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Sensitivity of Enzymatic Toxins from Corneal Isolate of Acanthamoeba Protozoan to Physicochemical Parameters

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Abstract

Acanthamoeba is a free-living amoeba that causes severe corneal infection (Acanthamoeba keratitis) and produces a variety of extracellular enzymes, called exoproteome. Since physicochemical characters are suggested being associated with therapeutic profile and clinical severity of the infection, we investigated the physicochemical properties of proteolysis mediated by amoebic exoproteome. Corneal scraping was collected from a patient who showed typical symptoms of acute Acanthamoeba keratitis. Axenic amoeba was phylogenetically identified by 18S rDNA sequencing analysis. Effects of pH, temperature and diamidines on proteolysis mediated by exoproteome were assessed using zymography assays. Proteolytic enzymes were most active at pH 7.0 and 37 °C. Calcium ions decreased enzymatic activity. The main components of amoebic exoproteome were characterized as serine proteases. We demonstrated for the first time that commercial antimicrobial diamidines used for Acanthamoeba keratitis therapy inhibit enzymatic activity of amoebic exoproteome. Results showed the thermostability of Acanthamoeba proteases, which suggest a long-term effect of these virulence factors at the central and peripheral cornea with possible role in degradation of extracellular matrix components. Our findings open new perspectives about the complementary and unreported properties of antimicrobial compounds of the diamidine class on the inhibition of enzymatic activity and presumptive control of amoebic infection in the cornea.

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References

  1. Altschul SF, Madden TL, Schäffer AA et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Belmonte C, Tervo TT, Gallar J (2011) Sensory innervation of the eye. In: Levin LA, Nilsson SFE, Hoeve JV, Wu SM (eds) Adler’s physiology of the eye, 11th edn. Elsevier Inc., New York, pp 363–384

    Chapter  Google Scholar 

  3. Carvalho FR, Foronda AS, Mannis MJ et al (2009) Twenty years of Acanthamoeba keratitis. Cornea 28:516–519

    Article  CAS  PubMed  Google Scholar 

  4. Carrijo LC, Andrich F, de Lima ME et al (2005) Biological properties of the venom from the scorpionfish (Scorpaena plumieri) and purification of a gelatinolytic protease. Toxicon 45:843–850

    Article  CAS  PubMed  Google Scholar 

  5. Carrijo-Carvalho LC, Sant’ana VP, Foronda AS et al (2017) Therapeutic agents and biocides for ocular infections by free-living amoebae of Acanthamoeba genus. Surv Ophthalmol 62:203–218

    Article  PubMed  Google Scholar 

  6. Clarke DW, Niederkorn JY (2006) The pathophysiology of Acanthamoeba keratitis. Trends Parasitol 22:175–180

    Article  CAS  PubMed  Google Scholar 

  7. Dart JK, Saw VP, Kilvington S (2009) Acanthamoeba keratitis: diagnosis and treatment update 2009. Am J Ophthalmol 148:487–499

    Article  PubMed  Google Scholar 

  8. Freeman RD, Fatt I (1973) Environmental influences on ocular temperature. Invest Ophthalmol 12:596–602

    CAS  PubMed  Google Scholar 

  9. Frees D, Brøndsted L, Ingmer H (2013) Bacterial proteases and virulence. Subcell Biochem 66:161–192

    Article  CAS  PubMed  Google Scholar 

  10. 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

    Article  CAS  PubMed  Google Scholar 

  11. Grass GM, Wood RW, Robinson JR (1985) Effects of calcium chelating agents on corneal permeability. Invest Ophthalmol Vis Sci 26:110–113

    CAS  PubMed  Google Scholar 

  12. Heussen C, Dowdle EB (1980) Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal Biochem 102:196–202

    Article  CAS  PubMed  Google Scholar 

  13. Ksiazek M, Karim AY, Bryzek D et al (2015) Mirolase, a novel subtilisin-like serine protease from the periodontopathogen Tannerella forsythia. Biol Chem 396:261–275

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lorenzo-Morales J, Khan NA, Walochnik J (2015) An update on Acanthamoeba keratitis: diagnosis, pathogenesis and treatment. Parasite 22:10

    Article  PubMed  PubMed Central  Google Scholar 

  15. Maciver SK, Asif M, Simmen MW et al (2013) A systematic analysis of Acanthamoeba genotype frequency correlated with source and pathogenicity: T4 is confirmed as a pathogen-rich genotype. Eur J Protistol 49:217–221

    Article  PubMed  Google Scholar 

  16. Mafra CS, Carrijo-Carvalho LC, Chudzinski-Tavassi AM et al (2013) Antimicrobial action of biguanides on the viability of Acanthamoeba cysts and assessment of cell toxicity. Invest Ophthalmol Vis Sci 54:6363–6372

    Article  CAS  PubMed  Google Scholar 

  17. Mares-Guia M, Shaw E (1965) Studies on the active center of trypsin. The binding of amidines and guanidines as models of the substrate side chain. J Biol Chem 240:1579–1585

    CAS  PubMed  Google Scholar 

  18. Martinez AJ, Visvesvara GS (1997) Free-living, amphizoic and opportunistic amebas. Brain Pathol 7:583–598

    Article  CAS  PubMed  Google Scholar 

  19. Nielsen MK, Nielsen K, Hjortdal J et al (2014) Temperature limitation may explain the containment of the trophozoites in the cornea during Acanthamoeba castellanii keratitis. Parasitol Res 113:4349–4353

    Article  PubMed  Google Scholar 

  20. Page FC (1976) An illustrated key to freshwater and soil amoebae. Freshwater Biological Association, Ambleside

    Google Scholar 

  21. Panjwani N (2010) Pathogenesis of Acanthamoeba keratitis. Ocul Surf 8:70–79

    Article  PubMed  PubMed Central  Google Scholar 

  22. Purslow C, Wolffsohn J (2007) The relation between physical properties of the anterior eye and ocular surface temperature. Optom Vis Sci 84:197–201

    Article  PubMed  Google Scholar 

  23. Sant’ana VP, Carrijo-Carvalho LC, Foronda AS et al (2015) Cytotoxic activity and degradation patterns of structural proteins by corneal isolates of Acanthamoeba spp. Graefes Arch Clin Exp Ophthalmol 253:65–75

    Article  PubMed  Google Scholar 

  24. Schroeder JM, Booton GC, Hay J et al (2001) Use of subgenic 18S ribosomal DNA PCR and sequencing for genus and genotype identification of acanthamoebae from humans with keratitis and from sewage sludge. J Clin Microbiol 39:1903–1911

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Schuster FL, Visvesvara GS (2004) Opportunistic amoebae: challenges in prophylaxis and treatment. Drug Resist Updat 7:41–51

    Article  CAS  PubMed  Google Scholar 

  26. Singh B, Fleury C, Jalalvand F et al (2012) Human pathogens utilize host extracellular matrix proteins laminin and collagen for adhesion and invasion of the host. FEMS Microbiol Rev 36:1122–1180

    Article  CAS  PubMed  Google Scholar 

  27. Souza Carvalho FR, Carrijo-Carvalho LC, Chudzinski-Tavassi AM et al (2011) Serine-like proteolytic enzymes correlated with differential pathogenicity in patients with acute Acanthamoeba keratitis. Clin Microbiol Infect 17:603–609

    Article  PubMed  Google Scholar 

  28. Tamura K, Stecher G, Peterson D et al (2013) MEGA6: molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30:2725–2729

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Tu EY (2014) Acanthamoeba keratitis: a new normal. Am J Ophthalmol 158:417–419

    Article  PubMed  Google Scholar 

  30. Visvesvara GS (2010) Parasite culture: Acanthamoeba and Naegleria spp. In: Garcia LS (ed) Clinical microbiology procedures handbook, 3rd edn. ASM Press, Washington, DC, pp 9.9.2.1–9.9.2.8

    Google Scholar 

  31. Xin Y, Sun Z, Chen Q et al (2015) Purification and characterization of a novel extracellular thermostable alkaline protease from Streptomyces sp. M30. J Microbiol Biotechnol 25:1944–1953

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by São Paulo Research Foundation (Grant Numbers 2008/53969-0 to D.F., 2014/18926-02 to V.P.S., 2011/51626-1 and 2012/15603-0 to F.R.S.C.) and Coordination for the Improvement of Higher Education Personnel (Grant Numbers 23038.001063/2012-01 and PNPD to L.C.C.C). The sponsor or funding organization had no role in the design or conduct of this research.

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Authors and Affiliations

  1. Department of Ophthalmology and Visual Sciences, Paulista School of Medicine, Federal University of Sao Paulo, Botucatu Street, 821, Vila Clementino, Sao Paulo, SP, 04023-062, Brazil

    Viviane P. Sant’Ana, Annette S. Foronda, Denise de Freitas, Linda C. Carrijo-Carvalho & Fábio Ramos de Souza Carvalho

  2. Centro Universitário do Espírito Santo (UNESC), Colatina, Espirito Santo, Brazil

    Linda C. Carrijo-Carvalho & Fábio Ramos de Souza Carvalho

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  1. Viviane P. Sant’Ana
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  2. Annette S. Foronda
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  3. Denise de Freitas
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  4. Linda C. Carrijo-Carvalho
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  5. Fábio Ramos de Souza Carvalho
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Correspondence to Fábio Ramos de Souza Carvalho.

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Sant’Ana, V.P., Foronda, A.S., de Freitas, D. et al. Sensitivity of Enzymatic Toxins from Corneal Isolate of Acanthamoeba Protozoan to Physicochemical Parameters. Curr Microbiol 74, 1316–1323 (2017). https://doi.org/10.1007/s00284-017-1319-6

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  • DOI: https://doi.org/10.1007/s00284-017-1319-6

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