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. i The search for Lactobacillus proteins that bind to host targets A thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Turitea New Zealand. Zoe Amber Erridge 2014 ii Abstract Interactions between microorganisms and host cells in the gastrointestinal tract are crucial to the host’s health. Probiotic bacteria, such as the lactobacilli provide numerous benefits to human health thought to be mediated by bacterial proteins called effectors. Lactobacillus rhamnosus HN001 (L. rhamnosus HN001) is a cheese- fermenting isolate with probiotic characteristics and Lactobacillus reuteri 100-23 (L. reuteri 100-23) is a coloniser of the rodent forestomach. Whereas L. rhamnosus HN001 was shown to reduce eczema in children, L. reuteri 100-23 reduces inflammation in mice. The effector proteins for these strains are largely unknown. In this thesis, phage display technology was used to search for proteins that bind specific ligands. Shot-gun genomic phage display library of L. rhamnosus HN001 was affinity screened on fibronectin as bait, leading to enrichment of specific recombinant clones. Analysis of 10 candidate clones, however, determined that these are not genuine binders, but may have been selected due to a potential growth advantage during amplification steps of the library. The L. reuteri 100-23 genomic shot-gun phage display library was subjected to two affinity screens on two baits: fibronectin and murine stomach tissue. The aim of the screen on the murine stomach tissue was to identify keratin-binding proteins, as this strain naturally colonises the murine keratinous forestomach. Whereas no enrichment was detected in the screen on fibronectin as a bait, a strong enrichment of a phagemid displaying a short peptide, IGINS, derived from a cell-surface protease of L. reuteri 100-23 was identified. Identifying and characterising probiotic bacterial proteins that positively influence health will lead to a greater understanding of gastrointestinal tract interactions. Ultimately, this aids development of probiotic use as therapeutic agents. iii Foreword and Acknowledgements Through perseverance we move forward in life. Perseverance is achieved with ‘One More Try’. This mantra comes from two hardworking men and also an angel who is dearly loved by me. They never give up in the face of hardship. This thesis is not only the product of two years study and experimentation into gastrointestinal interactions, but a reflection on a lifelong journey and pursuit of learning. My interest in science began at primary school. This involved learning about space, dinosaurs, geology and uncovering the remains of ancient civilisations. My interest in the genetic and chemical sciences began in high school. At University, by majoring in genetics and microbiology, I became highly interested in microorganisms and their significant influence. Therefore, this thesis is the product of all those years, hours and energy studying science and its role in our community. There are some significant people who have had a positive impact in my life. In particular, I would like to thank the following people: Peter Flynn, for being the greatest maths teacher I have known; Vicki Bothwell, for being the greatest English teacher I have known; Jasna Rakonjac, my supervisor, for her expertise and guidance; Marie McGlynn, my mother, for raising me to do my best and follow my dreams; Catherine Dyhrberg, for her long lasting friendship and advice; Misha Collins, for being a role model for universal love and kindness; Luke Phoenix, for his support and inspiring me to be stronger in all walks of life. I would also like to thank my lab mates at D.401, especially Wesley Wen and Julian Spagnuolo for their advice; Dragana Gagic, for constructing the vectors and phage display libraries; Linda Johnson, Richard Johnson, Gwenda Gopperth, Sharon Wright, Lindsay Wright and other members of my family. In memory of my father who would be proud of what I have achieved. iv Table of Contents Abstract.................................................................................................................... ii Foreword and Acknowledgments............................................................................ iii Table of Contents.................................................................................................... iv Abbreviations........................................................................................................... vi List of Figures.......................................................................................................... vii List of Tables........................................................................................................... vii 1.0 Introduction..................................................................................................... 1 1.1 The gastrointestinal tract................................................................................... 1 1.2 Probiotic bacteria............................................................................................... 3 1.3 Lactobacillus ..................................................................................................... 4 1.4 The search for lactobacilli effector molecules................................................... 5 1.5 Lactobacillus rhamnosus................................................................................... 7 1.6 Lactobacillus reuteri.......................................................................................... 8 1.7 Phage display technology.................................................................................. 9 1.8 Project aims....................................................................................................... 12 2.0 Materials and Methods................................................................................... 13 2.1 Materials............................................................................................................ 13 2.1.1 Bacterial strains, plasmids and phage............................................................. 13 2.1.2 Media, buffers and other solutions................................................................. 15 2.1.3 Animal organs used......................................................................................... 18 2.2 Methods............................................................................................................. 18 2.2.1 Bacterial growth and storage conditions........................................................ 18 2.2.2 Competent cells............................................................................................... 18 2.2.3 Transformation............................................................................................... 19 2.2.4 Phage preparation and propagation................................................................ 19 2.2.5 Amplification of phagemid particles (PPs) ................................................... 19 2.2.6 Helper phage stock.......................................................................................... 20 2.2.7 Titration of phage or PPs ............................................................................... 20 2.2.8 Densitometry.................................................................................................. 21 2.2.9 Pilot study....................................................................................................... 21 2.2.10 Affinity-screening of L. rhamnosus HN001 and L. reuteri 100-23 libraries with superfibronectin............................................................................................... 22 v 2.2.11 Blocking phage experiment.......................................................................... 22 2.2.12 Affinity-screening of L. reuteri 100-23 using mouse stomachs as bait........ 23 2.2.13 Analysis of selected inserts........................................................................... 23 2.2.14 Quantification of DNA................................................................................. 24 2.2.15 Restriction digestion of DNA....................................................................... 24 2.2.16 Agarose gel electrophoresis.......................................................................... 25 2.2.17 Sequencing.................................................................................................... 25 2.2.18 Binding assays of purified clonal recombinant PPs..................................... 25 3.0 Results............................................................................................................... 27 3.1 Pilot study.......................................................................................................... 27 3.2 Affinity-screening of the libraries with super fibronectin as bait...................... 28 3.3 Recombinant phagemids enriched for by affinity-screening on super fibronectin................................................................................................................ 29 3.4 Identification of inserts from the enriched recombinant phagemids................. 30 3.5 Screening of L. reuteri 100-23 phage display library for binding to murine stomach tissue.......................................................................................................... 33 3.6 Analysis of enriched recombinant phagemids................................................... 34 3.7 Analysis of dominant recombinant phagemids................................................. 36 3.8 Competition assays of IGINS-displaying PPs binding to the mouse stomach tissue......................................................................................................................... 38 3.9 Identity of the clone 7........................................................................................ 39 4.0 Discussion.......................................................................................................... 41 5.0 Conclusions....................................................................................................... 44 6.0 Future Directions............................................................................................. 45 6.1 Further characterisation of identified proteins................................................... 45 6.2 Further investigation into fibronectin binders.................................................... 45 6.3 Identify adhesin(s) that bind(s) CaCo-2 cells.................................................... 45 6.4 Identify Adhesin(s) that bind(s) signalling proteins of Toll and Nod pathways................................................................................................................... 45 7.0 References......................................................................................................... 47 vi Abbreviations Amp Ampicillin BLAST Basic Local Alignment Search Tools BSA Bovine Serum Albumin Cm Chloramphenicol DCs Dendritic Cells E. coli Escherichia coli ECM Extracellular Matrix GIT Gastrointestinal Tract IECs Intestinal Epithelial Cells IL_(number) Interleukin (number) (i.e Interleukin 17) Kn Kanamycin L. rhamnosus GG Lactobacillus rhamnosus GG L. rhamnosus HN001 Lactobacillus rhamnosus HN001 L. reuteri 100-23 Lactobacillus reuteri 100-23 M/PAMPs Microbial/Pathogen Associated Molecular Patterns NFkB Nuclear Factor kappa B NEC Necrotizing enterocolitis O.D. Optical Density PSA Polysaccharide A PBS Phosphate Buffer Saline PBST Phosphate Buffered Saline with Tween-20 PEG Polyethylene Glycol pmol Pico mole PP Phagemid Particles RF Replicative Form SDS Sodium Dodecyl Sulfate SOC Super Optimal broth with Catabolite repression ssDNA single stranded Deoxyribose Nucleic Acid TDPs Transducing Phagemid Particles 2xYT Yeast Extract Tryptone Broth vii LIST OF FIGURES Figure 1. Library screening for fibronectin-binding proteins............................. 11 Figure 2. Phagemid vector, pYW01, used to construct the shot-gun phage display libraries.................................................................................... 15 Figure 3. Nde I restriction digest of pooled phagemid DNA during affinity enrichment experiment........................................................................ 26 Figure 4. Nde I restriction analysis of individual recombinant clones isolated from enriched phagemid bands............................................................ 28 Figure 5. Monitoring enrichment for specific recombinant phagemids during the library panning on murine stomach tissue..................................... 32 Figure 6. Sph I and Eco RI restriction analysis of individual recombinant cones isolated from an enriched phagemid band ................................ 34 viii LIST OF TABLES Table 1 Bacterial strains used in this study...................................................... 13 Table 2 Plasmids and phage used in this study................................................. 13 Table 3 Oligonucleotides used for sequencing................................................. 23 Table 4 Pilot fibronectin-binding assay............................................................ 24 Table 5 Lactobacillus reuteri 100-23 and Lactobacillus rhamnosus HN001 phage display with super fibronectin................................................... 25 Table 6 Amino acid sequence of recombinant clones....................................... 29 Table 7 Lactobacillus reuteri 100-23 phage display using keratinous mouse lining..................................................................................................... 31 Table 8 Enrichment assays with Clone 7.......................................................... 36