prevents the trophozoites from invading the corneal tissue. However, a 30 min pre-treatment did not completely eliminate the amoebae population, possibly due to insufficient time for pre-treatment of the trophozoites. The corneal epithelium was intact in the infected mouse corneas treated simultaneously and 30 min after infection. Staurosporine eliminated the entire amoebic population, preventing adhesion and infection of amoebae to the epithelium. Therefore, this study demonstrated that Staurosporine could be used to develop a new line of eye drops for the treatment of superficial AK or early stages of amoebic infection. Nevertheless, our study has limitations. For example, in patients with AK in the chronic stage of the disease, where the infection is located at deeper levels of the corneal epithelium, further studies are needed to determine whether the effect of Staurosporine could be observed deeper in the corneal tissue and completely eliminate the amoebic infection. Conclusions In summary, Staurosporine induced structural alterations of the actin and tubulin cytoskeleton in trophozoites of Acanthamoeba castellanii . In addition, the proteomic analysis of treated trophozoites with Staurosporine showed that various membrane protein kinases and cysteine proteases were significantly downregulated, mainly involved in the pathogenicity, growth, tissue invasion as well as in the encystation pathway of Acanthamoeba. Furthermore, almost 16% of the total identified proteins were overexpressed. We have observed the upregulation of Profilin, implicated in the regulation of actin polymerization and affects the structure of the cytoskeleton. On the other hand, the mechanisms of pathogenicity of amoebic infection reported in previous ex vivo investigations were very similar to our findings using a mouse model, which validate this animal model for the study of the pathogenesis of AK. Beside this, Staurosporine eliminated the entire amoebic population and prevented amoeba adhesion and infection to the epithelium of mouse corneas, demonstrating that Staurosporine could be used to develop a new line of eye drops for the treatment of superficial AK or early stages of amoebic infections. Declarations Conflicts of Interest The authors declare no conflict of interest. Funding This study was supported funded by the Consorcio Centro de Investigación Biomédica (CIBER) de Enfermedades Infecciosas (CIBERINFEC); Instituto de Salud Carlos III, 28006 Madrid, Spain (CB21/13/00100); Cabildo Insular de Tenerife 2023–2028 proyect CC20230222, CABILDO.23; and Ministerio de Sanidad, Spain. R. L. R.-E. (TESIS2020010117) and C. J. B.-E. (TESIS2020010057) were funded by a grant from the Agencia Canaria de Investigación, Innovación y Sociedad de la Información, co-funded with 85% by Fondo Social Europeo (FSE). R. S. is supported by CePaViP, provided by ERDF and MEYS CR (CZ.02.1.01/0.0/0.0/16_019/0000759). Author Contribution Methodology, R.L.R.-E., I.S., L. S.-V., C. J. B.-E. and R.S.; software, R.L.R.-E., I.S. and R.S.; validation, A. R. D.-M, J. J. F., M. O.-M., R.S., J.E.P. and J.L.-M.; formal analysis, M. O.-M., R.S., J.E.P. and J.L.-M.; investigation, R.L.R.-E., I.S., L. S.-V., C. J. B.-E. and R. S.; resources, J.E.P. and J.L.-M.; data curation, R.L.R.-E. and I.S.; writing—original draft preparation, R.L.R.-E. and I.S.; writing—review and editing, L. S.-V., M. O.-M, R.S. J.E.P. and J.L.-M.; conceptualization, M. O.-M., , R.S., J.E.P. and J.L.-M.; visualization, J.E.P. and J.L.-M.; supervision, J.E.P. and J.L.-M.; project administration, J.E.P. and J.L.-M.; funding acquisition, A. R. D.-M, J. J. F., M. O.-M., R.S., J.E.P. and J.L.-M. All authors have read and agreed to the published version of the manuscript. 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Antioxidants 12:2081. https://doi.org/10.3390/antiox12122081 Xu X, Xu S, Wan J, Wang D, Pang X, Gao Y, Ni N, Chen D, Sun X (2023) Disturbing cytoskeleton by engineered nanomaterials for enhanced cancer therapeutics. Bioact Mater 29:50–71. https://doi.org/10.1016/j.bioactmat.2023.06.016 Henriquez FL, Ingram PR, Muench SP, Rice DW, Roberts CW (2008) Molecular Basis for Resistance of Acanthamoeba Tubulins to All Major Classes of Antitubulin Compounds. Antimicrob Agents Chemother 52:1133–1135. https://doi.org/10.1128/AAC.00355-07 OMURA S, IWAI Y, HIRANO A, NAKAGAWA A, AWAYA J, TSUCHIYA H, TAKAHASHI Y, ASUMA R (1977) A new alkaloid AM-2282 of Streptomyces origin taxonomy, fermentation, isolation and preliminary characterization. J Antibiot (Tokyo) 30:275–282. https://doi.org/10.7164/antibiotics.30.275 Nakano H, Ōmura S (2009) Chemical biology of natural indolocarbazole products: 30 years since the discovery of staurosporine. J Antibiot (Tokyo) 62:17–26. https://doi.org/10.1038/ja.2008.4 OMURA S, SASAKI Y, IWAI Y, TAKESHIMA H (1995) Staurosporine, a Potentially Important Gift from a Microorganism. J Antibiot (Tokyo) 48:535–548. https://doi.org/10.7164/antibiotics.48.535 Cartuche L, Sifaoui I, Cruz D, Reyes-Batlle M, López-Arencibia A, Javier Fernández J, Díaz-Marrero AR, Piñero JE, Lorenzo-Morales J (2019) Staurosporine from Streptomyces sanyensis activates Programmed Cell Death in Acanthamoeba via the mitochondrial pathway and presents low in vitro cytotoxicity levels in a macrophage cell line. Sci Rep 9:11651. https://doi.org/10.1038/s41598-019-48261-7 Cartuche L, Reyes-Batlle M, Sifaoui I, Arberas-Jiménez I, Piñero JE, Fernández JJ, Lorenzo-Morales J, Díaz-Marrero AR (2019) Antiamoebic Activities of Indolocarbazole Metabolites Isolated from Streptomyces sanyensis Cultures. Mar Drugs 17. https://doi.org/10.3390/md17100588 Hedberg KK, Birrell GB, Habliston DL, Griffith OH (1990) Staurosporine induces dissolution of microfilament bundles by a protein kinase C-independent pathway. Exp Cell Res 188:199–208. https://doi.org/10.1016/0014-4827(90)90160-C Xie JL, O’Meara TR, Polvi EJ, Robbins N, Cowen LE (2017) Staurosporine Induces Filamentation in the Human Fungal Pathogen Candida albicans via Signaling through Cyr1 and Protein Kinase A. https://doi.org/10.1128/mSphere.00056-17 . mSphere 2: Olguín-Albuerne M, Domínguez G, Morán J (2014) Effect of Staurosporine in the Morphology and Viability of Cerebellar Astrocytes: Role of Reactive Oxygen Species and NADPH Oxidase. Oxid Med Cell Longev 2014:1–13. https://doi.org/10.1155/2014/678371 Hong Y, Kang J-M, Joo S-Y, Song S-M, Lê HG, Thái TL, Lee J, Goo Y-K, Chung D-I, Sohn W-M, Na B-K (2018) Molecular and Biochemical Properties of a Cysteine Protease of Acanthamoeba castellanii . Korean J Parasitol 56:409–418. https://doi.org/10.3347/kjp.2018.56.5.409 Saurav S, Manna SK (2022) Profilin upregulation induces autophagy through stabilization of AMP-activated protein kinase. FEBS Lett 596:1765–1777. https://doi.org/10.1002/1873-3468.14372 Pernier J, Shekhar S, Jegou A, Guichard B, Carlier M-F (2016) Profilin Interaction with Actin Filament Barbed End Controls Dynamic Instability, Capping, Branching, and Motility. Dev Cell 36:201–214. https://doi.org/10.1016/j.devcel.2015.12.024 Omaña-Molina M, González-Robles A, Salazar-Villatoro LI, Cristóbal-Ramos AR, González-Lázaro M, Salinas-Moreno E, Méndez-Cruz R, Sánchez-Cornejo M, De la Torre-González E, Martínez-Palomo A (2010) Acanthamoeba castellanii : Morphological analysis of the interaction with human cornea. Exp Parasitol 126:73–78. https://doi.org/10.1016/j.exppara.2010.02.004 Shing B, Balen M, McKerrow JH, Debnath A (2021) Acanthamoeba Keratitis: an update on amebicidal and cysticidal drug screening methodologies and potential treatment with azole drugs. Expert Rev Anti Infect Ther 19:1427–1441. https://doi.org/10.1080/14787210.2021.1924673 Megha K, Sharma M, Sharma C, Gupta A, Sehgal R, Khurana S (2022) Evaluation of in vitro activity of five antimicrobial agents on Acanthamoeba isolates and their toxicity on human corneal epithelium. Eye 36:1911–1917. https://doi.org/10.1038/s41433-021-01768-8 Wang Y, Jiang L, Zhao Y, Ju X, Wang L, Jin L, Fine RD, Li M (2023) Biological characteristics and pathogenicity of Acanthamoeba . Front Microbiol 14. https://doi.org/10.3389/fmicb.2023.1147077 Liu JX, Werner J, Kirsch T, Zuckerman JD, Virk MS (2018) Cytotoxicity evaluation of chlorhexidine gluconate on human fibroblasts, myoblasts, and osteoblasts. J Bone Jt Infect 3:165–172. https://doi.org/10.7150/jbji.26355 Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterialproteomicanalysis.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3878546","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":268183451,"identity":"6db9aef1-9060-47c2-a8ef-f8f0e64c77b7","order_by":0,"name":"Rubén L. Rodríguez-Expósito","email":"","orcid":"","institution":"Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias (IUETSPC), Universidad de La Laguna (ULL)","correspondingAuthor":false,"prefix":"","firstName":"Rubén","middleName":"L.","lastName":"Rodríguez-Expósito","suffix":""},{"id":268183452,"identity":"cee85805-68fe-489b-a4e2-77dc78ea7d77","order_by":1,"name":"Ines Sifaoui","email":"","orcid":"","institution":"Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias (IUETSPC), Universidad de La Laguna (ULL)","correspondingAuthor":false,"prefix":"","firstName":"Ines","middleName":"","lastName":"Sifaoui","suffix":""},{"id":268183453,"identity":"439e3238-809d-4db3-959e-22ea634b65df","order_by":2,"name":"Lizbeth Salazar-Villatoro","email":"","orcid":"","institution":"Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional","correspondingAuthor":false,"prefix":"","firstName":"Lizbeth","middleName":"","lastName":"Salazar-Villatoro","suffix":""},{"id":268183454,"identity":"48afa265-2ff1-40dc-86f5-6e03e67436b6","order_by":3,"name":"Carlos J. Bethencourt-Estrella","email":"","orcid":"","institution":"Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias (IUETSPC), Universidad de La Laguna (ULL)","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"J.","lastName":"Bethencourt-Estrella","suffix":""},{"id":268183455,"identity":"bf725e60-858d-4777-a082-e3b368867528","order_by":4,"name":"José J. Fernández","email":"","orcid":"","institution":"Instituto Universitario de Bio-Orgánica Antonio González (IUBO AG), Universidad de La Laguna (ULL)","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"J.","lastName":"Fernández","suffix":""},{"id":268183456,"identity":"e0decc7d-f084-42e4-aabf-654839e7d19d","order_by":5,"name":"Ana R. Díaz-Marrero","email":"","orcid":"","institution":"Instituto Universitario de Bio-Orgánica Antonio González (IUBO AG), Universidad de La Laguna (ULL)","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"R.","lastName":"Díaz-Marrero","suffix":""},{"id":268183457,"identity":"c16dd806-920f-493f-915d-0fb7f0f08d83","order_by":6,"name":"Robert Sutak","email":"","orcid":"","institution":"Charles University","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Sutak","suffix":""},{"id":268183458,"identity":"28209f96-c4fa-404a-8871-a220a6e397df","order_by":7,"name":"Maritza Omaña-Molina","email":"","orcid":"","institution":"UNAM, Estado de México","correspondingAuthor":false,"prefix":"","firstName":"Maritza","middleName":"","lastName":"Omaña-Molina","suffix":""},{"id":268183459,"identity":"9f71c572-cdb3-4fbc-9506-c56f55d75a75","order_by":8,"name":"José E. Piñero","email":"","orcid":"","institution":"Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"E.","lastName":"Piñero","suffix":""},{"id":268183460,"identity":"d5c9035c-c3e5-4529-989e-2d9e300bfd48","order_by":9,"name":"Jacob Lorenzo-Morales","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYBACxgbmBjCDn4G58QADAzMxWhghWiSBDOK0gDSBKYMDxGphnnaw8XFFxR174xuJQC0V1okN7O0P8NsxO7HZ8MyZZ8xmYC1n0hMbeM4YENLSJtnYdpgNrIWx7XBig0QOAZ+Atfw7zGM8A6TlH1CL/HOCDgNqaTgsYSAB0tIAsoWBoMOaDRuOHTaQOPOw4UDCsXTjNp4c/FoMZycffNhQc9ievz354IMPNday/ezH8TvMsAGZlwDEbHjVA4E8IQWjYBSMglEwChgAtNxOV4Pg7EMAAAAASUVORK5CYII=","orcid":"","institution":"Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III","correspondingAuthor":true,"prefix":"","firstName":"Jacob","middleName":"","lastName":"Lorenzo-Morales","suffix":""}],"badges":[],"createdAt":"2024-01-19 11:14:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3878546/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3878546/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50105754,"identity":"26947cd1-1f76-405d-8740-ea93231cdf15","added_by":"auto","created_at":"2024-01-24 15:49:05","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":299589,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of the effect of IC\u003csub\u003e90\u003c/sub\u003e of Staurosporine on the actin cytoskeleton of \u003cem\u003eAcanthamoeba\u003c/em\u003e \u003cem\u003ecastellanii \u003c/em\u003etrophozoites 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 (Image A). Treated cells emitted a lower orange fluorescence, and trophozoites showed disorganized and degraded acanthopodium (Image B). All images (63x) were obtained using an inverted confocal light microscope Leica DMI 4000 B (Scale Bar represents 10 µm).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3878546/v1/17d84f3f5ceb1d2184ac7357.jpeg"},{"id":50105752,"identity":"c54f17ac-7fad-474d-82a5-f4bf0e2b7116","added_by":"auto","created_at":"2024-01-24 15:49:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":177045,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of the intracellular organization of tubulin microtubules using tubulin antibodies. Tubulin microtubules in control cells showed intense red fluorescence and demonstrated a normal conformation (A; scale bar represents 5 µm). Trophozoites of \u003cem\u003eAcanthamoeba\u003c/em\u003e \u003cem\u003ecastellanii\u003c/em\u003e incubated with the IC\u003csub\u003e90\u003c/sub\u003e of Staurosporine for 24 h showed disorganization or destruction of the tubulin microtubules (B; scale bar represents 2 µm). Mounting DAPI solution for DNA staining show a blue fluorescence. Images (63x) were obtained by using the inverted confocal light microscope Leica DMI 4000 B.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3878546/v1/908fb7b6782145f5bff57376.png"},{"id":50106271,"identity":"d2a02d9b-2dcf-4de4-af10-062d2f49d4dd","added_by":"auto","created_at":"2024-01-24 15:57:05","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":83859,"visible":true,"origin":"","legend":"\u003cp\u003eVolcano graph expressing a logarithmic Student’s t-test \u003cem\u003ep-\u003c/em\u003evalue as a function of Staurosporine protein control fold change.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3878546/v1/dbb609fbb3c877c4a28bcfa9.jpeg"},{"id":50106272,"identity":"44a0b833-6eb2-46ba-8685-fdc97de48724","added_by":"auto","created_at":"2024-01-24 15:57:05","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1933358,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopy analysis of the effect of \u003cem\u003eAcanthamoeba castellanii\u003c/em\u003e trophozoites and/or Staurosporine on mouse corneas during 3 h. The co-incubation of mouse corneas with Staurosporine at IC\u003csub\u003e90\u003c/sub\u003e shows no evidence of damage or cell disorganization (A; Group 1; Scale Bar represents 10 µm). Mouse corneas co-cultured with trophozoites of \u003cem\u003eA. castellanii \u003c/em\u003e(B; Group 2; Scale Bar represents 10 µm) 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 \u003cem\u003eA. castellanii \u003c/em\u003epretreated with Staurosporine 30 min prior (C; Group 3; Scale Bar represents 10 µm) showed fewer trophozoites adhered to the corneal epithelium (Black arrows), which did not migrate towards 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), cellular debris of \u003cem\u003eA. castellanii \u003c/em\u003etrophozoites 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; Group 5; Scale Bar represents 10 µm), trophozoites were complete destroyed (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).\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3878546/v1/004d324efd648ac1702ab5ea.jpeg"},{"id":50349711,"identity":"84132fde-874d-4b90-a3b6-2dd97add9649","added_by":"auto","created_at":"2024-01-30 07:26:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":876160,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3878546/v1/4ad1c572-11ee-4b15-b518-4be36d7170a7.pdf"},{"id":50105755,"identity":"b5c080a4-8995-4e3f-9523-a0e6eb665164","added_by":"auto","created_at":"2024-01-24 15:49:05","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1691582,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialproteomicanalysis.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3878546/v1/28f6d4c68c1ccb98939486f0.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Staurosporine as a potential treatment for Acanthamoeba keratitis using mouse cornea as an ex vivo model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFree-living amoebae are ubiquitous single-celled living organisms isolated from multiple habitats including water and soil. Until present, six different genera were reported to be pathogenic to humans and animals, which the most common being \u003cem\u003eAcanthamoeba\u003c/em\u003e genus. This amoeba could cause a threatening eye infection known as \u003cem\u003eAcanthamoeba\u003c/em\u003e keratitis: a rare eye infection but commonly associated with contact lens wearers [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Although, \u003cem\u003eAcanthamoeba\u003c/em\u003e keratitis constitutes 2% of the corneal infections, its incidence has been consistently increasing [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Actual treatment protocols are based on the combination of cationic antiseptics such as polyhexamethylene biguanide (0.02%) or chlorhexidine (0.02%) and aromatic diamidines such as propamidine (0.1%) or hexamidine (0.1%) [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Still those current therapies are not fully effective in eliminating the parasite because of their variable efficacy among different genotypes, the appearance of highly resistant cyst form, or due to their toxicity generated by a prolonged administration. This leads to an urgent need to discover new drugs and/or drug posology.\u003c/p\u003e \u003cp\u003eOn the one hand, \u003cem\u003ein vitro\u003c/em\u003e assays are fundamental for drug discovery against the present infection, because they are simple, reproductible and much more economical than the \u003cem\u003ein vivo\u003c/em\u003e assay, but still inefficient to predict the drug\u0026rsquo;s action inside the host organism. On the other hand, \u003cem\u003ein vivo\u003c/em\u003e assays are more clinically relevant and the drug effect and side effect could be much more accurate, yet \u003cem\u003ein vivo\u003c/em\u003e assays are much more expensive and harder to control as they include multitude of variables. \u003cem\u003eEx vivo\u003c/em\u003e assays are important preclinical tools between \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e assays. In these \u003cem\u003eex vivo\u003c/em\u003e systems, the cytoarchitecture and intracellular connections and metabolic processes could be conserved, mimicking the \u003cem\u003ein vivo\u003c/em\u003e environment [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Rabbits, hamsters and mice were used as models for \u003cem\u003ein vivo\u003c/em\u003e assay of \u003cem\u003eAcanthamoeba\u003c/em\u003e Keratitis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. To the best of our knowledge, only the hamster cornea has been used as an \u003cem\u003eex vivo\u003c/em\u003e model for \u003cem\u003eAcanthamoeba\u003c/em\u003e keratitis [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The aim of the present study was to evaluate Staurosporine as potential treatment for \u003cem\u003eAcanthamoeba\u003c/em\u003e keratitis using mouse cornea as an \u003cem\u003eex vivo\u003c/em\u003e model and to investigate its model of action by comparative proteomic analysis.\u003c/p\u003e"},{"header":"Study design/ Material and Methods","content":"\u003cp\u003e \u003cb\u003eAcanthamoeba\u003c/b\u003e \u003cb\u003estrain\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTrophozoites of \u003cem\u003eA. castellanii\u003c/em\u003e (genotype T4) isolated from a clinical case of AK from \u0026ldquo;Asociaci\u0026oacute;n para evitar la ceguera en M\u0026eacute;xico\u0026rdquo;, Luis S\u0026aacute;nchez Bulnes Hospital, Mexico City. The strain was reported to be invasive in the GAE murine model [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The strain was grown axenically in PYG medium (0.75% (w/v) proteose peptone, 0.75% (w/v) yeast extract and 1.5% (w/v) glucose) containing 40 \u0026micro;g gentamicin ml\u0026thinsp;\u0026minus;\u0026thinsp;1 (Biochrom AG, Cultek, Granollers, Barcelona, Spain) at 26\u0026deg;C. After 72 hours of incubation (end of growth logarithmic phase), culture was centrifuged (2500 rpm during 5 min) and trophozoites were harvested for the subsequent assays.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eassay\u003c/b\u003e\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFluorescent staining of actin distribution\u003c/h2\u003e \u003cp\u003eFor direct fluorescent staining, trophozoites of \u003cem\u003eA. castellanii\u003c/em\u003e were treated first with the IC\u003csub\u003e90\u003c/sub\u003e (2.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015 \u0026micro;M) of the Staurosporine. After 30 min of incubation, cells were fixed with formaldehyde and deposit on pre-coated coverslip. Later, cells were treated with Triton (0.1%) for 30 min followed by Phalloidin\u0026ndash;tetramethylrhodamine B isothiocyanate (Phalloidin-TRITC; Sigma-Aldrich, Madrid) for another 30 min at room temperature. Finally, cells were washed with PBS and later examined by \u003cem\u003eZ\u003c/em\u003e-stack imaging using an inverted light confocal microscope Leica DMI 4000 B with LAS X software, a 532 nm laser and Leica HCX PL Apo 63x Oil Objective were used. Untreated cells were considered as the negative control.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunofluorescence staining of tubulin of Acanthamoeba trophozoites.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe immunofluorescence staining of tubulin was conducted using the immunofluorescence staining procedure of the manufacture (Sigma-Aldrich) with slight modifications. Briefly, cells were treated first the IC\u003csub\u003e90\u003c/sub\u003e of Staurosporine for 30 min. 50 \u0026micro;L of the cell suspension were placed on a gelatine precoated coverslip for 30 min. Later they were fixed with paraformaldehyde (4%). After 15 min, cells were treated with Triton (0.3%) for 10 min followed by 3 washes with PBS 1X. At this stage, cells were treated with 5% BSA in PBS 1X/150 mM sucrose for 30 min and washed with glycine 100 mM in PBS 1X for 5 min. Later, the trophozoites were incubated with the first anti-tubulin antibody 1:2000 for 2 hours at room temperature (Monoclonal Anti-α-Tubulin antibody produced in mouse, Sigma-Aldrich, Madrid). After 3 washes with PBS 1X cells were incubated with the second antibody labeled with Alexa 594 (1:500) for 1 hour at room temperature in darkness (Goat anti-Mouse IgG (H\u0026thinsp;+\u0026thinsp;L) Highly Cross \u0026ndash; Adsorbed Secondary Antibody, Alexa Fluor Plus 594; Thermo Fisher Scientific, Rockford; USA). Finally, cells were washed with PBS 1X and mounted in DAPI (4\u0026prime;,6-Diamidino-2-phenylindole dihydrochloride; Sigma-Aldrich; Madrid) containing mounting solution. 3D and maximum projection imaging of the trophozoites were performed by Z-stack imaging using an inverted light confocal microscope Leica DMI 4000 B with LAS X software, a 405 nm laser and 532 nm laser and Leica HCX PL Apo 63x Oil Objective were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eProteomic analysis\u003c/h2\u003e \u003cp\u003eComparative label-free proteomic analysis was conducted described in our recent study [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. 10\u003csup\u003e6\u003c/sup\u003e cells of \u003cem\u003eA. castellanii\u003c/em\u003e were treated with the IC\u003csub\u003e50\u003c/sub\u003e of Staurosporine for 24 hours, washed 1 time with PBS and the pellets were subjected to further processing. Untreated cells were prepared as a control group. Both groups were prepared in three biological replicates. Protein identification was made using the data base of \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEx vivo\u003c/b\u003e \u003cb\u003einfection\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eCo-incubation and interaction of\u003c/b\u003e \u003cb\u003eA. castellanii\u003c/b\u003e \u003cb\u003etrophozoites with BALB/c mice cornea.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA total of 12 pathogen-free male BALB/c (\u003cem\u003eMus musculus\u003c/em\u003e) mice were used, with an average age of 21 to 28 days and an average body weight of 35 g. Experiments were based on protocol previously described in hamster cornea (\u003cem\u003eMesocricetus auratus\u003c/em\u003e) [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and were approved by Research Ethics and Animal Welfare Committee of the University of La Laguna, included in the project known as \u003cem\u003eEvaluation of the in vivo amoebicidal activity of eye drops containing active ingredients administered via the ocular route\u003c/em\u003e, with the reference number CEIBA2021-3074.\u003c/p\u003e \u003cp\u003eThe corneas of each mouse were removed and processed. Then, corneas were placed in 96-well polystyrene plates and were washed with 1x PBS twice. Five experimental groups were established in the 96-well plates. The \u003cb\u003eGroup 1\u003c/b\u003e included corneas incubated alone with Staurosporine at IC\u003csub\u003e90\u003c/sub\u003e of \u003cem\u003eA. castellanii\u003c/em\u003e. Corneas in \u003cb\u003eGroup 2\u003c/b\u003e were co-cultured alone with 10\u003csup\u003e6\u003c/sup\u003e \u003cem\u003eAcanthamoeba castellanii\u003c/em\u003e trophozoites. Corneas in \u003cb\u003eGroup 3\u003c/b\u003e were co-incubated with pretreated \u003cem\u003eA. castellanii\u003c/em\u003e with the IC\u003csub\u003e90\u003c/sub\u003e of Staurosporine for 30 min. \u003cb\u003eGroup 4\u003c/b\u003e consisted of corneas co-incubated simultaneous with trophozoites and with IC\u003csub\u003e90\u003c/sub\u003e of Staurosporine. Corneas of \u003cb\u003eGroup 5\u003c/b\u003e were co-cultured alone with \u003cem\u003eA. castellanii\u003c/em\u003e trophozoites and, after 30 min of incubation, amoebas were treated with the IC\u003csub\u003e90\u003c/sub\u003e of Staurosporine. The plates with all groups of mouse corneas were incubated in a humidity chamber at 36 \u0026ordm;C during 3 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eScanning electron microscopy\u003c/h2\u003e \u003cp\u003eAfter co-incubation, all groups of corneas were fixed at room temperature with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, dehydrated in increasing concentrations of ethanol and critical-point dried with liquid CO\u003csub\u003e2\u003c/sub\u003e using a Samdri 780 apparatus (Tousimis, Rockville Maryland, USA). Then, the corneas were coated with a thin layer (30 nm) of gold in a JEOL-JFC I 100 ion-sputtering device. Finally, all groups of corneas were observed using a JEOL-JSM 7100F scanning electron microscope (JEOL Ltd., Tokyo, Japan) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eStaurosporine-induced structural damage of the\u003c/b\u003e \u003cb\u003eAcanthamoeba\u003c/b\u003e \u003cb\u003ecytoskeleton.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn eukaryotic cells, the cytoskeleton is mainly composed of proteins like microfilaments, microtubules, and intermediate fibres. Microfilaments, composed essentially of actin filaments, play a crucial role in cellular motility and cell interaction with the extracellular and intracellular environment [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In \u003cem\u003eAcanthamoeba\u003c/em\u003e, targeting the actin network could prevent infection by inhibiting adhesion and cyst formation. A staining using the conjugate phalloidin-TRITC revealed the damage induced by Staurosporine on the distribution of actin cytoskeleton: treated cells emitted lower fluorescence and we observed partly degraded and disorganized acanthopodium, reflecting on the lower expression of actin protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs for the microtubules which are mainly composed of tubulin, they have been involved in motility, intracellular transport, and cell division [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Inhibiting this protein could prevent cells growth and has been suggested as a good target for amoebicidal agents. An indirect immunofluorescence assay for tubulin detection was conducted. After 24 h of incubation with the Staurosporine, we observed a decrease in cell shape with a uniformly distributed tubulin network. The affected cells emit lower fluorescence than the untreated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eProteomic analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo comprehend the effect of Staurosporine on \u003cem\u003eAcanthamoeba\u003c/em\u003e, a mass spectrometry-based proteomic approach was conducted. Two groups of trophozoite stage of \u003cem\u003eAcanthamoeba castellanii\u003c/em\u003e cells were prepared: Untreated and treated cells with IC\u003csub\u003e50\u003c/sub\u003e of the present molecule. After 24 hours of treatment total proteins were extracted. Proteomic profiling of both cultures resulted in the identification of 4566 proteins (Supplementary Material, Protein analysis). Compared to untreated cells, the proteomic analysis revealed that a total of 812 proteins were differentially expressed at least twofold, in which, 97 and 715 proteins were downregulated and upregulated, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Selected affected proteins discussed below are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChanges in the levels of selected proteins upon Staurosporine treatment at IC\u003csub\u003e50\u003c/sub\u003e for 24 hours.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProduct\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFold Change\u003c/p\u003e \u003cp\u003eStaurosporine/control\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACA1_376810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUniversal stress domain containing protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN.d. in control\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACA1_146430\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCatalase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8,87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACA1_392960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUniversal stress domain containing protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3,44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACA1_387720\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUniversal stress domain containing protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2,92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACA1_076370\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProfilin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2,69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACA1_265580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eManganese and iron superoxide dismutase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2,67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACA1_031660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCatalase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2,59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACA1_062010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUniversal stress domain containing protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2,41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACA1_236670\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUniversal stress protein (USP) family protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2,38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACA1_045080\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThioredoxin, putative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2,28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACA1_054650\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlutathione transferase family protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2,03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACA1_095580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSerine/threonine kinase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2,16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACA1_288400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDual specificity protein kinase shkB, putative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-3,73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eACA1_159080\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePapain family cysteine protease subfamily protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN.d. in Staurosporine\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eN.d.: not detected.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEx vivo\u003c/b\u003e \u003cb\u003eassay in mouse corneas infected with\u003c/b\u003e \u003cb\u003eA. castellanii\u003c/b\u003e \u003cb\u003eand treated with Staurosporine.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eScanning electron microscopy was used to analyse the effect of Staurosporine in the mouse corneas infected with trophozoites of \u003cem\u003eAcanthamoeba castellanii.\u003c/em\u003e Mouse corneas incubated alone with Staurosporine at IC\u003csub\u003e90\u003c/sub\u003e for \u003cem\u003eA. castellanii\u003c/em\u003e presented no corneal injury. Furthermore, epithelial cells showed a normal conformation and structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, A). In the group of mouse corneas cultured alone with trophozoites, amoebas adhere corneal surface and penetrated the junctions of the epithelial cells of the cornea, taking advantage of these junctions to produce infections of the epithelium (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, B). A contact-damage was observed without lysis, corneal epithelium showed de-epithelisation and destabilisation produced by trophozoites, and then, phagocytosis took place.\u003c/p\u003e \u003cp\u003eThe group co-cultured with trophozoites pretreated with the IC\u003csub\u003e90\u003c/sub\u003e of Staurosporine before incubation showed that epithelium cells were not damaged, and scarce amoebas were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, C). The pre-treatment with Staurosporine affected the adhesion of the amoebae to the epithelial cells, and consequently, prevents the amoebae from invading the corneal epithelium. Mouse corneas co-incubated simultaneously with trophozoites and Staurosporine at IC\u003csub\u003e90\u003c/sub\u003e revealed that corneal epithelium was intact without signs of cellular damage. Moreover, no healthy amoebae were observed on the epithelium. The simultaneous treatment with Staurosporine eliminated all the amoebas, preventing the amoebic invasion of the corneal epithelium (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, D).\u003c/p\u003e \u003cp\u003eMouse corneas co-cultured with trophozoites, and treated with Staurosporine after 30 min of incubation, showed no healthy adherent trophozoites on the corneal epithelium. The amoebae presented morphological alterations after Staurosporine treatment. The corneal epithelium showed cellular damage caused by the early stages of amoebic, but once Staurosporine was administered after 30 min of incubation, it acted directly on the trophozoites preventing a more severe invasion of the corneal epithelium (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eStaurosporine was first isolated by Omura, S. \u003cem\u003eet al.\u003c/em\u003e in 1977 from a Streptomyces strain [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Until present, several authors have confirmed its pharmacological activities like hypotensive, antiprotozoal, \u003cem\u003eanticoagulant\u003c/em\u003e and antifungal agent [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In a previous work, we have proved the amoebicidal activity of Staurosporine against various \u003cem\u003eAcanthamoeba\u003c/em\u003e strains [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The approach was based on molecule isolation and bio-guided fractionation from a \u003cem\u003eStreptomyces sanyensis\u003c/em\u003e extract. In addition, we have confirmed that the present indolocarbazole could induce program cell death in \u003cem\u003eAcanthamoeba castellanii\u003c/em\u003e Neff [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Yet, to confirm the obtained results and to indicate the mechanism of action, we opt to study the effect of the present molecules on the proteomic profile of \u003cem\u003eAcanthamoeba\u003c/em\u003e and to confirm its amoebicidal activity using and \u003cem\u003eex vivo\u003c/em\u003e approach.\u003c/p\u003e \u003cp\u003eThe main objectives of the present work were first to confirm the amoebicidal effect of Staurosporine on this clinical strain of \u003cem\u003eAcanthamoeba castellanii\u003c/em\u003e following protein expression in the early stage of treatment. Second to establish a new protocol to study the effect on amoebicidal drug on an amoebic keratitis murine \u003cem\u003eex vivo\u003c/em\u003e model, which turned out to be efficient since data similar to those observed and described in the cornea of hamster (\u003cem\u003eMesocricetus auratus\u003c/em\u003e) were obtained.\u003c/p\u003e \u003cp\u003eIn our previous work, we highlighted the morphological alterations induced by the Staurosporine on \u003cem\u003eAcanthamoeba\u003c/em\u003e in the early stage of treatment. To corroborate the effect of the present drug on the cytoskeleton of \u003cem\u003eAcanthamoeba\u003c/em\u003e, a specific staining of actin and tubulin was done. After 30 min of incubation with the present drug, we observed a dramatic alteration of the cell cytoskeleton; actin staining revealed the formation of long elongation. Various reports confirmed the present findings, Hedberg \u003cem\u003eet al.\u003c/em\u003e, (1990) have reported the alteration of the cytoskeleton of several cell lines, including PTK2 epithelial cells, Swiss 3T3 fibroblasts, and human foreskin fibroblasts, by the Staurosporine [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. While Xie \u003cem\u003eet al.\u003c/em\u003e, (2017) have observed the formation of filaments in the fungal pathogen \u003cem\u003eCandida albicans\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In fact, they have related the Staurosporine-induced filament to a defect in septin ring formation implicating cell cycles kinases as potential Staurosporine targets [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. As for the microtubules network, we observed that the present drug induces alteration in its rearrangement as a disorganisation network with the presence of concentrated points. All those events could be a result of a cell dismantling upon program cell death [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe proteomic analysis of cells treated with the Staurosporine (IC\u003csub\u003e50\u003c/sub\u003e) for 24 hours revealed that various membrane protein kinases were significantly downregulated, including dual specificity protein kinase shkB and Serine/threonine-protein kinase. Those proteins have been described as regulators of chemotaxis and phagocytosis processes in \u003cem\u003eAcanthamoeba.\u003c/em\u003e Inhibiting those proteins could reduce the pathogenicity and growth of \u003cem\u003eAcanthamoeba\u003c/em\u003e. Along with this effect, we notice the inhibition of cysteine protease implicated in the tissue invasion as well as in the encystation pathway [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn treated cells, almost 16% of the total identified proteins were overexpressed. Among those proteins, we have observed the upregulation of Profilin. This molecule is implicated in the regulation of actin polymerization and affects the structure of the cytoskeleton. Various reports have confirmed that at lower concentrations this protein enhances the actin polymerization while at high concentrations would prevent it and trigger the autophagy via the mTOR pathway [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Among the most upregulated proteins, we found proteins involved in cell survival under oxidative stress.\u003c/p\u003e \u003cp\u003eAlthough, we have confirmed \u003cem\u003ein vitro\u003c/em\u003e the effect of Staurosporine on \u003cem\u003eAcanthamoeba\u003c/em\u003e, these results are still insufficient to scale it up \u003cem\u003ein vivo\u003c/em\u003e. For this reason, one of the main objectives of the present study was to establish an \u003cem\u003eex vivo\u003c/em\u003e model to study the effect of drug therapy on AK infection. Oma\u0026ntilde;a-Molina \u003cem\u003eet al.\u003c/em\u003e, (2004) established an \u003cem\u003eex vivo\u003c/em\u003e model to study the cytopathic effect of \u003cem\u003eAcanthamoeba castellanii\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e on hamster corneas [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In this study, the \u003cem\u003eex vivo\u003c/em\u003e assay revealed that trophozoites of \u003cem\u003eAcanthamoeba castellanii\u003c/em\u003e infected mouse corneas, showing that the amoebas invaded corneal epithelium penetrating the junctions of the epithelial cells without lysis process, only phagocyting the detached cells. This type of invasion suggest that \u003cem\u003eAcanthamoeba\u003c/em\u003e infections are contact-dependent. In this sense, Oma\u0026ntilde;a-Molina \u003cem\u003eet al.\u003c/em\u003e, (2013) demonstrated that \u003cem\u003eA. castellanii\u003c/em\u003e and \u003cem\u003eA. polyphaga\u003c/em\u003e invasion and disruption of corneal epithelium in the hamster model is performed by the penetration of the amoebae through cell junctions either by the action of proteases and/or a mechanical effect exerted by trophozoites, suggesting that the contact-dependent activity is an important pathogenic mechanism of these strains of \u003cem\u003eAcanthamoeba\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Moreover, in a previous study Oma\u0026ntilde;a-Molina \u003cem\u003eet al.\u003c/em\u003e, (2010) co-cultivated \u003cem\u003eA. castellanii\u003c/em\u003e trophozoites with human corneas and reported mechanisms of pathogenicity of amoebic infections were very similar to the previous study using a hamster model and our findings using a mouse model, which validate these animal models for the study of the pathogenesis of AK [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, the agents normally recommended to treat AK need to be administered for a prolonged period, which often result in severe ocular surface toxicity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In this study, mouse corneas cultured alone with Staurosporine revelated that this compound causes no adverse effect on corneal epithelium compared to other drugs, maintaining the corneal tissues intact. Importantly, it has been shown that Staurosporine with only 30 min pre-treatment affects the adhesion of the amoebae to the corneal epithelium and prevents the trophozoites from invading the corneal tissue. However, a 30 min pre-treatment did not completely eliminate the amoebae population, possibly due to insufficient time for pre-treatment of the trophozoites. The corneal epithelium was intact in the infected mouse corneas treated simultaneously and 30 min after infection. Staurosporine eliminated the entire amoebic population, preventing adhesion and infection of amoebae to the epithelium. Therefore, this study demonstrated that Staurosporine could be used to develop a new line of eye drops for the treatment of superficial AK or early stages of amoebic infection.\u003c/p\u003e \u003cp\u003eNevertheless, our study has limitations. For example, in patients with AK in the chronic stage of the disease, where the infection is located at deeper levels of the corneal epithelium, further studies are needed to determine whether the effect of Staurosporine could be observed deeper in the corneal tissue and completely eliminate the amoebic infection.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, Staurosporine induced structural alterations of the actin and tubulin cytoskeleton in trophozoites of \u003cem\u003eAcanthamoeba castellanii\u003c/em\u003e. In addition, the proteomic analysis of treated trophozoites with Staurosporine showed that various membrane protein kinases and cysteine proteases were significantly downregulated, mainly involved in the pathogenicity, growth, tissue invasion as well as in the encystation pathway of \u003cem\u003eAcanthamoeba.\u003c/em\u003e Furthermore, almost 16% of the total identified proteins were overexpressed. We have observed the upregulation of Profilin, implicated in the regulation of actin polymerization and affects the structure of the cytoskeleton. On the other hand, the mechanisms of pathogenicity of amoebic infection reported in previous \u003cem\u003eex vivo\u003c/em\u003e investigations were very similar to our findings using a mouse model, which validate this animal model for the study of the pathogenesis of AK. Beside this, Staurosporine eliminated the entire amoebic population and prevented amoeba adhesion and infection to the epithelium of mouse corneas, demonstrating that Staurosporine could be used to develop a new line of eye drops for the treatment of superficial AK or early stages of amoebic infections.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported funded by the Consorcio Centro de Investigaci\u0026oacute;n Biom\u0026eacute;dica (CIBER) de Enfermedades Infecciosas (CIBERINFEC); Instituto de Salud Carlos III, 28006 Madrid, Spain (CB21/13/00100); Cabildo Insular de Tenerife 2023\u0026ndash;2028 proyect CC20230222, CABILDO.23; and Ministerio de Sanidad, Spain. R. L. R.-E. (TESIS2020010117) and C. J. B.-E. (TESIS2020010057) were funded by a grant from the Agencia Canaria de Investigaci\u0026oacute;n, Innovaci\u0026oacute;n y Sociedad de la Informaci\u0026oacute;n, co-funded with 85% by Fondo Social Europeo (FSE). R. S. is supported by CePaViP, provided by ERDF and MEYS CR (CZ.02.1.01/0.0/0.0/16_019/0000759).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMethodology, R.L.R.-E., I.S., L. S.-V., C. J. B.-E. and R.S.; software, R.L.R.-E., I.S. and R.S.; validation, A. R. D.-M, J. J. F., M. O.-M., R.S., J.E.P. and J.L.-M.; formal analysis, M. O.-M., R.S., J.E.P. and J.L.-M.; investigation, R.L.R.-E., I.S., L. S.-V., C. J. B.-E. and R. S.; resources, J.E.P. and J.L.-M.; data curation, R.L.R.-E. and I.S.; writing\u0026mdash;original draft preparation, R.L.R.-E. and I.S.; writing\u0026mdash;review and editing, L. S.-V., M. O.-M, R.S. J.E.P. and J.L.-M.; conceptualization, M. O.-M., , R.S., J.E.P. and J.L.-M.; visualization, J.E.P. and J.L.-M.; supervision, J.E.P. and J.L.-M.; project administration, J.E.P. and J.L.-M.; funding acquisition, A. R. D.-M, J. J. F., M. O.-M., R.S., J.E.P. and J.L.-M. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe acknowledge Karel Harant and Pavel Talacko from Laboratory of Mass Spectrometry, Biocev, Charles University, Faculty of Science, where proteomic and mass spectrometric analysis had been done.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHenriquez FL (2009) Review of \u003cem\u003eAcanthamoeba\u003c/em\u003e: Biology and Pathogenesis by Naveed Ahmed Khan. 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J Bone Jt Infect 3:165\u0026ndash;172. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7150/jbji.26355\u003c/span\u003e\u003cspan address=\"10.7150/jbji.26355\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Acanthamoeba, ex vivo, mouse cornea, Staurosporine, proteomic analysis, PCD","lastPublishedDoi":"10.21203/rs.3.rs-3878546/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3878546/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCurrently, six different genera were reported to be pathogenic to humans and animals, which the most common being \u003cem\u003eAcanthamoeba\u003c/em\u003e genus. \u003cem\u003eAcanthamoeba\u003c/em\u003e is a ubiquitous genus of amoebae that can trigger severe and progressive ocular disease kwon as \u003cem\u003eAcanthamoeba\u003c/em\u003e Keratitis (AK). Furthermore, actual treatment protocols are based on the combination of different compounds that are not fully effective in eliminating the parasite in ocular infections. Therefore, this leads to an urgent need to develop new compounds to treat \u003cem\u003eAcanthamoeba\u003c/em\u003e infections. In the present study, we have evaluated Staurosporine as a potential treatment for \u003cem\u003eAcanthamoeba\u003c/em\u003e keratitis using mouse cornea as an \u003cem\u003eex vivo\u003c/em\u003e model, and to investigate its model of action by comparative proteomic analysis. Staurosporine altered the conformation of actin and tubulin cytoskeleton of treated trophozoites of \u003cem\u003eA. castellanii.\u003c/em\u003e In addition, proteomic analysis of the effect of Staurosporine on treated trophozoites revelated that this molecule induced an overexpression and a down-regulation of proteins related to functions vital for \u003cem\u003eAcanthamoeba\u003c/em\u003e infections. Additionally, obtained results in this study on the \u003cem\u003eex vivo\u003c/em\u003e assay using mouse corneas validate this animal model for the study of the pathogenesis of AK. Finally, Staurosporine eliminated the entire amoebic population and prevented adhesion and infection of amoebae to the epithelium of treated mouse corneas.\u003c/p\u003e","manuscriptTitle":"Staurosporine as a potential treatment for Acanthamoeba keratitis using mouse cornea as an ex vivo model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-24 15:49:00","doi":"10.21203/rs.3.rs-3878546/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c3445740-7a5e-4960-9a9d-6ed2c6fd7fa5","owner":[],"postedDate":"January 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-30T07:18:20+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-24 15:49:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3878546","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3878546","identity":"rs-3878546","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}