Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. DDiirreecctt sseelleeccttiioonn aanndd pphhaaggee ddiissppllaayy ooff tthhee LLaaccttoobbaacciilllluuss rrhhaammnnoossuuss HHNN000011 sseeccrreettoommee A thesis presented to Massey University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Dragana Jankovic 2008 Acknowledgments ii Acknowledgments I would like to thank the following people for the time, help, and support they have given me during my PhD work. Firstly and primarily I would like to thank my supervisor Dr Jasna Rakonjac for her encouragement, help, support, and expedience which were most appreciated. Her great scientific enthusiasm and amazing energy have been and will always be the inspiration for me. Also many thanks go to my cosupervisors Dr Mark Lubbers and Dr Michael Collett who were always a well of new ideas and discussion topics off all kinds. Their support was invaluable. I thank my cosupervisor Dr John Tweedie for helpful discussions and critical comments on my work. Special thanks to my office mates from Helipad lab, especially David Sheerin and Nicholas Bennett whose tolerance, patience and entertaining discussions were sometimes the only thing that kept me sane. Thanks to everyone at the Institute of Molecular BioSciences for their support. Thanks to them I looked forward to every coffee break to discuss new and interesting current issues. It was a pleasure and privilege working with such a great group of people. A special thanks goes to my sisters, Tatijana and Vesna, and my nephews Fedja and Filip for all their support and encouragement, without which I would never have made it this far. Finally all this would not have been possible without amazing support from my husband Milan, my mum and my daughter Mina whose presence and encouragement was always with me. Contents iii Contents Acknowledgments ii Contents iii List of Figures viii List of Tables x Abbreviations xi Abstract xiii Chapter IA: Probiotic effects of lactic acid bacteria with special reference to Lactobacillus rhamnosus HN001 1.1 Probiotic bacteria ................................................................................................................ 1 1.1.2 Selection of probiotic strains ..................................................................................... 2 1.2 Lactic Acid Bacteria ........................................................................................................... 2 1.2.1 General characteristics of lactic acid bacteria .......................................................... 2 1.2.2 Lactobacilli ................................................................................................................. 4 1.2.2.1 General characteristics ....................................................................................... 4 1.2.2.2 General features of the Lactobacillus genomes ............................................... 6 1.2.2.3 Cell surface of Lactobacilli ............................................................................... 7 1.3 Lactobacillus rhamnosus HN001 .................................................................................... 10 1.3.1 History ....................................................................................................................... 10 1.3.2 HN001 genome ......................................................................................................... 10 1.3.3 Probiotic characteristics of L. rhamnosus HN001 ................................................. 11 1.4 Colonisation of the human GIT by lactobacilli............................................................... 12 1.4.1 Intestinal epithelium ................................................................................................. 12 1.4.2 Aggregation .............................................................................................................. 15 1.4.3 Adhesion to human GIT........................................................................................... 16 1.4.4 Characterised adhesins of Lactobacillus species.................................................... 18 1.4.4.1 Mucus binding proteins ................................................................................... 18 1.4.4.2 Proteins mediating adhesion to the intestinal epithelium and extracellular matrix ................................................................................................................ 21 1.4.4.3 Unconventional Lactobacillus surface proteins and non protein molecules mediating adhesion .......................................................................................... 22 Contents iv Chapter IB: Secretome Proteins ...................................................................... 24 1.1 Definition of the secretome .............................................................................................. 25 1.2 Secretion pathways ........................................................................................................... 25 1.2.1 Sec-dependent and Sec-independent secretion systems of Gram-negative and Gram-positive bacteria ......................................................................................................... 26 1.3 Transport across the cytoplasmic membrane .................................................................. 28 1.3.1 Secretome protein sorting ........................................................................................ 28 1.3.2 Membrane-targeting, translocation and release of secretome proteins ................. 32 1.4 Anchoring of proteins to the plasma membrane and cell wall....................................... 35 1.4.1 Transmembrane anchors .......................................................................................... 35 1.4.2 Lipoprotein anchors.................................................................................................. 36 1.4.3 LPXTG anchors ........................................................................................................ 37 1.4.4 Noncovalent binding of surface proteins to the cell wall ...................................... 38 1.5 Secretomes of Lactobacillus species ............................................................................... 39 1.5.1 Membrane-targeting and anchoring to the cell envelope ....................................... 39 1.5.2 Functional groups of Lactobacillus secretome proteins ........................................ 40 Chapter IC: Phage Display .............................................................................. 42 1.1 Phage display - overview ................................................................................................. 43 1.2 The Ff phage lifecycle ...................................................................................................... 46 1.3 The phagemid-based phage display system .................................................................... 49 1.4 Methods for identification of secretome proteins ........................................................... 52 1.4.1 Examples of bacterial adhesins identified using phage display technology ........ 54 Aims of the Project ................................................................................................................ 56 Chapter II: Materials and Methods ................................................................. 57 2.1 Materials ............................................................................................................................ 58 2.1.1 Laboratory Chemicals and enzymes ....................................................................... 58 2.1.2 Buffers, solutions, and media .................................................................................. 58 2.1.3 Bacterial strains, plasmids and phage ..................................................................... 59 2.1.4 Oligonucleotides ....................................................................................................... 61 2.2 Methods ............................................................................................................................. 62 Contents v 2.2.1 General molecular biology methods ....................................................................... 62 2.2.2 Bacterial growth conditions ..................................................................................... 62 2.2.3 Nucleic acid techniques ........................................................................................... 63 2.2.3.1 Isolation of total genomic DNA from L. rhamnosus HN001 ........................ 63 2.2.3.2 Plasmid DNA isolation .................................................................................... 63 2.2.3.3 Polymerase chain reaction (PCR) amplification ............................................ 64 2.2.3.4 Dot blot hybridisations..................................................................................... 67 2.2.4 Phage protocols ........................................................................................................ 68 2.2.4.1 Phage infection, growth and purification ....................................................... 68 2.2.4.2 Phage enumeration ........................................................................................... 68