Unravelling the Role of Keratins in Bacterial Infections Rui Filipe Cruz
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D DOUTORAMENTO BIOMÉDICAS CIÊNCIAS Unravelling the Role of Keratins in in Keratins of Role the Unravelling Bacterial Infections Cruz Rui Filipe 2018 Rui Filipe Cruz. Unravelling the Role of Keratins in Bacterial D.ICBAS 2018 Infections Unravelling the Role of Keratins in Bacterial Infections Rui Filipe Cruz INSTITUTO DE CIÊNCIAS BIOMÉDICAS ABEL SALAZAR RUI FILIPE DA SILVA E CRUZ UNRAVELLING THE ROLE OF KERATINS IN BACTERIAL INFECTIONS 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 – Doutora Sandra Manuela Rodrigues Sousa Cabanes Categoria – Investigador auxiliar 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 Many people helped me through this long journey. Inside and outside the lab, I could always count with their support, kindness, patience, wisdom and laughter. To all of my travel companions, my most humble and sincere thank you. Rui Cruz 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 esclarece que na elaboração desta tese foram incluídos dados das publicações abaixo indicadas, e declara ter participado ativamente na conceção e execução das experiências que estiveram na origem dos mesmos, assim como na sua interpretação, discussão e redação. According to the relevant national legislation, the author clarifies that this thesis includes data from the publications listed below, and declares that he participated actively in the conception and execution of the experiments that produced such data, as well as in their interpretation, discussion and writing. PUBLICAÇÕES / PUBLICATIONS Cruz, R., Castro, I.P., Almeida, M.T., Moreira, A., Cabanes, D., and Sousa, S. (2018) Epithelial keratins modulate cMet expression and signalling and promote InlB-mediated Listeria monocytogenes infection of HeLa cells. Front Cell Infect Microbiol. doi: 10.3389/fcimb.2018.00146. (Publication accepted and manuscript production ongoing) FUNDING This work received funding from Norte-01-0145-FEDER-000012 - Structured program on bioengineered therapies for infectious diseases and tissue regeneration, supported by Norte Portugal Regional Operational Programe (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). Publication fees were supported by ICBAS, University of Porto. The author was supported by an FCT Doctoral Fellowship grant (SFRH/BD/90607/2012) through FCT/MEC co-funded by QREN (Quadro de Referência Estratégico Nacional) and POPH (Programa Operacional Potencial Humano). The supervisor (SS) was supported by FCT Investigator program (COMPETE, POPH, and FCT). TABLE OF CONTENTS ABSTRACT ..........................................................................................................................13 RESUMO .............................................................................................................................15 LIST OF ABBREVIATIONS ..................................................................................................17 CHAPTER l – INTRODUCTION ...........................................................................................21 A. Listeria monocytogenes ................................................................................................23 A.1. Overview ...........................................................................................................23 A.2. General characteristics ......................................................................................24 A.3. Listeriosis...........................................................................................................25 A.3.1. Transmission and Pathogenesis ..............................................................25 A.3.2. Clinical manifestations and epidemiology.................................................26 A.3.3. Treatment ................................................................................................27 A.4. Cellular infection cycle .......................................................................................27 A.4.1. Adhesion and entry ..................................................................................28 A.4.1.1. InlA mediated entry ...........................................................................29 A.4.1.2. InlB mediated entry ...........................................................................31 A.4.2. Escape from internalization vacuole .........................................................34 A.4.3. Intracellular life, actin-based motility and intercellular spread ...................35 A.4.4. Cytoskeleton manipulation by Listeria infection ........................................37 B. Intermediate filaments ...................................................................................................39 B.1. General properties .............................................................................................39 B.2. Distribution in cells and tissues ..........................................................................41 B.3. Structure, assembly and regulation ....................................................................43 B.4. Mechanical functions of IFs ...............................................................................47 B.5. Non-mechanical functions of IFs ........................................................................49 B.5.1. Vectorial processes .................................................................................49 B.5.2. Cellular adhesion, invasion and migration ................................................51 B.5.3. Microbial infection ....................................................................................52 B.6. Keratins as multifunctional cytoskeleton components ........................................53 B.6.1. Overview..................................................................................................53 B.6.2. Cell cycle .................................................................................................53 B.6.3. Protein synthesis and cell growth .............................................................54 B.6.4. Apoptosis modulation...............................................................................54 B.6.5. Stress protection ......................................................................................55 B.6.6. Gene expression ......................................................................................57 B.7. Keratins in infection ...........................................................................................59 CHAPTER II – PROJECT PRESENTATION ........................................................................61 CHAPTER III – RESULTS ...................................................................................................65 Part I – Epithelial keratins modulate cMet expression and signaling and promote InlB- mediated Listeria monocytogenes infection of HeLa cells .............................................69 I.1. Abstract ...............................................................................................................72 I.2. Introduction .........................................................................................................73 I.3. Results ................................................................................................................75 I.3.1. K8 and K18 favor InlB/cMet-mediated L. monocytogenes cellular invasion ...........................................................................................................................75 I.3.2. K8 and K18 accumulate at InlB-mediated internalization sites ...................76 I.3.3. K8 and K18 modulate actin dynamics at InlB-mediated entry sites ............78 I.3.4. K8 and K18 control HGF/cMet-mediated signaling ....................................80 I.3.5. cMet expression is dependent on K8 and K18 ..........................................82 I.3.6. K18 controls the expression of other transmembrane receptors ................84 I.3.7. Protein synthesis and stability do not depend on K18 expression..............86 I.3.8. K18 promotes transcripts stability ..............................................................88 I.4. Discussion...........................................................................................................90 I.5. Materials and Methods ........................................................................................93 I.5.1. Reagents and antibodies ...........................................................................93 I.5.2. Bacterial Strains and Cell Lines .................................................................93 I.5.3. Bacterial infections ....................................................................................93 I.5.4. Transfection of siRNA Duplexes ................................................................94 I.5.5. Immunoblotting .........................................................................................94 I.5.6. Immunoprecipitation assays