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The Thermo-Responsive Regulation of Yersinia Pestis Immune Protective Protein (F1) by the Caf1r Transcription Factor

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The Thermo-Responsive Regulation of Yersinia Pestis Immune Protective Protein (F1) by the Caf1r Transcription Factor The thermo-responsive regulation of Yersinia pestis immune protective protein (F1) by the Caf1R transcription factor Abdulmajeed Dhafer Majeed Al-jawdah Thesis submitted in partial fulfillment of the requirements of the regulations for the degree of Doctor of Philosophy Newcastle University Faculty of Medical Sciences Institute for Cell and Molecular Biosciences August 2019 i Acknowledgements First of all, I would like to express my very great gratitude to my lord, Allah who guided me and helped me to finish this study. I would like to express my deep gratefulness to my best supervisor Professor Jeremy H. Lakey for his patient supervision, enthusiastic encouragement and valuable assessments at all stages of this research work. I am more than grateful to Dr Daniel T. Peters for his smart advice and beneficial assistance at all stages of this study. Many thanks to Dr Helen Waller for her continuous professional technical support and for Dr Jeremy Brown and Dr Kevin Waldron for their wise advice and worthy evaluations. Many thanks for my sponsor, Higher Committee for Education Development in Iraq for their generous funding of my study. I wish to express my warm thanks to my parents, my wife, my brothers, my sister and my friends for their continuous support and bright encouragement. Finally, special thanks for Dr Azzeldin Madkour, Dr Nico Paracini, Dr Iglika G. Ivanova and Prof Neil D Perkins and other lab members for their appreciated assistance during this work. ii Abstract Pathogenic bacteria can sense the temperature of the host body to provoke the production of required virulence factors that allow them to defend themselves from the host immune system. The molecular mechanism of such temperature-sensitive regulation might be at the transcriptional or translational level involving DNA, RNA or protein. In one example, when Yersinia pestis, (the plague causative bacterium) infects the flea where the temperature ≈ 25oC it does not produce the Capsular Antigen F1 (Caf1). However, the bacterium can sense the change in temperature when it invades the human body and produces the Caf1 polymer as a coat to avoid the phagocytosis. The gene transcription of the caf1 operon is increased in response to body temperature (37oC). Although Caf1R is thought to be the responsible factor for such transcriptional upregulation, there are no clear results explaining the mode of action of Caf1R. In this study it was shown that caf1R gene deletion stops the secretion of Caf1 polymer. Caf1R is the transcription factor responsible for the immediate thermal upregulation of caf1 operon genes. Using molecular biology techniques, it was found that the caf1 operon genes caf1M, caf1A and caf1 are transcribed as a single long mRNA. The deletion of one of three predicted promoter regions upstream of this region (between caf1R and caf1M) was seen to have a significant effect on the transcription of caf1 operon genes and protein production. Finally, Caf1R was exploited to generate a novel thermal responsive expression system, which can be induced by switching the temperature from 25oC to 35oC instead of using a chemical inducer like IPTG or L-arabinose. Although this expression system needs more development to be usable in biotechnology applications, gfp expression from this system was shown to be highly temperature dependent. iii List of abbreviations LPS lipopolysaccharides CT cytotoxin FGS F1-G1 loop Short FGL F1-G1 loop Long Caf1 Capsular antigen F1 Ig Immunoglobulin SDS Sodium Dodecyl Sulfate PAGE Poly Acrylamide Gel Electrophoresis IPTG Isopropyl β-D-1-thiogalactopyranoside CAPS N-cyclohexyl-3-aminopropanesulfonic acid OD600 Optical Density at 600 nm RT-PCR Real-Time Polymerase Chain Reaction TB medium Terrific Broth medium LB medium Luria-Bertani medium nt Nucleotide bp Base pair RBS Ribosome Binding Site RACE Rapid Amplification of cDNA Ends GSP Gene Specific Primer GSP-RT Gene Specific Primer for Reverse Transcription HTH Helix-Turn-Helix SHS2 Strand-helix-Strand-Strand WT Wild-Type iv 3D Three Dimensional Y. pestis Yersinia pestis E. coli Escherichia coli EDTA Ethylenediamine tetraacetic acid DTT Dithiothreitol PBS Phosphate Buffered Saline SOC medium Super Optimal broth with Catabolite repression medium PMF Peptide Mass Fingerprinting 6His-tag Six-Histidine-tag v List of contents Acknowledgements .................................................................................. ii Abstract ................................................................................................... iii List of abbreviations ................................................................................ iv List of contents ........................................................................................ vi List of figures ........................................................................................... xi 1. Chapter One: Introduction .................................................................. 2 1.1. The plague causing bacterium Yersinia pestis .................................. 2 1.1.1. Y. pestis biology ............................................................................ 2 1.1.2. Y. pestis in the flea body ............................................................... 3 1.1.3. Y. pestis in the mammalian host ................................................... 4 1.2. Gram-negative bacteria ................................................................... 6 1.2.1. Gram-negative vs. Gram-positive bacteria .................................... 6 1.2.2. Pathogenicity of Gram-negative ................................................... 8 1.2.3. Uses of Gram-negative bacteria in biotechnology ....................... 10 1.2.4. The outer membrane of Gram-negative bacteria ........................ 11 1.3. Surface structures of Gram-negative bacteria ................................ 13 1.4. Chaperone-usher secretion system ................................................ 15 1.4.1. Caf1M (chaperone) from Y. pestis as an example of FGL subfamily ………………………………………………………………………………………………….18 1.4.2. Caf1A outer membrane protein (usher) from Y. pestis as an example of FGL subfamily ....................................................................... 20 1.5. Capsular Antigen Fraction 1 (Caf1) in Y. pestis ............................... 22 1.5.1. F1 capsule biogenesis ................................................................. 22 1.5.2. The role of F1 capsule in virulence and immunity ....................... 26 1.6. The Caf1R protein and its role in Caf1 expression .......................... 28 1.7. AraC family transcriptional regulators ........................................... 30 1.8. The F1 capsule (Caf1) protein and its special properties ................. 32 vi 1.9. The aim of this study ..................................................................... 34 2. Chapter Two: Materials and Methods .............................................. 37 2.1. Introduction .................................................................................. 37 2.2. Suppliers ........................................................................................ 37 2.3. Bacterial strains ............................................................................. 38 2.4. Plasmids construction .................................................................... 39 2.5. TB medium preparation ................................................................. 50 2.6. LB medium preparation ................................................................. 50 2.7. LB medium for agar plate .............................................................. 51 2.8. Q5-site directed mutagenesis ........................................................ 51 2.9. In-Fusion HD cloning ...................................................................... 52 2.10. Polymerase Chain Reaction (PCR) using Phusion DNA Polymerase54 2.11. Plasmid DNA purification from liquid culture medium ................. 54 2.12. Plasmid DNA purification from agarose gel .................................. 55 2.13. Transformation of bacterial cells ................................................. 56 2.14. The analysis of flocculent layer production .................................. 56 2.15. Caf1R purification ........................................................................ 56 2.16. Bacterial culture preparation for RT-PCR and western blot .......... 59 2.17. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) ................... 60 2.18. Western blot ................................................................................ 60 2.19. RNA extraction ............................................................................ 61 2.20. cDNA synthesis by reverse transcription reaction ........................ 63 2.21. Real-Time Polymerase Chain Reaction (RT-PCR) ........................... 63 2.22. Rapid amplification of 5’ cDNA ends (5’ RACE) ............................. 64 2.23. The preparation of DNA samples for sequencing ......................... 66 2.24. Bioinformatics ............................................................................. 66 2.25. Plate reader assays ...................................................................... 67 2.26. Statistics ...................................................................................... 67 vii 3. Chapter Three: Caf1R control of the Caf1 polymer secretion by recombinant Escherichia coli .................................................................. 69 3.1. Introduction .................................................................................
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