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UNIVERSITY OF SOUTHAMPTON Faculty of Medicine Clinical and Experimental Sciences Volume 1 of 1 Microevolution of Neisseria lactamica during prolonged colonisation of the nasopharynx by Anish Kumar Pandey Thesis for the degree of PhD UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF MEDICINE Microevolution of Neisseria lactamica during prolonged colonisation of the nasopharynx Authors: Anish Pandey1, Supervisors: David W Cleary1, Jay R Laver1, Andrew Gorringe2 & Robert C Read (Primary) 1 1. Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton 2. Public Health England, Porton Down. Abstract Carriage of Neisseria lactamica occurs naturally at high frequency in infants and low frequency in young adults. There is an inverse epidemiological relationship between N. lactamica carriage and disease caused by Neisseria meningitidis (meningococcus). Serogroup B meningococci remain the dominant cause of invasive meningococcal disease in the developed world and have frustrated the production of polysaccharide-conjugate vaccines. While two, recombinant, OMV based vaccines (Richmond et al., 2012; Vernikos and Medini, 2014) have been created and elicit immunological responses, they are less effective on infants (one of the groups most at risk of IMD) and have limited effect on meningococcal carriage and subsequently, on herd immunity. A human experimental challenge study in which healthy, young adult volunteers were inoculated with N. lactamica Y92-1009 showed that carriage of N. lactamica both displaced and inhibited reacquisition of wild type N. meningitidis, and although rare, co-colonization of the two species was also observed in a small number of cases (Deasy et al., 2015). This study provided the opportunity to investigate whether there is a genomic basis for N. lactamica’s effect on meningococcal carriage as the mechanism for this interaction remains unknown. Secondly, the use of whole genome sequencing, paired with mutation analysis via the breseq pipeline (Barrick et al., 2014) will comment on the mutability of N. lactamica, a potential bacterial medicine, during 6 months of in vivo, human challenge. Thirdly, this allows us to track the within-host microevolution of an identically administered commensal Neisseria spp. over the course of 6 months of carriage (chapter 5) Isolates obtained from individuals who were co-colonised by N. meningitidis and N. lactamica for a prolonged period were examined for evidence of the effect of recombination (r/m) as well as loci affected by it (chapter 6). In addition to the majority of volunteers who solo carried N. lactamica Y92-1009. Recombination was determined for; volunteers in which inoculated N. lactamica was the sole Neisseria spp. detected, seven, artificially inoculated, N. lactamica/meningococcal co-carriers and two extra volunteers who were naturally co-colonised. Using ClonalFrameML (Didelot and Wilson, 2015), we detected minimal homologous recombination events among N. lactamica Y92-1009 and no examples of interspecific allele transferred with co-colonising meningococci. In contrast, we found evidence of a dynamic, interspecific relationship and a number of recombination events occurring among co-colonised volunteers with naturally acquired Neisseria. A separate, short term clinical trial utilizing multiple colony sampling (chapter 4) examined the difference in mutational profiles of longitudinal samples N. lactamica strain Y92-1009 sourced from in vitro conditions versus in vivo conditions over one month. Larger numbers of SNPs, nonsense and recurring mutations were observed among the in vitro cohort and the quantity/diversity of phase variable mutations was more pronounced among the in vivo cohort. Chapters 4 and 5 are supported by a highly-accurate reference genome. The sequencing, assembly, annotation and characterisation of the first complete N. lactamica Y92-1009 genome is described in chapter 3 (Pandey et al., 2017). This chapter also revealed the presence of a large but uncharacterised prophage sequence in the strain. The very first example of a species encompassing, pan genomic analysis of N. lactamica (chapter 7) revealed that N. lactamica Y92- 1009 possess fewer unique genes/alleles than other members of the species with no virulence factors detected among the results. In conclusion, the N. lactamica Y92-1009 genome is a self-curated system with plastic elements that (like other Neisseria spp.) could facilitate rapid changes in expression via its phase variable elements. However, it appears to have remained genetically stable during the 6-month course of carriage in human volunteers. Demonstrating little recombination, no interspecific gene transfer with co-colonising meningococci and an average mutation rate for a Neisseria species. While efforts need to be made to improve the acquisition and retention of carriage, N. lactamica appears to be a safe, naturally competent, potential bacterial therapeutic, capable of a broad- spectrum reduction of meningococcal carriage. (637 words) Computational Microbiology Thesis for the degree of Doctor of Philosophy_ Microevolution of Neisseria lactamica during prolonged colonisation of the nasopharynx MICROEVOLUTION OF NEISSERIA LACTAMICA DURING PROLONG COLONISATION OF THE NASOPHARYNX Anish Kumar Pandey Table of Contents Table of Contents ......................................................................................................................................... i List of Tables ............................................................................................................................................. vii List of Figures ............................................................................................................................................. ix DECLARATION OF AUTHORSHIP ................................................................................................................. xi Acknowledgements .................................................................................................................................. xiii Definitions and Abbreviations ..................................................................................................................... 1 Chapter 1: Introduction ........................................................................................................................... 2 1.1 Neisseria meningitidis, Neisseria lactamica and the Nasopharynx .................................................. 2 1.1.1 Typing ........................................................................................................................................... 3 1.1.2 Meningococcal Vaccines: A brief history ...................................................................................... 3 1.1.3 The search for a Serogroup B vaccine .......................................................................................... 6 1.1.4 Recombinant protein OMV Vaccines ........................................................................................... 7 1.1.5 N. lactamica .................................................................................................................................. 9 1.1.6 N. lactamica Y92-1009: an answer to meningococcal serogroup B disease burden? ................ 10 1.2 The nasopharynx: the niche and the microbiota ............................................................................ 11 1.2.1 Summary of metagenomic research on the nasophayngeal microbiota in healthy, adult humans. ................................................................................................................................................... 13 1.3 Sequencing ...................................................................................................................................... 15 1.3.1 Overview and definitions ........................................................................................................... 15 1.3.2 Read length, Genome assembly, Quality Control and Annotation ............................................ 15 1.3.3 Historical background and first-generation sequencing ............................................................ 17 1.3.4 Second generation (Next generation) Sequencing ..................................................................... 17 1.3.5 Third generation of sequencing ................................................................................................. 21 1.3.6 Challenges for and limitations of bacterial WGS and bioinformatics ......................................... 23 1.3.7 Genome-wide association studies .............................................................................................. 23 1.4 Mutations and mutational analysis ................................................................................................ 24 1.4.1 Point mutations and single nucleotide polymorphisms ............................................................. 24 Figure 1-5 Overview of sources of genetic stability, instability and selective pressure on the genomes of Neisseria spp ............................................................................................................................................... 25 i 1.4.2 Phase variation ........................................................................................................................... 27 1.4.3 Phase variable, sub-unit modifiers in Neisseria: pgl and lgt loci ................................................ 30 1.4.4 Sources of variation:
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