Abstract
The members of the genus Acanthamoeba are ubiquitous, free-living amoebae found in various environments. The amoebae can cause severe complications in both, immunocompetent and immunocompromised individuals. The aim of this study was to characterize extracellular proteases of Acanthamoeba isolates from different sources belonging to genotype T4 as well as the determination of the pathogenicity potential to correlate pathogenicity with protease activity and protease banding pattern. A total of 19 isolates (11 clinical and 8 environmental) were cultured axenically, then the pathogenicity of the isolates was assessed by osmo- and thermo- tolerance tests. An applied colorimetric method using azocasein as a substrate was used for the extracellular protease activity assay. Protease characterization was carried out by zymography analysis with and without protease inhibitors. Assessment of the pathogenicity potential using physical parameters revealed that 2 (25%), 2 (25%), and 4 (50%) of the environmental isolates were potential pathogens, weak potential pathogens, and non-pathogens, respectively. According to our results, this protease activity assay can be a powerful tool for differentiating pathogenic and non-pathogenic strains of Acanthamoeba.
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References
Alfieri SC, Correia CE, Motegi SA, Pral EM (2000) Proteinase activities in total extracts and in medium conditioned by Acanthamoeba polyphaga trophozoites. J Parasitol 86:220–227. https://doi.org/10.1645/0022-3395(2000)086[0220:PAITEA]2.0.CO;2
Alizadeh H, Neelam S, Niederkorn JY (2007) Effect of immunization with the mannose-induced Acanthamoeba protein and Acanthamoeba plasminogen activator in mitigating Acanthamoeba keratitis. Invest Ophthalmol Vis Sci 48:5597–5604. https://doi.org/10.1167/iovs.07-0407
Armand B, Motazedian MH, Asgari Q (2016) Isolation and identification of pathogenic free-living amoeba from surface and tap water of Shiraz City using morphological and molecular methods. Parasitol Res 115:63–68. https://doi.org/10.1007/s00436-015-4721-7
Blaschitz M, Köhsler M, Aspöck H, Walochnik J (2006) Detection of a serine proteinase gene in Acanthamoeba genotype T6 (Amoebozoa: Lobosea). Exp Parasitol 114:26–33. https://doi.org/10.1016/j.exppara.2006.02.004
Cao Z, Jefferson DM, Panjwani N (1998) Role of carbohydrate-mediated adherence in cytopathogenic mechanisms of Acanthamoeba. J Biol Chem 273:15838–15845
Cho JH, Na BK, Kim TS, Song CY (2000) Purification and characterization of an extracellular serine proteinase from Acanthamoeba castellanii. IUBMB Life 50:209–214. https://doi.org/10.1080/152165400300001534
da Rocha-Azevedo B, Costa e Silva-Filho F (2007) Biological characterization of a clinical and an environmental isolate of Acanthamoeba polyphaga: analysis of relevant parameters to decode pathogenicity. Arch Microbiol 188:441–449. https://doi.org/10.1007/s00203-007-0264-3
Fuerst PA, Booton GC, Crary M (2015) Phylogenetic analysis and the evolution of the 18S rRNA gene typing system of Acanthamoeba. J Eukaryot Microbiol 62:69–84. https://doi.org/10.1111/jeu.12186
Gast RJ, Ledee DR, Fuerst PA, Byers TJ (1996) Subgenus systematics of Acanthamoeba: four nuclear 18S rDNA sequence types. J Eukaryot Microbiol 43:498–504
Hurt M, Neelam S, Niederkorn J, Alizadeh H (2003) Pathogenic Acanthamoeba spp. secrete a mannose-induced cytolytic protein that correlates with the ability to cause disease. Infect Immun 71:6243–6255
Joslin CE, Tu EY, Shoff ME, Booton GC, Fuerst PA, McMahon TT, Anderson RJ, Dworkin MS, Sugar J, Davis FG, Stayner LT (2007) The association of contact lens solution use and Acanthamoeba keratitis. Am J Ophthalmol 144:169–180. https://doi.org/10.1016/j.ajo.2007.05.029
Khan NA (2006) Acanthamoeba: biology and increasing importance in human health. FEMS Microbiol Rev 30:564–595. https://doi.org/10.1111/j.1574-6976.2006.00023.x
Khan NA, Jarroll EL, Panjwani N, Cao Z, Paget TA (2000) Proteases as markers for differentiation of pathogenic and nonpathogenic species of Acanthamoeba. J Clin Microbiol 38:2858–2861
Khan NA, Jarroll EL, Paget TA (2001) Acanthamoeba can be differentiated by the polymerase chain reaction and simple plating assays. Curr Microbiol 43:204–208. https://doi.org/10.1007/s002840010288
Kim WT, Kong HH, Ha YR, Hong YC, Jeong HJ, HS Y, Chung DI (2006) Comparison of specific activity and cytopathic effects of purified 33 kDa serine proteinase from Acanthamoeba strains with different degree of virulence. Korean J Parasitol 44:321–330
Köhsler M, Mrva M, Walochnik J (2016) Acanthamoeba. In: Walochnik J, Duchêne M (eds) Molecular Parasitology. Springer, Vienna, pp 285–324
Kong HH, Kim TH, Chung DI (2000) Purification and characterization of a secretory serine proteinase of Acanthamoeba healyi isolated from GAE. J Parasitol 86:12–17. https://doi.org/10.1645/0022-3395(2000)086[0012:PACOAS]2.0.CO;2
Marciano-Cabral F, Cabral G (2003) Acanthamoeba spp. as agents of disease in humans. Clin Microbiol Rev 16:273–307
Ramírez-Rico G, Martínez-Castillo M, de la Garza M, Shibayama M, Serrano-Luna J (2015) Acanthamoeba castellanii proteases are capable of degrading iron-binding proteins as a possible mechanism of pathogenicity. J Eukaryot Microbiol 62:614–622. https://doi.org/10.1111/jeu.12215
Sarath G, Zeece MG, Penheiter AR (2001) Protease assay methods. In: Beynon R, Bond JS (eds) Proteolytic enzymes: a practical approach, 2nd edn. Oxford University Press, New York, pp 45–76
Serrano-Luna Jde J, Cervantes-Sandoval I, Calderón J, Navarro-García F, Tsutsumi V, Shibayama M (2006) Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii. Can J Microbiol 52:16–23. https://doi.org/10.1139/w05-114
Siddiqui R, Khan NA (2012) Biology and pathogenesis of Acanthamoeba. Parasit Vectors 5:6. https://doi.org/10.1186/1756-3305-5-6
Sissons J, Alsam S, Goldsworthy G, Lightfoot M, Jarroll EL, Khan NA (2006) Identification and properties of proteases from an Acanthamoeba isolate capable of producing granulomatous encephalitis. BMC Microbiol 6:42. https://doi.org/10.1186/1471-2180-6-42
Strober W (2001) Trypan blue exclusion test of cell viability. Curr Protoc Immunol Appendix3: B. https://doi.org/10.1002/0471142735.ima03bs21
Tawfeek GM, Bishara SA, Sarhan RM, ElShabrawi Taher E, ElSaady Khayyal A (2016) Genotypic, physiological, and biochemical characterization of potentially pathogenic Acanthamoeba isolated from the environment in Cairo, Egypt. Parasitol Res 115:1871–1881. https://doi.org/10.1007/s00436-016-4927-3
Toth M, Fridman R (2001) Assessment of gelatinases (MMP-2 and MMP-9) by gelatin zymography. In: Brooks SA, Schumacher U (eds) Metastasis research protocols, Volume I: analysis of cells and tissues, methods in molecular medicine, vol 57. Humana Press, Totowa, pp 163–174
Visvesvara GS, Balamuth W (1975) Comparative studies on related free-living and pathogenic amebae with special reference to Acanthamoeba. J Protozool 22:245–256. https://doi.org/10.1111/j.1550-7408.1975.tb05860.x
Walochnik J, Haller-Schober E, Kölli H, Picher O, Obwaller A, Aspöck H (2000) Discrimination between clinically relevant and nonrelevant Acanthamoeba strains isolated from contact lens-wearing keratitis patients in Austria. J Clin Microbiol 38:3932–3936
Acknowledgments
This study is part of a PhD thesis that supported financially by Tarbiat Modares University. The authors would like to thank all staffs of the Parasitology Department of Medical Sciences Faculty of Tarbiat Modares University for their kind assistance.
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Mahdavi Poor, B., Dalimi, A., Ghafarifar, F. et al. Characterization of extracellular proteases of Acanthamoeba genotype T4 isolated from different sources in Iran. Parasitol Res 116, 3373–3380 (2017). https://doi.org/10.1007/s00436-017-5656-y
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DOI: https://doi.org/10.1007/s00436-017-5656-y