Defences of Helicobacter species against host antimicrobials Adelina Margarida Lima Pereira Rodrigues Parente Dissertation presented to obtain the Ph.D degree in Biochemistry Instituto de Tecnologia Química e Biológica António Xavier | Universidade Nova de Lisboa Oeiras, June, 2016 Defences of Helicobacter species against host antimicrobials Adelina Margarida Lima Pereira Rodrigues Parente Dissertation presented to obtain the Ph.D degree in Biochemistry Instituto de Tecnologia Química e Biológica António Xavier | Universidade Nova de Lisboa Oeiras, June 2016 From left to right: Mónica Oleastro (4th oponente), Gabriel Martins (3 rd opponent), Marta Justino (Co-supervisor) , Miguel Viveiros (2 nd opponent), Adelina Margarida Parente , Lígia Saraiva (supervisor) , Cecília Arraiano (president of the jury), and Maria do Céu Figueiredo (1 st opponent). nd 22 June 2016 Second edition, June 2016 Molecular Mechanisms of Pathogen Resistance Laboratory Instituto de Tecnologia Química e Biológica António Xavier Universidade Nova de Lisboa 2780-157 Portugal ii “Science knows no country, because knowledge belongs to humanity, and is the torch which illuminates the world” Louis Pasteur iii iv Acknowledgments Firstly, I would like to express my gratitude to the person that allowed me the opportunity to perform a PhD and who also contributed the most for my accomplishment of this thesis by constantly supporting me during these last years. I thus thank my supervisor, Dr. Lígia Saraiva , for her permanent availability whenever I needed guidance, for all the excellent ideas and advices related to my practical work and lastly for all the patience and enthusiasm! I also thank Dr. Lígia for her rigour and enormous help in the writing of this thesis. Above all, I would like to thank Dr. Lígia for the friendship, personal advices, generosity and all the care demonstrated for me. To my co-supervisor and friend Dr. Marta Justino , I would like to thank for all she taught me in the lab, especially in the early years of my thesis, her ideas and helpful discussions. To Dr. Célia Romão and Dr. Mónica Oleasto for being part of my scientific thesis commission and for the interesting discussions, their suggestions and advices. I thank Dr. Alessandro Giuffrè, Dr. Elena Forte and Dr. Micol Falabella for performing the experiments of the peroxynitrite activities presented in Chapter VI, the analysis of results and the preparation of the manuscript. I have to thank Dr. Alessandro for his helpful advices and for his constant availability and sympathy in answering to all my e-mails. To Dr. Gabriel Martins for his help in the confocal experiments, treatment of images, for the careful revision and help in the preparation of the paper presented in Chapter V. Thank you for your hospitality at IGC. To Prof. Miguel Teixeira , for performing the EPR experiments and for his helpful explanations on such a complex topic. v I want to thank Dr. Ivo Gomperts Boneca for the excellent receptions in his laboratory, his guidance and helpful discussions. I feel privileged to have learnt from an expert in the field and who has always been so generous to me. I also want to thank the members of Dr. Ivo’s group for their constant help in anything I needed, their helpful discussions and work advices as well as for their companionship. In special, I thank Chantal Ecobichon and Martine Fanton D´Andon who became very special friends and made me feel at home in Institute Pasteur. I want to make a special thanks to my group colleagues. To Susana Lobo , Lígia Nobre and Sandra Carvalho , for all their great support, motivation and teachings from such excellent scientists and outstanding friends. To Marco Videira and Liliana Silva , I thank their help and excellent companionship. I thank João Monteiro for his “marathons” and all the dedication employed in the lab in favour of our work. His help was precious to complete this thesis. To Inês Ladeira , for her participation in this work during her final degree project. I also want to thank the former members of Molecular Mechanisms of Pathogen Resistance group. A special thanks to Filipa Tavares de Oliveira , to whom I directly worked with and learnt a lot, and to Mafalda Figueiredo , Sara Sousa Santos , and Joana Baptista de Matos for their help and friendship. Thank you all for your help and for the good moments shared over the last years. I could not ask for better friends and colleagues to work with. Lastly, I also want to include in the thank you list the people of the 3 rd floor and, in particular, to Catarina Paquete , Américo Duarte and Ivo Saraiva , that so nicely received me in “the aquarium”. To Fundação para a Ciência e Tecnologia (FCT) for the financial support crucial for the accomplishment of this work and by awarding a PhD grant (SFRH/BD/81192/2011). Agradeço aos meus amigos e família , em especial ao Joaquim , à Sara e Luís , à Margarida Gomes , aos meus sogros e aos meus padrinhos pelo seu apoio vi e amizade. À minha querida amiga Rute agradeço pelos longos anos de amizade e apoio. Ao João , por me fazer muito feliz, por ser o meu melhor amigo, por todo o seu amor incondicional, e por me ajudar sempre em tudo o que eu preciso. Aos meus pais que sempre me deram tudo o que tinham e que me apoiaram e apoiam incondicionalmente em todos os momentos. Nada que eu faça pode igualar tal dedicação. This thesis is dedicated to my parents and husband vii viii Thesis Publications This dissertation is based on the following original publications, listed by chronological order: Justino MC, Parente MR , Boneca IG, Saraiva LM. FrxA is an S-nitrosoglutathione reductase enzyme that contributes to Helicobacter pylori pathogenicity. FEBS J. 2014 Oct. 281(19):4495-505. Parente MR , Monteiro JT, Martins GG, Saraiva LM. Helicobacter pullorum induces in murine macrophages nitric oxide release that promotes phagocytosis and killing. Microbiology. 2016 Jan 13. doi: 10.1099/mic.0.000240. Manuscript in preparation, which results are presented in Chapter VI: Margarida R. Parente , Elena Forte, Micol Falabella, João T. Monteiro, Ivo G. Boneca, Miguel Teixeira, Alessandro Giuffrè and Lígia M. Saraiva (2016) The nitrosative stress defences of the enterohepatic Helicobacter pullorum. Publications not included in this thesis: Tavares AF, Parente MR , Justino MC, Oleastro M, Nobre LS, Saraiva LM. The bactericidal activity of carbon monoxide-releasing molecules against Helicobacter pylori. PLoS One. 2013 Dec. 26;8(12):e83157. ix x Abstract Bacteria of the genus Helicobacter are related to gastrointestinal and hepatic disease in humans. Helicobacter pylori infects the gastric mucosa of a large percentage of the human population and has been reported to cause gastric ulcer and stomach cancer. Other Helicobacter related species, such as the enterohepatic Helicobacter pullorum , although firstly discovered in chickens are now known to be associated with human digestive disorders such as gastroenteritis, inflammatory bowel disease, hepatobiliary disease and hepatic cancer. During host invasion, all pathogens are exposed to the antimicrobials produced by the innate immunity’s phagocytes such as nitric oxide (NO) and derivate reactive nitrogen species (RNS), which constitute one of the main strategies employed by the host to eliminate pathogens. Due to the high importance of H. pylori as a human pathogen and the more recent recognition of H. pullorum as an emergent human pathogen, the so far unknown mechanisms used by these bacteria to circumvent the innate immune system need to be addressed. Therefore, the objectives of this work were: i) investigation of the role of H. pylori nitroreductases in the defence against the host induced nitrosative stress, ii) evaluation of the sensitivity of H. pullorum to antimicrobials, iii) assessment of the ability of H. pullorum to activate, infect and survive within macrophages, and iv) identification and characterization of the enzymes putatively involved in H. pullorum defence against host antimicrobials. H. pylori nitroreductases activate metronidazole, which is the antibiotic commonly used for H. pylori treatment. In this work, a new function of these enzymes was explored. For this purpose, an H. pylori strain deleted in the nitroreductase frxA gene was constructed, its phenotype and the expression of the frxA gene was analysed under NO stress conditions. Inactivation of the frxA gene resulted in a strain that is more sensitive to nitrosative stress. Furthermore, H. pylori frxA is induced in response to nitrosative stress generators. Studies on the capacity of the wild type and frxA deficient cell extracts to degrade the toxic compound nitrosoglutathione (GSNO) xi showed that in the absence of frxA H. pylori cells have significantly lower GSNO reductase activity. Accordingly, experiments on the activity of the purified FrxA protein towards nitrocompounds and S-nitrosothiols revealed that not only FrxA reduces metronidazole, but exhibits a GSNO reductase activity. The interaction of H. pylori frxA mutant with macrophages and mice was also analysed. FrxA was shown to contribute to the survival of H. pylori within macrophages and to the bacterium´s virulence during mice colonization. The resistance of the enterohepatic H. pullorum to nitrosative stress was also analysed. H. pullorum was exposed at different growth phases to nitrosative stress generated by several NO donors. Treatment with NO lowered the H. pullorum viability, in a growth-phase-dependent manner and, H. pullorum cells suffered a decrease in the mean bacterial cell size. In the second part of this study, the interaction between H. pullorum and host cells was analysed. Confocal microscopy revealed that H. pullorum is internalized by murine macrophages, triggering the production of NO that promoted phagocytosis and bacterial clearance. Interaction between H. pullorum and macrophages was shown to stimulate secretion of pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6 and MIP-2, indicating that H. pullorum triggers a host inflammatory response.