Identified Listeria Monocytogenes Virulence Regulators
Total Page：16
File Type：pdf, Size：1020Kb
Listeria monocytogenes DOUTORAMENTO EM CIÊNCIAS BIOMÉDICAS DOUTORAMENTO Molecular characterization of newly newly of characterization Molecular identified Jorge Nuno Pinheiro D 2017 virulence regulators Jorge Nuno Pinheiro. Molecular characterization of newly D.ICBAS 2017 identified Listeria monocytogenes virulence regulators Molecular characterization of newly identified Listeria monocytogenes virulence regulators Jorge Nuno Martins Campos Pinheiro INSTITUTO DE CIÊNCIAS BIOMÉDICAS ABEL SALAZAR JORGE NUNO MARTINS CAMPOS PINHEIRO MOLECULAR CHARACTERIZATION OF NEWLY IDENTIFIED LISTERIA MONOCYTOGENES VIRULENCE REGULATORS Tese de Candidatura ao grau de Doutor em Ciências Biomédicas, submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Instituição de acolhimento – Instituto de Biologia Molecular e Celular - Instituto de Investigação e Inovação em Saúde Orientador – Doutor Didier Jacques Christian Cabanes Categoria – Investigador principal Afiliação – Instituto de Biologia Molecular e Celular - Instituto de Investigação e Inovação em Saúde Coorientador – Professor Doutor Rui Appelberg Gaio Lima Categoria – Professor catedrático Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto De acordo com o disposto no ponto n.º 2 do Art.º 31º do Decreto-Lei n.º 74/2006, de 24 de Março, aditado pelo Decreto-Lei n.º 230/2009, de 14 de Setembro, o autor declara que na elaboração desta tese foram incluídos dados da publicação abaixo indicada. O autor participou ativamente na conceção e execução dos trabalhos que estiveram na origem dos mesmos, assim como na sua interpretação, discussão e redação. According to the relevant national legislation, the author declares that this thesis includes data from the publication indicated below. The author participated actively in the conception and execution of the work that originated that data, as well as in their interpretation, discussion and writing. PUBLICAÇÃO / PUBLICATION Pinheiro, J., Reis, O., Vieira, A., Moura, I.M., Zanolli Moreno, L., Carvalho, F., Pucciarelli, M.G., García-del Portillo, F., Sousa, S., and Cabanes, D. (2017). Listeria monocytogenes encodes a functional ESX-1 secretion system whose expression is detrimental to in vivo infection. Virulence 8, 993–1004. Acknowledgments I would to like to briefly acknowledge some people who contributed to the work developed during my PhD, to the writing of this thesis or just made it all a lot more fun! My gratitude to My supervisor Didier Cabanes, for allowing me to develop my PhD project in his group, for all the valuable teachings, criticism and the permanent good mood and sense of humor. Sandra Sousa, for always being available to help and actively contribute to our discussions. For all the good advice and finding good solutions. Professor Rui Appelberg for accepting to be the co-supervisor of my thesis. Past and present members of the GM2 group for the important contributions in all the discussion sessions and for all the good times in the lab! Johnny for the great collaboration and bringing new ideas. Paula, Tânia and Mafalda for the technical support, scientific discussions and for always doing a bit more than needed just to help us. Rita Pombinho! The PhD-Buddy! For all these years of incredibly good mood, all the nonsense, the Friday-madness, that wonderful music you were always choosing for me (not), all those media plates you always had ready whenever I really needed (not), all those fun times during lunch (not), etc… Thank you for the unconditional help Rita! Obrigado À mãe, ao pai À avó, à famelga À Sofia À “Irmandade do Chamade” Aos FullTrack Ao Bubba TABLE OF CONTENTS Abstract .................................................................................................................. 9 Resumo ................................................................................................................. 11 List of abbreviations ............................................................................................ 13 CHAPTER I - INTRODUCTION ............................................................................. 17 Listeria monocytogenes ............................................................................................ 19 Historical overview and taxonomy ....................................................................... 19 General features and physiology ........................................................................ 20 Listeria pathogenesis ................................................................................................ 23 Listeriosis ............................................................................................................ 23 Cell infection cycle .............................................................................................. 27 Arsenal of virulence ............................................................................................ 28 Virulence regulation .................................................................................................. 32 Regulation of expression of virulence genes in bacteria ...................................... 32 Virulence regulation in Lm .................................................................................. 34 GntR family of transcription regulators ................................................................... 38 Discovery and general features .......................................................................... 38 Classification and mode of action ....................................................................... 39 The Accessory Gene Regulator (Agr) system ......................................................... 42 Discovery and general features .......................................................................... 42 Agr system in Lm ................................................................................................ 44 Secretion systems and bacterial virulence .............................................................. 47 The ESX-1 secretion system ............................................................................... 48 CHAPTER II - PROJECT PRESENTATION ......................................................... 51 Project goals .............................................................................................................. 53 Selection of Lm putative virulence transcription regulators .................................. 54 CHAPTER III - RESULTS ...................................................................................... 57 Part I - MouR controls the expression of the Listeria monocytogenes Agr system and mediates virulence ................................................................................ 59 Part II - Identification and characterization of Lmo0443, a novel Listeria monocytogenes virulence regulator ......................................................................... 99 Part III - Listeria monocytogenes encodes a functional ESX-1 secretion system whose expression is detrimental to in vivo infection ............................... 117 CHAPTER IV – GENERAL DISCUSSION .......................................................... 135 CHAPTER V – REFERENCES ............................................................................ 141 Abstract Listeria monocytogenes is a bacterial foodborne pathogen that causes listeriosis, an invasive and potentially fatal infection in susceptible animals and humans. To infect, survive and proliferate in the host L. monocytogenes requires tight and coordinate networks of virulence regulation. In this work, we identify and characterize MouR and Lmo0443, two novel regulators of L. monocytogenes, and through RNAseq transcriptomic analysis we identify the genes under their transcriptional control. Importantly, we describe MouR as the central activator of the Agr system (agrBDCA). We also reveal how MouR binds to the promoter DNA of the agr locus, modulates biofilm formation and chitinase activity and is required for full virulence in the mouse infection model. By resolving its crystal structure, we were able to classify MouR as a dimeric DNA-binding VanR (FadR subfamily) regulator of the GntR family of transcriptional factors. Lmo0443 is shown here as the transcriptional repressor of lmo0444, an uncharacterized gene from the stress survival islet 1 (SSI-1) which is implicated in stress tolerance. Here we demonstrate that Lmo0443 is required for successful mouse infection, revealing its role in virulence, and propose a novel role for Lmo0443 and Lmo0444 in cell wall dynamics. In this work we also demonstrate how L. monocytogenes regulates virulence through control of protein secretion. The ESX-1 secretion system, responsible for the secretion of WXG100 proteins and implicated in virulence of Mycobacterium tuberculosis, was previously thought to be dispensable for L. monocytogenes virulence. Here we reveal how the secretion of EsxA by the ESX-1 system has a detrimental effect on L. monocytogenes virulence. 9 Resumo Listeria monocytogenes é um agente bacteriano alimentar patogénico causador da listeriose, uma infeção invasiva e potencialmente fatal em animais e humanos suscetíveis. A infeção, sobrevivência e proliferação da L. monocytogenes no hospedeiro requer uma rede bem coordenada de regulação de virulência. Neste trabalho identificamos e caracterizamos MouR e Lmo0443, dois novos reguladores da L. monocytogenes, e através de análise transcriptómica por RNAseq identificamos os genes sob o controlo transcripcional