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Endolysins As Antibacterial Agents: from Engineering Approaches to the Uncovering of Holin As a Key Factor Influencing Lytic Activity

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Endolysins As Antibacterial Agents: from Engineering Approaches to the Uncovering of Holin As a Key Factor Influencing Lytic Activity UNIVERSIDADE DE LISBOA FACULDADE DE FARMÁCIA Endolysins as Antibacterial Agents: from Engineering Approaches to the Uncovering of Holin as a Key Factor Influencing Lytic Activity Orientador: Prof. Doutor Carlos Jorge Sousa de São-José Ana Sofia Conceição Fernandes Tese especialmente elaborada para a obtenção do grau de doutor em Farmácia, especialidade de Microbiologia. 2016 UNIVERSIDADE DE LISBOA FACULDADE DE FARMÁCIA Endolysins as Antibacterial Agents: from Engineering Approaches to the Uncovering of Holin as a Key Factor Influencing Lytic Activity Orientador: Prof. Doutor Carlos Jorge Sousa de São-José Ana Sofia Conceição Fernandes Tese especialmente elaborada para a obtenção do grau de doutor em Farmácia, especialidade de Microbiologia. Júri Presidente: Doulora Matilde da Luz dos Santos Duque da Fonseca e Castro Vogais: - Doutor Adriano José Alves de Oliveira Henriques. Professor Associado Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa; - Doutor Ricardo Manuel Soares Parreira, Professor Auxiliar Instituto de Higiene e Medicina Tropical da Universidade Nova de Lisboa; - Doutor Mario Nuno Ramos d´Almeida Ramirez. Professor Associado corn Agregação Faculdade de Medicina da Universidade de Lisboa; - Doutor José Antonio Frazão Moniz Pereira. Professor Catedrótico Faculdade de Farmácia da Universidade de Lisboa; - Doutora Madalena Maria Vilela Pirnentel, Professora Associada Faculdade de Farmácia da Universidade de Lisboa; - Doutor Carlos Jorge Sousa de São José. Professor Auxiliar Faculdade de Farmácia da Universidade de Lisboa, Orientador. 2016 A autora da investigação descrita nesta dissertação, Ana Sofia Conceição Fernandes, teve o apoio da Fundação para a Ciência e a Tecnologia através da concessão da bolsa de doutoramento SFRH/BD/81802/2011, com financiamento comparticipado pelo Fundo Social Europeu e por fundos nacionais do Ministério da Ciência, Tecnologia e Ensino Superior (MCTES). O trabalho apresentado nesta dissertação foi desenvolvido no Centro de Patogénese Molecular – Unidade de Retrovírus e Infeções Associadas (CPM-URIA) e no Instituto de Investigação do Medicamento (iMed.ULisboa) da Faculdade de Farmácia da Universidade de Lisboa (FFUL), sob a orientação do Prof. Dr. Carlos São-José. Os resultados descritos nesta dissertação estão incluídos nos manuscritos publicados ou submetidos para publicação abaixo indicados: Fernandes, S. and São-José, C. (2016) Probing the function of the two holin- like proteins of bacteriophage SPP1. Submitted to Virology. Fernandes, S. and São-José, C. (2016) More than a hole: The holin lethal function may be required to fully sensitize bacteria to the lytic action of canonical endolysins. Molecular Microbiology. Accepted for publication on June 21, 2016. DOI: 10.1111/mmi.13448. Fernandes, S., Proença, D., Cantante, C., Silva, F.A., Leandro, C., Lourenço, S., et al. (2012) Novel chimerical endolysins with broad antimicrobial activity against methicillin-resistant Staphylococcus aureus. Microbial Drug Resistance 18: 333-43. DOI: 10.1089/mdr. De acordo com o disposto no ponto 1 do artigo n° 45 do Regulamento de Estudos Pós- Graduados da Universidade de Lisboa, deliberação n° 4624/2012, publicado em Diário da República – II Série n° 65 – 30 de Março de 2012, a autora desta dissertação declara que participou na conceção e execução do trabalho experimental, interpretação dos resultados obtidos e redação dos manuscritos. TABLE OF CONTENTS AGRADECIMENTOS ................................................................................................................ v RESUMO ................................................................................................................................... vii ABSTRACT ................................................................................................................................. xi ABBREVIATIONS ................................................................................................................... xv CHAPTER 1. GENERAL INTRODUCTION 1.1 Bacteriophages: general features and life cycle .................................................................... 3 1.2 Bacterial cell envelope: barrier to virus entry and release .................................................... 4 1.3 Phage release from infected cells .......................................................................................... 8 1.3.1 Lysis players: holin/endolysin molecular diversity ................................................... 8 1.3.2 Lysis mechanisms in tailed phages ......................................................................... 16 1.3.2.1 Holin-dependent export of endolysins ................................................................ 19 1.3.2.2 Holin-independent export of endolysins ............................................................. 19 1.4 Lysis regulation: insights from autolysis in Gram-positive bacteria ................................... 23 1.4.1 The phenomenon of bacterial autolysis: first dissipate pmf and then lyse .............. 24 1.4.2 Autolysis regulation ................................................................................................ 26 1.5 Exploration of endolysins as antibacterial agents ............................................................... 28 1.5.1 Engineering of endolysins ....................................................................................... 29 1.5.2 Enzibiotic therapy: aren’t we missing something? .................................................. 31 1.6 Thesis goals ......................................................................................................................... 33 1.7 References ........................................................................................................................... 33 CHAPTER 2. NOVEL CHIMERICAL ENDOLYSINS WITH BROAD ANTIMICROBIAL ACTIVITY AGAINST METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS Acknowledgments ....................................................................................................................... 55 Abstract ....................................................................................................................................... 57 2.1 Introduction ........................................................................................................................... 59 2.2 Results ............................................................................................................................... 60 2.2.1 Production of chimerical endolysins targeting S. aureus ............................................. 60 2.2.2 Lytic action of the chimerical endolysins against clinical S. aureus ............................ 63 2.2.3 Lys168-87 and Lys170-87 have a synergistic lytic effect in liquid media ................... 66 i 2.2.4 Activity of the chimerical endolysins against other Gram-positive pathogenic bacteria ........................................................................................................................................... …..67 2.3 Discussion ............................................................................................................................. 69 2.4 Materials and methods .......................................................................................................... 72 2.4.1 Bacteria, culture media and growth conditions ............................................................ 72 2.4.2 Identification and bioinformatics analysis of phage endolysins ................................... 73 2.4.3 Construction and cloning of lys168-87 and lys170-87 chimerical genes ..................... 73 2.4.4 Production and purification of the chimerical endolysins Lys168-87 and Lys170-87 ............................................................................................................................................... ..74 2.4.5 Evaluation of the lytic action of chimerical endolysins against bacterial pathogens .... 75 2.5 References ............................................................................................................................. 76 2.6 Supplementary materials ....................................................................................................... 81 2.6.1 References of tables and supplementary materials ....................................................... 93 CHAPTER 3. MORE THAN A HOLE: THE HOLIN LETHAL FUNCTION MAY BE REQUIRED TO FULLY SENSITIZE BACTERIA TO THE LYTIC ACTION OF CANONICAL ENDOLYSINS Acknowledgments ..................................................................................................................... 101 Abstract ..................................................................................................................................... 103 3.1 Introduction ......................................................................................................................... 105 3.2 Results ................................................................................................................................. 107 3.2.1 Actively growing B. subtilis cells can resist to the lytic action of secreted SPP1 endolysin ............................................................................................................................... 107 3.2.2 LySPP1-mediated lysis from without is enhanced in conditions leading to CM depolarization .......................................................................................................................
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