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Unraveling the Biotechnological Potential of the Secretome of Burkholderia Cepacia Complex, with Focus on Its Antimicrobial Activity

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Unraveling the Biotechnological Potential of the Secretome of Burkholderia Cepacia Complex, with Focus on Its Antimicrobial Activity Unraveling the biotechnological potential of the secretome of Burkholderia cepacia complex, with focus on its antimicrobial activity Carina Andreia Ribeiro Galhofa Thesis to obtain the Master of Science Degree in Biotechnology Supervisors: Prof. Doctor Isabel Maria de Sá Correia Leite de Almeida; Doctor Patrick de Oliveira Freire Examination Committee Chairperson: Doctor Arsénio do Carmo Sales Mendes Fialho Supervisor: Prof. Doctor Isabel Maria de Sá Correia Leite de Almeida Members of the Committee: Doctor Inês Batista Guinote December 2017 Acknowledgements The wish to become a future microbiologist and to study the “little bugs” that were able to cause diseases in individuals way bigger than them has been present since I was a child. As such, I would like to acknowledge those that helped me from the beginning to the end of this work, making this possible. To Prof. Dr.Isabel Sá Correia, I would like to express my gratitude not only for advising, but also for accepting me in this project which I enjoyed to do so much. To Dr. Patrick Freire, my advisor within BioMimetx, not only for all the guidance and patience but, most of all, for the motivation and believing in my work and Dr. Carla Coutinho for the support, for bearing my inexperience and for all the tips given that prevented me from exploding the laboratory…and the centrifuge. To the funding of IBB (Institute for Bioengineering and Biosciences) and BioMimetx, that gave me all the resources and conditions that enabled the conduction of my work. To Amir Hassan, for staying up late at the lab waiting for me to finish my work and for the “Goooooodddd woooorkkkkk” motivational screams. Words cannot describe how grateful I am for all your help and how a good part of this thesis wouldn’t be done without your unconditional patience, motivation and incomprehensible wake up calls, usually in arabic. Special thanks go to Inês Leonardo, for the whole work performed that guided this project and for all the support and time spent with the lessons and tips that enabled me to start my work. To the BioMimetx team for the warm welcome. In particular, to Dr. Inês Guinote, Raquel Marques, Sílvia Ribeiro, Susana Saraiva and Tânia Chança for the cheering, help and good times, making my work so much easier and delightful. To João Nascimento, José Teles Reis, Kamran Azmaliyev, Kcénia Bougrova, Lauren Lopes, Renato Dimas and Sónia Santos, for always being there for me when it seemed to get harder and to Emilia Wójcik for all the polish good vibes, the wonderful friendship and for believing in me from the absolute beginning. Without her this couldn’t be possible, ever! To my lab colleagues at BSRG (Biological Sciences Research Group) for the nice times spent at the laboratory and the library. It was a great pleasure to share all the thesis adventures with you. At last, the most special thanks to my family, in particular, to my parents and my sister for all the love and support. Thank you for feeding my love for science and for bearing my enthusiasm for my work even though you may not have found it that interesting. To my teachers and professors, from my first grade to my masters, for the inspiration and for the enlightenment that enabled me to get this far… ...My most sincere thank you. i “What better microbial challenge to unite agricultural and medical microbiologists than an organism that reduces an onion to a macerated pulp, protects other crops from bacterial and fungal disease, devastates the health and social life of cystic fibrosis patients, and not only is resistant to the most famous of antibiotics, penicillin, but can use it as a nutrient!" -J. R. W. Govan, 1998 ii Abstract In this project, developed in collaboration with BioMimetx, a start-up company dedicated to the development of innovative biological biocides, the secretomes of B. cenocepacia IST01 were explored for potential biotechnological applications. Culture supernatants of IST01 were harvested in mid exponential, transition to the stationary, early and late stationary phases and tested against Escherichia coli, Enterococcus faecalis, Listeria monocytogenes and Staphylococcus aureus. The highest bactericidal effectiveness was exhibited by the culture supernatants of the late and the early stationary phases, that were also effective against the multidrug resistant isolates Burkholderia cenocepacia, Pseudomonas aeruginosa and Methicillin-resistant Staphylococcus aureus. These two culture supernatant samples presented a distinct protein migration pattern when compared with the samples of the mid exponential and transition to the stationary phase, in analyses by SDS-PAGE, and the highest proteolytic activity values among the tested samples. Taking these results into account, early stationary phase supernatants were selected for further fractionation, by centrifugal ultrafiltration and separation of the molecules in the samples according to their different molecular weights for the characterization of the mix that is of value to activity. However accounted for over 78% of the total secretome and was the only one which exhibited antimicrobial activity, probably consisting in a mixture of secondary metabolites and small molecular proteins or peptides. The results obtained in this project are encouraging as they support a possible future use of the Bcc secretomes as an antimicrobial solution cannot be lifted. Key words: Burkholderia cepacia complex, bacterial secretomes, antimicrobial activity, lipolytic activity, proteolytic activity iii Resumo Neste projecto, desenvolvido em colaboração com a BioMimetx, uma start-up que se dedica ao desenvolvimento de biocidas biológicos, os secretomas de B. cenocepacia IST01 foram explorados e caracterizados para potenciais aplicações biotecnológicas. Sobrenadantes de cultura do isolado foram recolhidos a meio da fase exponencial, transição para a fase estacionária, no início da fase estacionária e na fase estacionária tardia e testados contra Escherichia coli, Enterococcus faecalis, Listeria monocytogenes e Staphylococcus aureus. As amostras da fase estacionária tardia e do início da fase estacionária demonstraram ser as mais eficazes, de forma bactericida, contra os alvos testados, sendo as últimas capazes ainda de inibir o crescimento de isolados conhecidos pela sua multiresistência, nomeadamente, Burkholderia cenocepacia, Pseudomonas aeruginosa e Staphylococcus aureus resistente à meticilina. Estas duas amostras apresentaram um perfil de proteínas, obtido por SDS-PAGE, distinto das restantes amostras recolhidas e os valores de actividade proteolítica mais elevados. Assim sendo sobrenadantes recolhidos no início da fase estacionária foram seleccionados para fraccionamento, por ultrafiltração, permitindo o isolamento de uma fracção do secretoma, composta por moléculas de peso molecular abaixo dos 3 kDa, que correspondeu a 78% do secretoma total e a única capaz de inibir o crescimento dos alvos testados, podendo ser composta por metabolitos produzidos durante o crescimento e pequenas proteínas. Os resultados obtidos neste projecto encorajam, assim, a possibilidade do futuro uso dos secretomas do Bcc para aplicações biotecnológicas, em particular como uma solução antibacteriana. Palavras chave: Complexo de Burkholderia cepacia, secretoma bacteriano, actividade antibacteriana, actividade proteolítica, actividade lipolítica iv Abbreviations AHL- Acyl homoserine lactones AMP- Antimicrobial peptides ATCC- American type culture collection Bcc- Burkholderia cepacia complex B. cenocepacia- Burkholderia cenocepacia BCL- Burkholderia cepacia lipase BHI- Brain heart infusion BSA- Bovine serum albumin BSRG/IBB- Biological sciences research group /Institute for bioengineering and biosciences CF- Cystic fibrosis CFU- Colony forming units CNCM- Collection nationale de cultures de microorganismes DSM- Deutsche sammlung von mikroorganismem E. coli- Escherichia coli E. faecalis- Enterococcus faecalis FITC- Fluorecein Isothiocyanide HGH- Horizontal gene transfer IST- Instituto superior técnico LB- Luria-Bertani broth LES- Liverpool epidemic strain L. monocytogenes- Listeria monocytogenes MGE- Mobile genetic elements MBC- Minimal bactericidal concentration MIC- Minimal inhibitory concentration MRSA- Methicillin resistant Staphylococcus aureus MWCO- Molecular weight cut-off NCTC- National collection of type culture P. aeruginosa- Pseudomonas aeruginosa OD- Optical density QS- Quorum sensing QQ- Quorum quenching S. aureus- Staphylococcus aureus SDS- Sodium dodecyl sulfate TBS- Tris-buffered saline TCE- Trichloroethylene v Index of figures Figure 1- Beneficial and harmful effects of Burkholderia cepacia complex bacteria ............................ 11 Figure 2- Scheme of the fractionation process, by centrifugal ultrafiltration used for the recovery of the fractions of the IST01 culture medium, according to the molecular weights. ........................................ 22 Figure 3- B. cenocepacia IST01 growth curve obtained in LB media, at 37ºC. The arrows indicate the times, in hours, chosen to collect IST01 culture supernatants. The values represent the average of two independent bacterial cultivations. ........................................................................................................ 23 Figure 4- Protein quantification, in g/L of culture medium, of lyophilized B. cenocepacia IST01 culture supernatants, collected at different phases of the growth curve, by the Bradford method. The data represents the average of three independent experiments performed
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