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Mechanisms of Pathogenesis of African Pneumococcal Serotype 1 Isolates During Nasopharyngeal Carriage and Invasive Disease

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Mechanisms of Pathogenesis of African Pneumococcal Serotype 1 Isolates During Nasopharyngeal Carriage and Invasive Disease Mechanisms of pathogenesis of African pneumococcal serotype 1 isolates during nasopharyngeal carriage and invasive disease Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Laura Bricio Moreno December 2015 Abstract Background: Streptococcus pneumoniae is a human pathogen responsible for serious diseases such as pneumonia, septicaemia and meningitis. There are over 90 pneumococcal serotypes, with serotype 1 being a major cause of invasive disease worldwide. Despite its high invasiveness, serotype 1 is rarely isolated during nasopharyngeal carriage. The aim of this study is to understand the various mechanisms that determine the pathogenicity of serotype 1. Methods: In vitro techniques were used to determine adhesion to and invasion of epithelial cells by serotype 1, and to assess its ability to avoid phagocytosis as well as to uncover the mechanisms involved. Furthermore, two in vivo models of infection in mice were used to assess serotype 1 virulence and ability to colonise and carry in the nasopharynx. Immunological techniques including flow cytometry and enzyme-linked immunosorbent assays were applied to determine the host immune responses to serotype 1. Furthermore, the gene expression of key virulence factors and metabolic pathways was studied in serotype 1 and compared to the less virulent serotype 2 laboratory reference strain D39. Results: Serotype 1 is highly virulent, causing the death of 80-100% of infected mice by 48h post-infection when using an invasive pneumonia model. In a nasopharyngeal carriage model in mice, serotype 1 is able to establish colonisation, although at a lower density and for a shorter period of time than other serotypes. Serotype 1 is not able to induce an early recruitment of T regulatory cells or early production of the cytokines TGF-β1 and IL-10. The induction of these modulatory cells and cytokines is associated with maintenance of carriage and provides protection to the host during pneumococcal pneumonia. Therefore, the failure to induce immune-modulatory cytokines and regulatory T cells may lead to the clearance of serotype 1 in the nasopharynx and reduced protection against pneumococcal dissemination. Moreover, the gene expression analysis of key virulence factors and metabolic pathways showed that serotype 1 is less adapted to be a successful coloniser but is, in contrast, very successful at becoming invasive. Conclusions: The unique characteristics of the type 1 capsule are likely to influence the immune responses against this serotype; however, differences in the expression of non-capsular determinants may also have a key role in the invasiveness of serotype 1. Although serotype 1 is not successful at maintaining nasopharyngeal carriage, it is highly successful at becoming invasive. Acknowledgements I would like to express my sincere gratitude to my supervisors Prof. Aras Kadioglu and Dr. Dean Everett for their support throughout the past four years. Their vast knowledge in the pneumococcal area has guided me through my research allowing me to widen my knowledge in the subject and to produce this thesis. Moreover, I am immensely grateful for their guidance and encouragement during the ups and downs of my PhD study. I would like to thank my family who have worked very hard to give me the opportunity to get to this stage in my career, even if that meant living in a different country. They have all given me great moral support, and at times, I even got some “scientific advice” from my father. I would like to thank all my friends who have been there for me, especially when I needed a break, or even a few formatting lessons. You know who you are. Last but not least, I would like to thank all the members in the Kadioglu lab for helping me during all the stages of the PhD. I am very grateful for their technical support and for the great discussions we have had throughout the years, some of which have led to important experiments included in this thesis. However, I am the most grateful for their time and moral support during the many frustrating times. Again, you know who you are. I would like to finish by mentioning a Spanish saying that I have had to remember many times during the past four years: “La paciencia es la madre de la ciencia” Table of contents 1. Introduction ............................................................................................................ 5 1.1. Streptococcus pneumoniae ................................................................................ 5 1.2. Epidemiology ..................................................................................................... 8 1.3. Pneumococcal virulence factors ...................................................................... 10 1.3.1. Polysaccharide capsule ............................................................................. 11 1.3.2. Pneumolysin.............................................................................................. 15 1.3.3. Choline-binding proteins (CBP) ............................................................... 20 1.3.3.1. Pneumococcal surface protein A (PspA) ........................................... 21 1.3.3.2. Choline –binding protein A (CbpA) .................................................. 22 1.3.3.3 Autolysin ............................................................................................. 22 1.3.4. Hyaluronate lyase (Hyl) ............................................................................ 23 1.3.5. Neuraminidases ......................................................................................... 23 1.3.6. PavA.......................................................................................................... 25 1.3.7. Enolase ...................................................................................................... 26 1.3.8. PsaA .......................................................................................................... 26 1.3.9. Immunoglobulin A1 Protease ................................................................... 27 1.3.10. Pili and sortases ...................................................................................... 28 1.3.11. Gene expression of virulence factors ...................................................... 30 1.4. Pneumococcal vaccines ................................................................................... 32 1.4.1 Pneumococcal polysaccharide vaccines .................................................... 34 1.4.2. Conjugate pneumococcal vaccines ........................................................... 35 1.4.3. Serotype replacement ................................................................................ 37 1.4.4. Potential vaccine candidates ..................................................................... 37 1.5. Host immune response to pneumococcal infection ......................................... 40 1.5.1. Immune responses during nasopharyngeal colonisation .......................... 40 1.5.2. Immune responses during invasive pneumococcal disease ...................... 42 1.6. The pneumococcus in animal models of infection .......................................... 46 1.7. Pneumococcal Serotype 1 ................................................................................ 48 1.7.1. Epidemiology of serotype 1 ...................................................................... 48 1.7.2. Consequences of low carriage rates in serotype 1 distribution ................. 50 1.7.3. Epidemiology of serotype 1 in Africa....................................................... 52 1.7.4. Immune response to serotype 1 ................................................................ 53 1.7.5. Serotype 1 in animal models of infection ................................................. 54 1.8. Research aims .................................................................................................. 55 2. Materials and Methods ........................................................................................ 58 2.1. Pneumococcal bacterial strains ........................................................................ 58 2.2. Confirmation of serotype 1 isolates by PCR ................................................... 60 2.3. Determination of presence or absence of the pneumolysin gene ..................... 60 2.4. Haemolytic assays............................................................................................ 61 2.5. Growth curves .................................................................................................. 62 2.6. Tissue culture ................................................................................................... 62 2.7. Adhesion and invasion assays ......................................................................... 63 2.8. Capsule thickness ............................................................................................. 64 2.9. Opsonophagocytic killing assays ..................................................................... 65 2.10. Complement deposition ................................................................................. 66 2.11. Invasive pneumococcal disease model in mice ............................................. 67 2.12. Nasopharyngeal carriage model in mice ........................................................ 69 2.13. Assessment of long-term protection acquired during carriage ...................... 70 2.14. Nasopharyngeal competition experiments in
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