Ramírez ALS, Arias CM, Marulanda OJ

Language: Spanish
References: 24
Page: 261-672
PDF size: 456.84 Kb.

ABSTRACT

Introduction: the Secreted Aspartyl Protease (Sap) is considered a virulence factor in the infection process caused by Candida albicans. The frequency of candidiasis worldwide is increasing, hence the need for finding new drugs to fight this illness and also a quick and economic evaluation and screening methods to identify some Sap inhibition agents.
Objective: to standardize fluorescent method for identifying the Sap activity inhibition.
Methods: Sap production was induced in C. albicans cultures following the methodology described by Capobianco, their levels were evaluated by electrophoresis on SDS-PAGE gels at different periods of time. Sap activity was checked by spectrofluorometry, for which the reaction conditions were determined, varying the concentrations of copper and fluorexon, and the inhibition results were expressed as decreased fluorescence signal. Pepstatin A served as Sap inhibition control whereas pancreatin was the positive control of the protease activity.
Results: the concentration rates of 5.5 µM and 5.0 µM were found for fluorexon and copper, respectively; their optimal coupling times was 120 min and the maximum Sap activity was observed after 22 h of incubation.
Conclusions: the standardized conditions for the spectrofluorometric method allowed confirming the inhibition of Sap by Pepstatin A and showing that this is a viable method to evaluate inhibitors of this protease.

REFERENCES

1. Odds F. Candida species and virulence. ASM News. 1994;60:313-8.

2. Douglas LJ. Candida proteinases and candidosis. Crit Rev Biotechnol. 1988;8:121-9.

3. Cutler JE. Putative virulence factors of Candida albicans. Ann Rev Microbiol. 1991;45:187-218.

4. Ruchel R, de Bernardis F, Ray TL, Sullivan PA, Cole GT. Candida acid proteinases. J Med Vet Mycol. 1992;30(Suppl 1):123-32.

5. White TC, Kohler CA, Miyasaki SH, Agabian N. Expression of virulence factors in Candida albicans. Can J Botany. 1995;73(S1):1058-64.

6. Ray TL, Payne CD, Morrow BJ. Candida albicans acid proteinase: Characterization and role in candidiasis. Aspartic proteinase conference on structure and function of the aspartic proteinases: Genetics, structures, and mechanisms. California: Plenum Press; 1990. p. 173-83.

7. Hube B, Monod M, Schofield DA, Brown AJ, Gow NA. Expression of seven members of the gene family encoding secretory aspartyl proteinases in Candida albicans. Mol Microbiol. 1994;14:87-99.

8. White TC, Agabian N. Candida albicans secreted aspartyl proteinases: isoenzyme pattern is determined by cell type, and levels are determined by environmental factors. J Bacteriol. 1995;177:5215-21.

9. Aoki W, Kitahara N, Miura N, Morisaka H, Yamahoto Y, Kuroda K,et al. Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease. PloS One. 2012;7(2): e32513.

10. Cutfield SM, Dodson EJ, Anderson BF, Moody PC, Marshall CJ, Sullivan PA, et al. The crystal structure of a major secreted aspartic proteinase from Candida albicans in complexes with two inhibitors. Structure. 1995;3:1261-71.

11. Capobianco JO, Lerner CG, Goldman RC. Application of a fluorogenic substrate in the assay of proteolytic activity and in the discovery of a potent inhibitor of Candida albicans aspartic proteinase. Anal Biochem. 1992;204:96-102.

12. Goddard JP, Reymond JL. Recent advances in enzyme assays. Trends Biotechnol. 2004;22:363-70.

13. Breeuwer P, Drocourt JL, Bunschoten N, Zwietering MH, Rombouts FM, Abee T. Characterization of uptake and hydrolysis of fluorescein diacetate and carboxyfluoresceindiacetate by intracellular esterases in Saccharomyces cerevisiae, which result in accumulation of fluorescent product. Appl Environ Microbiol. 1995;61:1614-9.

14. Lomolino G, Lante A, Crapisi A, Spettoli P, Curioni A. Detection of Saccharomyces cerevisiae carboxy lesterase activity after native and sodium dodecyl sulfate electrophoresis by using fluorescein diacetate as substrate. Electrophoresis. 2001;22:1021-3.

15. Kierat RM, Kramer R. A fluorogenic and chromogenic probe that detects the esterase activity of trace copper(II). Bioorg Med Chem Lett. 2005;15:4824-7.

16. Takakusa H, Kikuchi K, Urano Y, Sakamoto S, Yamaguchi K, Nagano T. Design and synthesis of an enzyme-cleavable sensor molecule for phosphodiesterase activity based on fluorescence resonance energy transfer. J Am Chem Soc. 2002;124:1653-7.

17. Wang Q, Scheigetz J, Roy B, Ramachandran C, Gresser MJ. Novel caged fluorescein diphosphates as photoactivatable substrates for protein tyrosine phosphatases. Biochim Biophys Acta. 2002;1601:19-28.

18. Gee KR. Novel fluorogenic substrates for acid phosphatase. Bioorg Med ChemLett. 1999;9:1395-6.

19. Dostal J, Hamal P, Pavlickova L, Soucek M, Rumi T, Pichova I . Simple method for screening Candida species isolates for the presence of secreted proteinases: a tool for the prediction. J Clin Microbiol. 2003;41(2):712-6.

20. Kathryn Dean ES, Gerard K, Olivier R, Jean-Louis R. A Green Fluorescent Chemosensor for Amino Acids Provides a Versatile High-Throughput Screening (HTS) Assay for Proteases. Bioorg Med Chem Lett. 2003;13:1653-6.

21. Borg-von Zepelin M, Beggah S, Boggian K, Sanglard D, Monod M. The expression of the secreted aspartyl proteinases Sap4 to Sap6 from Candida albicans in murine macrophages. Mol Microbiol. 1998;28:543-54.

22. Ruchel R, Tgegeler R, Trost M. A comparison of secretory proteinase from different strains of Candida albicans. Saboraud. 1982;20:233-44.

23. Schaller M, Krnjaic N, Niewerth M, Hamm G, Hube B, Korting HC. Effect of antimycotic agents on the activity of aspartyl proteinases secreted by Candida albicans. J Med Microbiol. 2003;52:247-9.

24. Korting HC, Schaller M, Eder G, Hamm G, Bohmer U, Hube B. Effects of the human immunodeficiency virus (HIV) proteinase inhibitors saquinavir and indinavir on in vitro activities of secreted aspartyl proteinases of Candida albicans isolates from HIV-infected patients. Antimicrob Agents Chemother. 1999;43:2038-42.