The role of Type VI Secretion System in the virulence of Klebsiella pneumoniae Thesis Submitted for the degree of Doctor of Philosophy at the University of Leicester By David Mwin (B.Sc.) Department of Infection, Immunity, and Inflammation College of Medicine, Biological Sciences and Psychology University of Leicester October 2017 i David Mwin, Ph.D. Thesis “The role of Type VI secretion system in the virulence of Klebsiella pneumoniae”, University of Leicester, UK. Statement of originality The research work submitted in this thesis for the degree of Doctor of Philosophy PhD entitled “The role of Type VI secretion system in the virulence of Klebsiella pneumoniae” is based on work conducted by the author in the Department of Infection, Immunity, and Inflammation (College of Medicine and Biological Science) of the University of Leicester. during the period between February 2012 and August 2015. All the work recorded in this thesis is original unless otherwise acknowledged in the text or by references. No part of this work is submitted elsewhere for another degree in this or any other University. i David Mwin, Ph.D. Thesis “The role of Type VI secretion system in the virulence of Klebsiella pneumoniae”, University of Leicester, UK. Acknowledgement To God Almighty, I say thank you for Your Wisdom, Protection, and the Gift of life upon me throughout my studies and beyond. I am highly indebted to my family for their support, patience, and care during my studies. Thanks to the Government of Ghana for their generous sponsorship and provision of funds for this research work. I am very grateful to my Supervisors, Dr Yassine Amrani, Dr Shaun Heaphy, and Dr Kumar Rajakumar, for all their support and guidance during my studies. I am also very grateful for the support and constructive criticisms of Prof. Jose Bengoechea and Prof. Peter Andrew. To Dr Hasan Yelsilkaya and Dr Primerose Fresestone, I am immensely grateful for your mentorship. The team from the Biotechnology Core was beneficial during confocal and Electron microscopy, and I am very thankful for their assistance. I am very grateful to Dr Codula Stover and Dr Simon Bath for the provision of J744 and Acanthamoeba castellani, respectively. To my loving family whose undivided support and encouragement gave me the wings to saw higher during and beyond this journey, thank you for being such a pillar in my life. And finally, to all who in one way or the other supported me in completion of this work, I would like to say God bless you all. ii David Mwin, Ph.D. Thesis “The role of Type VI secretion system in the virulence of Klebsiella pneumoniae”, University of Leicester, UK. Dedication I dedicated this research work/ thesis to my; beautiful family (Mrs Ugomma Lauritta Mwin and our children), amazing mother (Marcelina Ngmenterebo) and late father, Godfred Ngmenterebo, whose support and memory inspire me for greater heights. iii David Mwin, Ph.D. Thesis “The role of Type VI secretion system in the virulence of Klebsiella pneumoniae”, University of Leicester, UK. Abstract The role of Type VI secretion system in the virulence of Klebsiella pneumoniae David Mwin The enterobacteria, especially the multidrug-resistant strains, pose a serious health threat with evolving virulence mechanisms against their host. The opportunistic pathogen, K. pneumoniae, is a leading cause of urinary tract infection, blood and pneumonia in patients at the hospital and remains a common isolate in community-acquired disease. Many bacteria, including K. pneumoniae, explore a range of factors /mechanisms such as secreted toxin and antibiotic resistance that may enhance their survival, virulence, and evasion of the host immune system. Type VI Secretion Systems (T6SSs) is a transmembrane “spring- loaded” toxin-translocating nanomolecular machinery recently characterized in over 25% of Proteobacteria. T6SS shares both structural and protein homology with phage tail and is considered to be reminiscent of the bacteriophage puncturing device. While available data suggest a diverse role of the toxin puncturing T6SS device in many Gram-negative bacteria, no experimental data have demonstrated the putative T6SS gene clusters’ role in the virulence of K. pneumoniae as at the time that this research work was conducted. In silico analysis, using a full range of bioinformatic tools were used to identify and map the T6SS gene cluster (T6SS1 and T6SS3) in K. pneumoniae. A library of molecular genetic tools was constructed via a novel strategy and used to disarm the resistance in MDR K. pneumoniae, which enhanced the safety and genetic manipulation of the strain for T6SS functional studies. Mainly, three T6SS mutants, ∆T1 (T6SS1 mutant), ∆T3 (T6SS3 mutant), and ∆T1∆T3 (T6SS1/T6SS3 mutant) generated via lambda red recombination allelic exchange and were examined for the role of the T6SS gene clusters in K. pneumoniae. The data obtained from the various assays and analysis suggest that K. pneumoniae via T6SS mediated antibacterial virulence against other competing bacteria and played a significant role T4SS-mediated conjugal transfer of mobile genetic elements. K. pneumoniae used T6SS to resist amoeba phagocytosis and enhance subsequent survival within the cell. Also, T6SS increased in vivo virulence in Galleria larvae, host cell invasion, survival, actin filament polymerisation and activation of host pro-inflammatory innate immunity. Thus, the putative T6SS gene clusters may be mediating a multipurpose virulence against host organism and other bacteria using pre-emptive contact-dependent strikes and toxin secretion. iv David Mwin, Ph.D. Thesis “The role of Type VI secretion system in the virulence of Klebsiella pneumoniae”, University of Leicester, UK. Abbreviations ABC ATP-binding cassette Mpf Mating pair formation ATP Adenosine triphosphate MGE Mobile genetic element ApE A plasmid editor MCS Multiple Cloning Site Blast Basic Local Alignment Tool SOE-PCR Spliced overlap extension-PCR bp Base pair EDTA Ethylenediaminetetraacetic acid CFU Colony-forming unit NEB New England Bio lab OD Optical Density CRKP Carbapenem-resistant Klebsiella pneumoniae DNA Deoxyribose nucleic acid Pfam Protein families dNTP Deoxy-ribonucleotide triphosphate PAI Pathogenicity island dsDNA Double-stranded DNA PCR Polymerase Chain Reaction DF Downstream flank PBS Phosphate buffered saline ddH2O Deionised distilled water RF Right Flank ESBL extended-spectrum beta-lactamases RNA Ribose nucleic acid FRT Flippase Recognition Target ST Sequence type g Gram DR Direct repeat PO Phenoloxidase SOE Splice overlap extension GC Guanine and Cytosine T1SS Type I secretion system GI Genomic island T2SS Type II secretion system HGT Horizontal gene transfer T3SS Type III secretion system ICE Integrative and conjugative element T4SS Type IV secretion system int Integrase T5SS Type V secretion system IR Inverted repeat T6SS Type VI secretion system IPTG Isopropyl-β-D-thiogalactopyranoside tRNA Transfer RNA Kb Kilobase λ Lambda Kp Klebsiella pneumoniae ⁰ C Degree Celsius μl Microliter TATOXB Tannic acid-treated oxblood LA Luria Bertani agar μg Microgram LAS Luria Bertani agar + sucrose M Micro Molar LB Luria Bertani broth cDNA Complementary DNA LB Luria Bertani broth + sucrose CDS Coding domain Sequence Mpf Mating pair formation DF Conserved downstream flan GP-RBS Guinea pig red blood cells DMSO Dimethyl sulfoxide NCBI National Centre for Biotechnology SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel Information electrophoresis kDa Kilodalton TAE Tris-acetate-EDTA SOC Super-optimal broth with Catabolite qRT-PCR Quantitative real-time polymerase chain Repression reaction mM Millimolar pmol Picomole v/v Volume per volume ratio w/v Weight per volume ratio v David Mwin, Ph.D. Thesis “The role of Type VI secretion system in the virulence of Klebsiella pneumoniae”, University of Leicester, UK. Table of Contents Statement of originality .................................................................................................................................. i Acknowledgement ........................................................................................................................................ ii Dedication .................................................................................................................................................... iii Abstract ........................................................................................................................................................ iv Abbreviations ................................................................................................................................................ v List of Figures .............................................................................................................................................. xi List of Tables ............................................................................................................................................. xiv Publications ................................................................................................................................................. xv Chapter 1: Introduction ................................................................................................................................. 2 1.1 Klebsiella Species ............................................................................................................................... 2 1.1.1 History and taxonomic structure .................................................................................................