Antifungal and antibacterial functions of medicinal leech recombinant destabilase-lysozyme and its heated-up derivative

Abstract

Antifungal activity of recombinant medicinal leech destabilase-lysozyme (rec.Dest-Lys) was investigated by using fungi: Botrytis cinerea, and Verticillium lateriticum, including yeasts Candida guillermoudii and Shizosaccharomyces pombe. Its antibacterial activity was investigated on gram-negative bacteria Pseudomonas fluorescens. These activities were assessed by radial agar diffusion assay, and scanning and transmission electron microscopy. Therefore, destabilase-lysozyme not only is endo-isopeptidase and lysozyme, but also has antifungal and antibacterial activities. Muramidase activity of rec.Dest-Lys disappeared after heat-treating at 90°C for 50 min without the loss of its antimicrobial activity. Furthermore we showed for the first time that the heated-up derivative of rec.Dest-Lys exhibited more potent activities against the above enumerated fungi and gram-negative bacteria than original protein.

Key words： recombinant destabilase-lysozyme (rec.Dest-Lys); antimicrobial activity; antifungal activity; medicinal leech; heated-up derivative of rec.Dest-Lys (T-rec.Dest-Lys)

Acknowledgments

This work was supported by the Russian Foundation for Basic Research (Grant 11-04-12048 ofi-m). The authors acknowledge V. N. Lazarev and S. A. Levitsky (Research Institute of Physicochemical Medicine, Moscow, Russia) for preparation of recombinant destabilase- lysozyme.

References

1 Thammasirirak S, Pukcothanung Y, Preecharram S, Daduang S, Patramanon R, Fukamizo T, Araki T. Antimicrobial peptides derived from goose egg white lysozyme. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 2010, 151(1): 84–91

PMID

2 Baskova I P, Zavalova L L. Polyfunctionality of destabilase, a lysozyme from a medicinal leech. Bioorganicheskaia Khimiia, 2008, 34(3): 337–343

PMID

3 Cantu D, Carl Greve L, Labavitch J M, Powell A L T. Characterization of the cell wall of the ubiquitous plant pathogen Botrytis cinerea. Mycological Research, 2009, 113(12): 1396–1403

PMID

4 Tokunaga M, Kusamichi M, Koike H. Ultrastructure of outermost layer of cell wall in Candida albicans observed by rapid-freezing technique. Journal of Electron Microscopy, 1986, 35(3): 237–246

PMID

5 Kalebina T S, Kulaev I S. The role of proteins in formation of molecular structure of yeast cell wall. Successes of biological chemistry (Moscow), 2001, 41: 105–130

6 Nishiyama Y, Nakaoka C, Hiratani T, Abe S, Uchida K, Yamaguchi H. Synergy of lysozyme and lanoconazole on the morphology of Candida albicans. Journal of Electron Microscopy, 2001, 50(1): 41–49

PMID

7 Ibrahim H R, Thomas U, Pellegrini A. A helix-loop-helix peptide at the upper lip of the active site cleft of lysozyme confers potent antimicrobial activity with membrane permeabilization action. The Journal of biological chemistry, 2001, 276(47): 43767–43774

PMID

8 Wu Y, Daeschel M A. Lytic antimicrobial activity of hen egg white lysozyme immobilized to polystyrene beads. Journal of Food Science, 2007, 72(9): M369–M374

PMID

9 Samaranayake Y H, MacFarlane T W, Samaranayake L P, Aitchison T C. The in vitro lysozyme susceptibility of Candida species cultured in sucrose supplemented media. Microbios, 1993, 74(298): 23–28

PMID

10 Lopera D, Aristizabal B H, Restrepo A, Cano L E, González A. Lysozyme plays a dual role against the dimorphic fungus Paracoccidioides brasiliensis. Revista do Instituto de Medicina Tropical de Sao Paulo, 2008, 50(3): 169–175

PMID

11 Zavalova L L, Yudina T G, Artamonova I I, Baskova I P. Antibacterial non-glycosidase activity of invertebrate destabilase-lysozyme and of its helical amphipathic peptides. Chemotherapy, 2006, 52(3): 158–160

PMID

12 Zavalova L L, Lazarev V N, Levitsky S A, Yudina T G, Baskova I P. Destabilase-lysozyme of the medicinal leech. Polyfunctionality of recombinant protein. Biochemistry, 2010, 75(9): 1173–1181

13 Yudina T G, Bogdanov A G. The electron microscopy and scanning probe (tunneling) microscopy in microbiology. In: Netrusov A I, ed. Practical exercises in Microbiology. Moscow, 2005, 83–93

14 Robson G D. Programmed cell death in the aspergilli and other filamentous fungi. Medical Mycology, 2006, 44(s1): 109–114

15 Ibrahim H R, Inazaki D, Abdou A, Aoki T, Kim M. Processing of lysozyme at distinct loops by pepsin: a novel action for generating multiple antimicrobial peptide motifs in the newborn stomach. Biochimica et Biophysica Acta, 2005, 1726(1): 102–114

PMID

16 Cong L, Yang X, Wang X, Tada M, Lu M, Liu H, Zhu B. Characterization of an i-type lysozyme gene from the sea cucumber Stichopus japonicus, and enzymatic and nonenzymatic antimicrobial activities of its recombinant protein. Journal of Bioscience and Bioengineering, 2009, 107(6): 583–588

PMID

17 Nash J A, Ballard T N, Weaver T E, Akinbi H T. The peptidoglycan-degrading property of lysozyme is not required for bactericidal activity in vivo. Journal of Immunology (Baltimore, MD.: 1950), 2006, 177(1): 519–526

PMID

18 Baskova I P, Zavalova L L, Berezhnoy S, Avdonin P, Afanasjeva G, Popov E, Gabbasov Z. Inhibition of induced and spontaneous platelet aggregation by destabilase from medicinal leech. Platelets, 2000, 11(2): 83–86

PMID

19 Zavalova L L, Baskova I P, Lukyanov S A, Sass A V, Snezhkov E V, Akopov S B, Artamonova I I, Archipova V S, Nesmeyanov V A, Kozlov D G, Benevolensky S V, Kiseleva V I, Poverenny A M, Sverdlov E D. Destabilase from the medicinal leech is a representative of a novel family of lysozymes. Biochimica et Biophysica Acta, 2000, 1478(1): 69–77

PMID