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Edinburgh Workshop 29-30th September 2010

Towards an understanding of global patterns of simple sequence repeat-mediated phase variation during host persistence of Campylobacter jejuni and Neisseria meningitidis. Chris Bayliss RCUK Research Fellow Department of Genetics University of Leicester.

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Edinburgh Workshop 29-30th September 2010

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  1. Towards an understanding of global patterns of simple sequence repeat-mediated phase variation during host persistence of Campylobacter jejuni and Neisseria meningitidis Chris Bayliss RCUK Research Fellow Department of Genetics University of Leicester Edinburgh Workshop 29-30th September 2010

  2. Outline • Overview of my research areas • Intro to SSRs and phase variation • Measuring mutation rates/patterns • Phase variation of C. jejuni genes in in vitro and in vivo models • Models of SSR-phase variation • Issues

  3. My Research: Phase Variation Experimental models/ Epidemiological samples In silico models Impact of phase variation rate on population structure Mechanistic studies Campylobacter jejuni In vitro models Colonisation of chickens Combined model Carriage samples Neisseria meningitidis Disease samples Selection of phase variants Hb receptors/reversible selection model Haemophilus influenzae R-M systems/Phage infection

  4. Consequences of Localised Hypermutation: Phase Variation SELECTION /MUTATION SELECTION /MUTATION MUTATION OFF ON ON Frequency = 10-2 to 10-4

  5. Streisinger Model

  6. Streisinger Model

  7. Streisinger Model Insertion

  8. Streisinger Model

  9. Streisinger Model Deletion

  10. In-Frame Repeats ATG………..CAAT(30)…..//………….TAG ON ATG………..CAAT(29)…..TAG OFF ATG………..CAAT(28)……..TAG OFF ATG………..CAAT(27)…..//………….TAG ON Promoter-Located Repeats -35 -10 ATTATA……..TA(10)…….ATTAAA…//…ATG ON ATTATA……..TA(9)…..ATTAAA…//…ATG OFF

  11. Functions of the Products of Repeat-Associated Genes Flagella Biosynthetic Enzymes Iron Acquisition Proteins Capsule Biosynthetic Enzymes LOS/LPS Biosynthetic Enzymes Adhesins Restriction Enzyme

  12. Long Tracts of Simple Sequence Repeats in Bacterial Genomes

  13. Length of PolyG/PolyC Repeat Tracts in C. jejuni Contingency Loci

  14. Phase Variation of Simple Sequence Contingency Loci SELECTION /MUTATION SELECTION /MUTATION OFF ON ON What are the mutation rates of SSRs? What are the determinants of SSR mutation rates? What are the fitness implications of differing switching rates? What are the roles of selective and non-selective bottlenecks? What are the implications of multiple SSCL?

  15. Campylobacter jejuni:- Phase Variation Frequencies

  16. Campylobacter jejuni * Gram –ve commensal of gasterointestinal tract of birds and widespread environmental contaminant * Major agent of foodborne gasteroenteritis * Implicated in autoimmmune diseases such as Guillain-Barre syndrome

  17. Reporter Constructs for Detecting Phase Variation in Campylobacter jejuni cj1139c cat lacZ G8 G8 lacZ G11 capA (cj0628/cj0629) T6-G11 Strain NCTC11168 ON CapA a-CapA antibodies (surface-located autotransporter)

  18. On-to-off ‘off’ variant Off-to-on ‘on’ variant

  19. Colony Blots of C. jejuni strain 11168 probed with anti-CapA ON-to-OFF Freq. -ve = 0.03 (filter 1, 9/8/07) OFF-to-ON Freq. +ve = 0.03 (filter 4, 23/7/07)

  20. MHA-VT plates MHA-VT-XGal plates

  21. No environmental factors

  22. Campylobacter jejuni:- In vitro/In vivo Passage

  23. PCR-Based Measurement of Repeat Tract Length FAM GGGGGGGGGG

  24. Multiple Passages of Growth in MHB Broth Inoculate 5mL MHB Inoculate 5mL MHB Inoculate 5mL MHB Inoculate 5mL MHB Inoculate 5mL MHB Pallet the cells Suspend inoculum Plate Dilutions Plate Dilutions Day 0 Day 1 Day 2 Day 3 Day 4 Pick 30 colonies Pick 30 colonies Colony Blotting Colony Blotting PCR Array PCR Array

  25. Analysis of Phase Variable Genes and Repeat Tracts CapA Frequency -ve Inoculum Output 0.29 0.24-0.36 0.29 0.27-0.36 Constant Inoculum (3.5x108cfu; 6 tubes) Variable Inoculum (from 3.5 x108 to 3.5x103cfu; 6 tubes)

  26. Drift, Bottlenecks, Selection and Hitch-Hiking 6 Genes = 64 Genotypes Selection Bottleneck 0685-on Random Drift Mutation/Bottleneck Mutation/Selection 0685-on 1139-off 1139-off Mutation/Bottleneck Mutation/Selection 0031-on

  27. Neisseria meningitidisPorA Phase Variation, Immune Evasion and Variant-Specific Immune Responses During Carriage

  28. Escape Assay Modified serum bactericidal assay using large inoculum (1x104-1x107 cfu) and multiple passages LPS phase variants with switches in expression of lgtG mediate escape of mAb B5 (translational switching) Escape dependent on size of inoculum, amount of antibody and rate of phase variation Bayliss et al. 2008 Infect. Immun. 76:5038

  29. PV of porA mediates immune escape in vitro 11C 10C *Variants examined had 10C residues in the porA repeat tract *Escape is due to pre-existing variants +/- mAb 1.2 10% human serum +/- mAb 1.2 10% human serum +/- mAb 1.2 10% human serum

  30. Correlation of porA PV Expression to Escape • Repeat tract changes to expression • Whole cell ELISA and lysate western blotting 10C 11C 9C *Level of PorA expression is highest when 11C repeat units is present in 8047 *~ 3 fold of reduction in expression of porA

  31. Week -4 Week 0 Week 4 Week 12 Week 24

  32. Phase Variation of NadA Volunteer 1st 2nd 3rd 4th V43 12 - 12 - V51 12 12 12 12 V52 12 12 12 - V54 14 14 12 - V58 12 12 - 12 V59 13 12 12 12 V88 11 9 9 9 V138 12 12 12 - OFF 9 and 12 rpts Number of tetranucleotide repeats All volunteers colonised with Y:P1.21,16:CC174

  33. Computer Models

  34. Multiple simple sequence contingency loci • Multiple loci = multiple potential genotypes • Haemophilus influenzae strain Rd has 12 genes containing tetranucleotide repeat tracts, a potential 4096 genotypes (if two genotypes per locus, i.e. ON and OFF) • Lic2 locus has three genotypes :- ON-Strong, ON-Weak and OFF (if all 12 loci had 3 genotypes then there is 531 441 potential genotypes)

  35. Computer Model 1 • Population founded by single organism which divides by binary fission • Three phase variable loci • Switching occurs in both directions at the same rates • Mutations occur during division giving one genotype of the parental phenotype and one mutant

  36. Effect of phase variation rate on the amount of genetic diversity produced in 20 generations Mutation rate (repeat number) 1x10-6 (< 6) 3.6x10-5 (10) 1.24x10-4 (22) 1000 900 800 700 600 Number of populations 500 400 300 200 100 0 1 2 3 4 5 6 7 8 8 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 Number of genotypes

  37. Effect of phase variation rate on the production of genotypes with multiple switches *Solution is when all three loci have switched from OFF to ON. *30 generations were used. *All cells of the parental genotype were removed at generation 20. *1000 replicates were performed Number of populations containing solution Mutation rate 3.6x10-5 21 1.24x10-4 370

  38. Model 2 Effect of Interval Between Selective Environments Environment A Selection for ON Phenotype Number of Generations 2,000-100,000 2,000-100,000 Environment B Selection for OFF Phenotype Variable Repeat Number 17 = ON = A 18 = OFF = B 19 = OFF = B 20 = ON = A etc 37 = OFF = B 38 = ON = A Mike Palmer and Marc Lipsitch

  39. Repeat Number 5 6 7 8 9 10 11 12 13 Evolution of Repeat Tracts in the Absence of Selection

  40. Evolution of Repeat Tracts with Selection and in a Fluctuating Environment Environmental switch period:- 20 000 generations Fitness advantage:- 0.1

  41. Environmental switch period:- 4 000 generations Fitness advantage:- 0.1

  42. Environmental switch period:- 2 000 generations Fitness advantage:- 0.1

  43. Environmental switch period:- 100 generations Fitness advantage:- 0.1

  44. Summary Computer Simulation Model • Selection is required to maintain large numbers of repeats in the repeat tracts • Repeat number is determined by the frequency of the environmental switch • Correlation between repeat number and environmental switch is also influenced by the conferred fitness advantage and mutational pattern

  45. Model 3 • Model phase shifts in multiple loci using known mutation rates (excludes mutational patterns) • Assumes each locus switches independently of other loci (can set PV rate for each gene, but not scalable with tract length changes) • Simple deterministic model, average of multiple trees from a Monte Carlo simulation, performed in Excel (maximum of 100 generations)

  46. Sample from Chicken B9 One Isolate B9.1 Note:- genotype is not directly correlated with phenotype (i.e. cj0045 is OFF with 9 or 10 repeats Coded phenotypes of all 30 colonies for B9

  47. Drift, Bottlenecks, Selection and Hitch-Hiking 6 Genes = 64 Genotypes Selection Bottleneck 0685-on Random Drift Mutation/Bottleneck Mutation/Selection 0685-on 1139-off 1139-off Mutation/Bottleneck Mutation/Selection 0031-on

  48. Modelling Changes in the Distribution of Phase Variants:- no selection 6 Phase variable genes = ON/OFF = 64 genotypes 0=off, 1=on Output = 100 generations Output 1 = all genes at G9 PV rate (0.0015) Output 2 = varied PV rates

  49. Scientific Issues • What factors to include in a model – mutation rate, mutational pattern, population size, fitness, frequency of environmental switching, bottlenecks, number of loci, number of generations • How to model – simulation of multiple populations or deterministic model of average solutions

  50. Logistical Issues • Data collection (sample bias) • Computational power • Biological and clinical relevance • Simultaneous data collection and modelling (local collaborators) • Relevance to systems biology • Requirement for a modelling community

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