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EVIDENCE OF CHITOSANASE INVOLVEMENT IN THE PROTECTION OF BACTERIA AGAINST THE ANTIMICROBIAL ACTIVITY OF THE CHITOSAN by Mariana Gabriela Ghinet Thesis presented to the Department of Biology in Fulfillment of the Requirements for the Degree of Philosophise Doctor (Ph. D.) FACULTE DES SCIENCES UNIVERSITE DE SHERBROOKE Sherbrooke, Quebec, Canada, August 2009 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 Ottawa ON K1A 0N4 Canada Canada Your file Vote reference ISBN: 978-0-494-64216-0 Our file Notre reference ISBN: 978-0-494-64216-0 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'lnternet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non­ support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission. In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these. While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis. 1*1 Canada LA CHITOSANASE EN TANT QUE FACTEUR DE PROTECTION DES BACTERIES CONTRE L'ACTIVITE ANTIMICROBIENNE DU CHITOSANE par Mariana Gabriela Ghinet These presentee au Departement de biologie en vue de l'obtention du grade de docteure es sciences (Ph. D.) FACULTE DES SCIENCES UNIVERSITE DE SHERBROOKE Sherbrooke, Quebec, Canada, Aout 2009 Le 21 mai 2010 lejury a accepte la these de Madame Mariana Ghinet dans sa version finale. Membres du jury Professeur Ryszard Brzezinski Directeur de recherche Departement de biologie Professeur Denis Lebel Membre Departement de biologie Monsieur Denis Groleau Membre externe Institut de recherche en biotechnologie Professeur Claude Dery President rapporteur Departement de biologie To my wonderful parents, Gheorghe and Sabina, who have raised me to be the person I am today. Despite the huge distance that separates us, you have been with me every step of the way, through good and bad times. To my brother Sebastian, my inspiration and model in life and to my sister- in-law Florenta, who encouraged me throughout the years. To my fiance, Johny, with whom I have spent the most wonderful years of my life, filled with love and happiness. I dedicate this Thesis. SUMMARY Chitosan, a biopolymer composed of P-(l,4)-linked D-glucosamine and JV-acetyl-D- glucosamine residues has multiple industrial applications. Recently, chitosan has gained great interest due to its antimicrobial activity. Chitosan has antimicrobial activity against a wide range of target organisms such as bacteria, fungi and viruses. This antimicrobial activity is based on its cationic character, and is mediated by the chitosan's positively charged amino groups interactions with negatively charged residues in the bacterial cell wall. Enzymes with chitosanase activity catalyzing the hydrolysis of glycoside linkages in chitosan are found in many organisms, including bacteria, fungi, and plants. In the last three decades, chitosanases have been intensively studied as tools for biotechnological transformation of chitosan. However, less is known about their physiological functions in chitosanase-producing microorganisms. Previous reports have characterized chitosanases as metabolic enzymes allowing bacteria to use chitosan as carbon and nitrogen sources. The aim of this research project was to examine chitosanases significance as possible resistance factors against the antimicrobial effect of chitosan. Our work, as well as previous studies realized in our laboratory, showed that expression of a heterologous chitosanase gene in the Gram-negative bacterium Escherichia coli (naturally devoid of chitosanase activity) increases the level of resistance against chitosan. Interestingly, the resistance level to chitosan was influenced by the relative activity of the heterologous chitosanase. The expression of inactive heterologous chitosanase did not confer any resistance to chitosan supporting our hypothesis that chitosanases may have a role in the protection against the antimicrobial effect of chitosan. In order to obtain more direct evidence sustaining our hypothesis, we inactivated the chitosanase gene from Streptomyces lividans TK24. Hence, we developed a new system for gene disruption and replacement in Streptomyces with cytosine deaminase as negative selection marker. The disruption of the chitosanase gene in S. lividans TK24 resulted in an 1 increased susceptibility of the mutant strain towards the toxic effect of chitosan. Our in vivo experiments showed that, in the presence of chitosan, growth of this mutant strain as well as its ability for xylose uptake were impaired compared to the wildtype strain. This represents the first genetical proof for the protective role of a chitosanase against the bactericidal effect of chitosan. In our quest to discover chitosanases with new characteristics, we determined the biochemical properties of the chitosanase CsnA from Streptomyces coelicolor A3(2). Our studies revealed that CsnA was, in many aspects, very similar to the chitosanase CsnN174 from Streptomyces sp. N174. An interesting feature of the CsnA is its secretion. The signal peptide of the CsnA has a Tat-dependent motif. The CsnA is the first studied chitosanase to be secreted via the Tat pathway. These studies also contributed to a better understanding of the chitosanase secretion. Evidence concerning the role of chitosanases in the protection of bacteria against the bactericidal effect of chitosan was also brought by the study of cell localization of the exo-P- D-glucosaminidase (CsxA) from Amycolatopsis orientalis. CsxA has a carbohydrate-binding module (CBM35) with an unusual affinity. This module appended to CsxA recognizes as substrate glucuronic acid, a component of the Gram-positive bacterie cell wall. Thereby, we analyzed by epifluorescence and confocal microscopy the cellular localization of the CsxA- CBM35 in Amycolatopsis orientalis cells grown in the presence of chitosan. The microscopy analysis showed that CsxA is anchored to the bacterial cell wall via CBM35's interaction with glucuronic acid. Due to its strategic localization, the exo-P-D-glucosaminidase from Amycolatopsis orientalis could also contribute to protection against the antimicrobial effect of chitosan. The results obtained during this study improved our knowledge concerning the physiological role of chitosanases in bacteria. Independent of their cellular localization, intracellular, cell wall-anchored or secreted, the chitosanases play an important role in bacterial resistance to chitosan. Furthermore, our results contribute to a better understanding of the chitosanase function versatility in bacteria. 11 SOMMAIRE Le chitosane, un biopolymere compose de residus de D-glucosamine et JV-acetyl-D- glucosamine lies par liaisons P-(l,4), possede des applications industrielles multiples. Recemment le chitosane a recu beaucoup d'interet grace a son activite antimicrobienne. Le chitosane montre une activite antimicrobienne envers des nombreux microorganismes comme les bacteries, les champignons et les virus. Cette activite antimicrobienne est basee sur son caractere cationique. Grace a ses groupements amines charges positivement, le chitosane interagit avec les residus charges negativement qui se trouvent au niveau de la paroi cellulaire bacterienne. Les enzymes possedant une activite chitosanase, catalysant l'hydrolyse des liens glycosidiques dans le chitosane, ont ete trouvees chez beaucoup d'organismes, incluant des bacteries, des champignons et des plantes. Au cours des trois dernieres decennies, les chitosanases ont ete intensivement etudiees comme outils pour la transformation biotechnologique du chitosane. Cependant, leurs fonctions physiologiques chez les micro­ organismes qui les produisent sont peu connues. Des etudes precedentes ont caracterise les chitosanases en tant qu'enzymes metaboliques permettant aux bacteries d'utiliser le chitosane comme sources de carbone et d'azote. Le but de ce projet de recherche etait d'examiner la signification des chitosanases comme facteurs possibles de resistance contre l'effet antimicrobien du chitosane. Nos travaux, ainsi que d'autres travaux effectues dans ce laboratoire, ont montre que l'expression d'un gene heterologue de chitosanase dans Escherichia coli, une bacterie
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