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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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slide1

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

outline
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
my research phase variation
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

slide4

Consequences of Localised Hypermutation:

Phase Variation

SELECTION

/MUTATION

SELECTION

/MUTATION

MUTATION

OFF

ON

ON

Frequency = 10-2 to 10-4

slide10

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

functions of the products of repeat associated genes
Functions of the Products of Repeat-Associated Genes

Flagella

Biosynthetic

Enzymes

Iron

Acquisition

Proteins

Capsule

Biosynthetic

Enzymes

LOS/LPS Biosynthetic Enzymes

Adhesins

Restriction

Enzyme

slide14

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?

campylobacter jejuni
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

slide17

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)

slide18

On-to-off

‘off’ variant

Off-to-on

‘on’

variant

colony blots of c jejuni strain 11168 probed with anti capa
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)

slide20

MHA-VT plates

MHA-VT-XGal plates

multiple passages of growth in mhb broth
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

analysis of phase variable genes and repeat tracts
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)

drift bottlenecks selection and hitch hiking
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

slide27
Neisseria meningitidisPorA Phase Variation, Immune Evasion and Variant-Specific Immune Responses During Carriage
escape assay
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

slide29

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

correlation of pora pv expression to escape
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

slide31

Week -4

Week 0

Week 4

Week 12

Week 24

phase variation of nada
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

multiple simple sequence contingency loci
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)
computer model 1
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
slide36

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

effect of phase variation rate on the production of genotypes with multiple switches
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

slide38

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

slide39

Repeat

Number

5

6

7

8

9

10

11

12

13

Evolution of Repeat Tracts in the Absence of Selection

slide40

Evolution of Repeat Tracts with Selection

and in a Fluctuating Environment

Environmental switch period:- 20 000 generations

Fitness advantage:- 0.1

summary computer simulation model
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
model 3
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)
slide46

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

drift bottlenecks selection and hitch hiking1
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

modelling changes in the distribution of phase variants no selection
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

scientific issues
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
logistical issues
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
slide51

Jean-Philipe Gautier

Jacques Marlet

Fadil Bidmos

Nathalie Ingouf

Rebecca Richards

Awais Anjum

Vladimir Manchev

Richard Haig

Julian Ketley

(University of Leicester)

Neil Oldfield

Del Ala’Aldeen

Karl Wooldridge

Michael Jones

Paul Barrow

(University of Nottingham)

Michael Tretyakov

Alexander Gorban

(University of Leicester)

Michael Palmer

Marc Lipsitch

Richard Moxon

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