Identification and Characterisation of an Old Yellow Enzyme (OYE) - NamA from Listeria monocytogenes by Majid Abdullah Bamaga A thesis submitted in partial fulfilment of the requirements of Edinburgh Napier University, for the award of Doctor of Philosophy September, 2014 Abstract The food-borne pathogene Listeria monocytogenes has been considered a significant threat to human health worldwide. It mainly infects individuals suffering insuffecint immunity such as pregnant women. During pregnancy, L. monocytogenes is capable of causing a serious damage to the mother and the fetus. It can spread to different organs including the placenta via adaptation to interacellular lifestyle. To maintain pregnancy, the levels of the hormones progesterone and β-estradiol increase and reduction in hormone levels was proposed to be associated with fetal death and abortion. The objectives of this project therefore were to investigate the role of pregnancy hormones on the growth and virulence of L. monocytogenes, and to identify bacterial genes with possible roles in binding to pregnancy hormones. It was obsereved that the growth of L. monocytogenes in the presence of progesterone under anaerobic condition was affected by the action of the hormone and the effect was dose/time-dependent of exposure as increasing concentrations showed greater effect on the bacterial growth. Interestingly, bacterial growth was restored within 24 h of exposure to the hormone. In parallel, a Tn917-LTV3 insertion library was constructed and a number of mutants isolated that had reduced growth in the presence of β-estradiol were identified. However, reduction in growth was not microbiologically significant. Furthermore, bioinformatics analysis was performed to identify listerial genes with possible role in hormones degradation. It was observed that L. monocytogenes encodes for a protein that is possibly involved in steroid degradation; therefore, gene expression and a clear-deletion mutant were performed to test this hypothesis. This revealed no significant role of this protein in the growth restoration observed in the presence of progesterone. Also, the deleted gene was investigated of its ability to reduce NADPH in the presence of a possible substrate (progesterone, β-estradiol). This showed that this gene could possess an enzymatic activity toward pregnancy hormones. An attempt to purify this protein for further investigation was performed and protein expression in a soluble form was unsuccessful. The findings presented in this thesis represent an important view when considering the relation between pregnancy hormones and L. monocytogenes; however, further investigations of hormone-degrading proteins from L. monocytogenes are needed. This knowledge may form the basis of a therapy to protect pregnant individuals. i Acknowledgment I wish to express my sincere gratitude to my supervisor Dr. Clare Taylor, for her continous support, advice, encouragement, and devotion of her priceless time throughout this project and providing me with this great opportunity to study and work in her laboratory. Many thanks to my second supervisor Dr. Sophie Foley, for her support and constructive criticism for which I am very greatful. I would also like to thank Kathryn Pitts, Sandra Dunbar, Farid Hemmati, David Conner and the rest of the technical staff of the Microbiology and Biotechnology division of Edinburgh Napier University, for their help and support. Special thanks to my colleague Agnieszka Piotrowska for her help, support and encouragement throughout my study. Finally, I would like to thank the Saudia Arabian Cultural Buearue for funding me throuought my study, and for their support. ii Table of Contents Abstract ........................................................................................................... i Acknowledgment ........................................................................................... ii List of Figures ............................................................................................. viii List of Tables ................................................................................................ xii List of abbreviations....................................................................................xiii Chapter I ......................................................................................................... 1 Introduction .................................................................................................... 1 1.1 Background ........................................................................................... 2 1.2 Placenta and its role during pregnancy ................................................. 4 1.3 Placental infection ................................................................................. 4 1.3.1 Toxoplasma gondii ..................................................................... 9 1.3.2 Campylobacter jejuni ................................................................ 10 1.3.3 Salmonella enterica .................................................................. 10 1.4 Influence of pregnancy hormones on immunity ................................... 11 1.5 Production of pregnancy hormones and formation from cholesterol . 12 1.6 Listeria monocytogenes - organism and characteristics ...................... 14 1.7 Listeriosis and prevalence of infection ................................................ 16 1.8 Pathogenesis of L. monocytogenes .................................................... 24 1.8.1 L. monocytogenes entry into host cells .................................... 24 1.8.2 Life cycle of L. monocytogenes ............................................... 29 1.8.3 Listeria virulence factors and their contribution to bacterial survival ................................................................................................ 30 1.9 Listeriosis during pregnancy ................................................................ 33 1.10 Hormone-pathogen interactions and its effects on bacterial growth .... 35 1.11 Aims .................................................................................................... 44 iii Chapter II ...................................................................................................... 45 Materials & Methods .................................................................................... 45 2.1 Bacterial strains ................................................................................... 46 2.2 Bacterial growth media ........................................................................ 46 2.3 Preparation of antibiotics ..................................................................... 46 2.4 Defined minimal medium for growth of L. monocytogenes .................. 47 2.5 Exposure of L. monocytogenes to pregnancy hormones .................... 47 2.5.1 Bacterial growth monitored by optical density and viable counting ............................................................................................... 47 2.6 SDS-PAGE analysis of proteins .......................................................... 48 2.6.1 Preparation of the resolving gel ................................................ 48 2.6.2 Preparation of the stacking gel ................................................. 48 2.6.3 Visualisation of proteins ............................................................ 49 2.7 Miniprep of plasmid DNA ..................................................................... 49 2.8 Agarose gel electrophoresis ................................................................ 51 2.9 Transformation of E. coli with plasmid DNA ........................................ 51 2.10 Preparation of chemically competent E. coli ................................... 51 2.11 Preparation of electrocompetent L. monocytogenes EGD-e ........ 52 2.12 Electrotransformation of L. monocytogenes with plasmid DNA .... 52 2.13 Tn917-mediated insertional mutagenesis .................................... 52 2.14 Primary screening of transposon mutants .................................... 53 2.15 Extraction of L. monocytogenes genomic DNA ............................ 54 2.16 Purification of RNA from L. monocytogenes ................................ 54 2.16.1 Semi-quantative RT-PCR for gene expression ........................ 55 2.17 Cloning and preparation of deletion-mutant in L. monocytogenes chromosomal DNA ..................................................................................... 55 2.18 PCR cycles ........................................................................................ 56 2.19 Screening of clones ............................................................................ 57 iv 2.20 Restriction endonuclease digest of DNA fragments ........................... 57 2.21 Ligation ................................................................................................ 57 2.22 Overexpression of the recombinant proteins ...................................... 58 2.23 Determination of recombinant protein solubility .................................. 59 2.24 Enzymatic activity assay .................................................................... 59 2.25 Bioinformatics analyses...................................................................... 60 2.26 Statistical analysis .............................................................................. 60 Chapter III ..................................................................................................... 62 Influence of pregnancy