Curli fimbriae of Salmonella Typhimurium induce an immune response in chickens producing IgY detectable in serum and yolk. Nicole Groves A thesis submitted as partial fulfilment of the requirements for the degree of Masters of Philosophy (BABS) School of Biotechnology and Biomolecular Sciences Faculty of Science March 2017 i ii iii Abstract Curli fimbriae of Salmonella spp., also known as GVVPQ fimbriae due to a highly conserved amino acid sequence at the N-terminus, play an important role in bacterial virulence in the host gut environment. Curli are essential in Salmonella biofilm formation, environmental resilience, and bacterial attachment and persistence in the host. The genes encoding curli fimbriae are found in the vast majority of Salmonella serovars, and expression can be induced in most of these serovars under stressful environmental conditions. The development of a curli fimbriae-based immunoassay to detect seroconversion in poultry would allow for quick identification of Salmonella colonisation of most serovars, and could be incorporated into current routine detection and monitoring programs in agricultural industries. Curli fimbriae were isolated from Salmonella Typhimurium phage type 135a and characterised by ELISA and mass spectrometry. Purified fimbriae were incorporated into a vaccine, based on an aluminium hydroxide adjuvant, which was administered to 13-week-old ISA Brown hens. Booster injections were given at 18 weeks and blood and egg samples were collected after this point. Crude IgY was purified from egg yolk samples using ammonium sulphate precipitation. The efficacy of the immunisation was determined by building a purified fimbriae-based indirect ELISA. Blood samples taken at 23 weeks had a significantly higher level of anti- curli fimbriae antibodies compared to the control birds and the samples taken at 18 weeks. Egg yolks from vaccinated birds contained elevated levels of antibodies to the fimbriae from weeks 21-24, after which the titre slowly declined to week 28. Western iv blotting using crude IgY purified from yolk showed that birds which had received the vaccine produced IgY specific to a wide array of Salmonella proteins, while the control birds did not. These results suggest that the vaccine elicited an immune response of birds to curli fimbriae, and, while no challenge studies were performed, the ELISA prototype was able to detect seroconversion. This implies that curli fimbriae are immunogenic, and this ELISA, if developed further, could be used to diagnose hypervirulent Salmonella infection in poultry, and potentially other agricultural animals. v Table of Contents 1 Introduction 1 1.1 Salmonella and Society 1 1.1.1Bacteriology of Salmonella 1 1.2 Salmonella and Disease 4 1.2.1 Virulence Factors 6 1.2.2 Biofilm Formation 13 1.2.3 Curli Fimbriae 16 1.3 Control and Detection of Salmonella 21 1.3.1 Prevention and Monitoring 21 1.3.2 Vaccination 24 1.4 Antibodies 28 1.4.1 Mammalian and Avian Antibodies 29 1.4.2 Research and Medicine 33 1.5 Aims and Significance 36 2 Materials and Methods 38 2.1 Purification of Curli Fimbriae 38 2.1.1 Expression of Curli 38 2.1.2 Digestion of Whole Cells 39 2.1.3 Differential Centrifugation 40 2.1.4 Lyophilisation and Quantification 40 2.2 Analysis of Extracted Fimbriae 41 2.2.1 Assessment of Purity by SDS-PAGE 41 vi 2.2.2 Mass Spectrometry 42 2.3 Vaccine Formulation 45 2.3.1 Vaccine Production 45 2.3.2 Vaccine Delivery 45 2.3.3 Sample Collection 46 2.3.4 Standard ELISA Blood Testing 47 2.4 Analysis of IgY 48 2.4.1 Crude Yolk IgY Purification 48 2.4.2 ELISA Development 49 2.4.3 Western Blotting 51 3 Results 56 3.1 Purification of Curli Fimbriae 56 3.1.1 Expression of Curli 56 3.1.2 Digestion of Whole Cells 58 3.1.3 Differential Centrifugation 58 3.1.4 Lyophilisation and Quantification 59 3.2 Analysis of Extracted Fimbriae 59 3.2.1 Assessment of Purity by SDS-PAGE 59 3.2.2 Mass Spectrometry 61 3.3 Vaccine Formulation 62 3.3.1 Vaccine Production and Delivery 62 3.3.2 Sample Collection 63 3.3.3 Standard ELISA Blood Testing 63 vii 3.4 Analysis of IgY 64 3.4.1 Crude Yolk IgY Purification 64 3.4.2 ELISA Development 65 3.4.3 Western Blotting 72 4 Discussion 80 4.1 Purification of Curli Fimbriae 80 4.1.1 Expression of Curli 80 4.1.2 Extraction of Fimbriae from Whole Cells 83 4.2 Analysis of Extracted Fimbriae 86 4.2.1 Assessment of Purity by SDS-PAGE and MS 86 4.3 Vaccine Formulation 92 4.3.1 Vaccine Production and Delivery 92 4.3.2 Standard ELISA Blood Testing 96 4.4 Analysis of IgY 96 4.4.1 Crude Yolk IgY Purification 96 4.4.2 ELISA Development 101 4.4.3 Western Blotting 106 5 Conclusions 111 References 113 viii Acknowledgements I would like to acknowledge all the people who supported me through this project, who helped me in my stressful moments, and celebrated my successes. In particular I would like to thank my parents, who gave me every kind of support I could need for the past two years. I also would like to thank my supervisors, Julian Cox and Chris Marquis, for giving me this opportunity, and for all of their invaluable guidance and advice over the last two years. Thank you for helping me develop as a scientist and encouraging me to think deeply and creatively about the questions and problems I encountered. Finally, thank you to all the staff at Birling Avian Laboratories, for your help over the years and for welcoming me even when time and bench space were limited. To Tony, who gave me the chance to work at Birling, for always pushing me to be better, and for always believing in me. And to Sue and Sarah, without whom I could not have gotten through the challenges I faced in this project, and who always had time to listen and offer advice no matter how busy they were. ix Abbreviations BCA Bicinchoninic acid BSA Bovine serum albumin CFA Colonisation factor antigen DTT Dithiothreitol ELISA Enzyme-linked immunosorbent assay EPS Exopolysaccharide FITC Fluorescein isothicyanate H-chain Heavy chain HRP Horseradish peroxidase L-chain Light chain LDS Lithium dodecyl sulfate LPS Lipopolysaccharide MES 2-(N-morpholino)ethanesulfonic acid MS Mass spectrometry m/z Mass-to-charge ratio OD650nm Optical density at 650nm OMP Outer membrane protein PBS Phosphate buffered saline RNA Ribonucleic acid RO Reverse osmosis SDS Sodium dodecyl sulfate SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis SPI Salmonella pathogenicity island spp. Species S/P Sample to Positive ratio TBST Tris-buffered saline with Tween 20 TGX Tris/glycine/SDS buffer Th1-cells Type 1 helper T-cells Th2-cells Type 2 helper T-cells TMB Tetramethylbenzidine T3SS Type 3 secretion system x 1 Introduction 1.1 Salmonella and Society Salmonella species (spp.) are responsible for 1.3 billion infections and approximately 3 million human deaths annually (Cox et al., 2011; Yue et al., 2012). The vast majority of this morbidity and mortality (between 55-96% depending on local food production standards) is caused through ingestion of contaminated food and water (World Health Organisation, 2007; Cox et al., 2011). While control and detection methods have been widely implemented through the food chain, their efficacy is questionable. There is a pressing need in the poultry industry for faster, reliable detection methods, and a wider variety of preventative methods to minimise the impact of Salmonella contamination on both human and animal health (Cox et al., 2011). To develop measures to reduce this mortality rate, the mechanisms and consequences of the disease must first be understood. 1.1.1 Bacteriology of Salmonella Salmonella is a mesophilic, rod-shaped, Gram-negative facultative bacterium from the Enterobacteriaceae family (Threlfall, 2008; Zhang et al., 2011). A typical Salmonella cell is shown schematically in Figure 1.1. Salmonella spp. are classified by species, subspecies, and serovar, for example Salmonella enterica subspecies enterica serovar Typhimurium (abbreviated to S. Typhimurium). There are two recognised species of Salmonella, S. enterica and S. bongori, though subspecies have only been identified for S. enterica (Hald, 2013). Between the Salmonella spp., over 2,500 serovars have been 1 identified and recognised internationally, with this number rising every year (World Health Organisation, 2013; Cosby et al., 2015). Some serovars are host-specific; for example, S. Typhi, S. Paratyphi and S. Sendai are specific to humans, while S. Gallinarum and S. Pullorum are specific to poultry. Other serovars may act as pathogens in a much wider range of hosts, while being best adapted to one in particular. Laying hens, for example, are particularly good reservoirs for S. Typhimurium and S. Infantis (Yue et al., 2012; Desin et al., 2013; Hald, 2013). Different serovars may also affect the same species differently; for example, S. Virchow in young children occasionally causes septicaemia, while elderly patients are more likely to experience septicaemia due to S. Enteritidis (Cox et al., 2011). Figure 1.1 – Typical Salmonella Typhimurium cell. (Source: de Jong et al., 2012a) 2 The cause of this specificity, while still not fully understood, may lie partially in the differing survivability of Salmonella serovars in certain environments. This survivability is influenced by the ability of a specific serovar to handle temperature stress and nutrient availability, and their capacity to attach to biotic and abiotic surfaces. It has been shown, for example, that S. Enteritidis is better suited to survival in food processing environments such as stainless steel surfaces than S. Typhimurium or S.