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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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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

Edinburgh Workshop 29-30th September 2010


Outline
Outline sequence repeat-mediated phase variation during host persistence of

  • 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 sequence repeat-mediated phase variation during host persistence of

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


Edinburgh workshop 29 30th september 2010

Consequences of Localised Hypermutation: sequence repeat-mediated phase variation during host persistence of

Phase Variation

SELECTION

/MUTATION

SELECTION

/MUTATION

MUTATION

OFF

ON

ON

Frequency = 10-2 to 10-4


Streisinger model
Streisinger Model sequence repeat-mediated phase variation during host persistence of


Streisinger model1
Streisinger Model sequence repeat-mediated phase variation during host persistence of


Streisinger model2
Streisinger Model sequence repeat-mediated phase variation during host persistence of

Insertion


Streisinger model3
Streisinger Model sequence repeat-mediated phase variation during host persistence of


Streisinger model4
Streisinger Model sequence repeat-mediated phase variation during host persistence of

Deletion


Edinburgh workshop 29 30th september 2010

In-Frame Repeats sequence repeat-mediated phase variation during host persistence of

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 sequence repeat-mediated phase variation during host persistence of

Flagella

Biosynthetic

Enzymes

Iron

Acquisition

Proteins

Capsule

Biosynthetic

Enzymes

LOS/LPS Biosynthetic Enzymes

Adhesins

Restriction

Enzyme


Long tracts of simple sequence repeats in bacterial genomes
Long Tracts of Simple Sequence Repeats in Bacterial Genomes sequence repeat-mediated phase variation during host persistence of


Length of polyg polyc repeat tracts in c jejuni contingency loci
Length of PolyG/PolyC Repeat Tracts in sequence repeat-mediated phase variation during host persistence of C. jejuni Contingency Loci


Edinburgh workshop 29 30th september 2010

Phase Variation of Simple Sequence Contingency Loci sequence repeat-mediated phase variation during host persistence of

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 phase variation frequencies
Campylobacter jejuni sequence repeat-mediated phase variation during host persistence of :- Phase Variation Frequencies


Campylobacter jejuni
Campylobacter jejuni sequence repeat-mediated phase variation during host persistence of

* 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


Edinburgh workshop 29 30th september 2010

Reporter Constructs for Detecting Phase Variation sequence repeat-mediated phase variation during host persistence of

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)


Edinburgh workshop 29 30th september 2010

On-to-off sequence repeat-mediated phase variation during host persistence of

‘off’ variant

Off-to-on

‘on’

variant


Colony blots of c jejuni strain 11168 probed with anti capa
Colony Blots of sequence repeat-mediated phase variation during host persistence 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)


Edinburgh workshop 29 30th september 2010

MHA-VT plates sequence repeat-mediated phase variation during host persistence of

MHA-VT-XGal plates


Edinburgh workshop 29 30th september 2010

No environmental factors sequence repeat-mediated phase variation during host persistence of


Campylobacter jejuni in vitro in vivo passage
Campylobacter jejuni sequence repeat-mediated phase variation during host persistence of :- In vitro/In vivo Passage


Edinburgh workshop 29 30th september 2010

PCR-Based Measurement of Repeat Tract Length sequence repeat-mediated phase variation during host persistence of

FAM

GGGGGGGGGG


Multiple passages of growth in mhb broth
Multiple Passages of Growth in MHB Broth sequence repeat-mediated phase variation during host persistence of

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 sequence repeat-mediated phase variation during host persistence of

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 sequence repeat-mediated phase variation during host persistence of

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


Edinburgh workshop 29 30th september 2010
Neisseria meningitidis sequence repeat-mediated phase variation during host persistence of PorA Phase Variation, Immune Evasion and Variant-Specific Immune Responses During Carriage


Escape assay
Escape Assay sequence repeat-mediated phase variation during host persistence of

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


Edinburgh workshop 29 30th september 2010

PV of sequence repeat-mediated phase variation during host persistence 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 sequence repeat-mediated phase variation during host persistence 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


Edinburgh workshop 29 30th september 2010

Week -4 sequence repeat-mediated phase variation during host persistence of

Week 0

Week 4

Week 12

Week 24


Phase variation of nada
Phase Variation of NadA sequence repeat-mediated phase variation during host persistence of

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


Computer models
Computer Models sequence repeat-mediated phase variation during host persistence of


Multiple simple sequence contingency loci
Multiple simple sequence contingency loci sequence repeat-mediated phase variation during host persistence of

  • 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 sequence repeat-mediated phase variation during host persistence of

  • 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


Edinburgh workshop 29 30th september 2010

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


Edinburgh workshop 29 30th september 2010

Model 2 genotypes with multiple switches

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


Edinburgh workshop 29 30th september 2010

Repeat genotypes with multiple switches

Number

5

6

7

8

9

10

11

12

13

Evolution of Repeat Tracts in the Absence of Selection


Edinburgh workshop 29 30th september 2010

Evolution of Repeat Tracts with Selection genotypes with multiple switches

and in a Fluctuating Environment

Environmental switch period:- 20 000 generations

Fitness advantage:- 0.1


Edinburgh workshop 29 30th september 2010

Environmental switch period:- 4 000 generations genotypes with multiple switches

Fitness advantage:- 0.1


Edinburgh workshop 29 30th september 2010

Environmental switch period:- 2 000 generations genotypes with multiple switches

Fitness advantage:- 0.1


Edinburgh workshop 29 30th september 2010

Environmental switch period:- 100 generations genotypes with multiple switches

Fitness advantage:- 0.1


Summary computer simulation model
Summary genotypes with multiple switchesComputer 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 genotypes with multiple switches

  • 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)


Edinburgh workshop 29 30th september 2010

Sample from Chicken B9 genotypes with multiple switches

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 genotypes with multiple switches

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 selection

  • 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 selection

  • 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


Edinburgh workshop 29 30th september 2010

Jean-Philipe Gautier selection

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