Towards modeling epigenetic phase variation of virulence

factors

Marjan van der Woude

Centre for Immunology and Infection

DEPARTMENT OF BIOLOGY

Haraga 2008

Infectious doseBottlenecks

Expression of Virulence factors

Cell division

Cell divisions C

ell d

ivis

ions

Phase variation: Heritable yet reversible gene expression

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QuickTime™ and aTIFF (Uncompressed) decompressor

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restreakrestreak

1 in 10-104 cells switch per generationlacZ reporter

QuickTime™ and aTIFF (LZW) decompressorare needed to see this picture.

1. Variable level of response within population2. Phase variation

Results in heterogeneous clonal population with cells expressing (ON) and not expressing gene(OFF).

Cells in a clonal population may never have identical phenotype

1.

2.

Why study population heterogeneity?

Host- pathogen /commensal interactions, interaction with (abiotic) environment, biofilms, resistance

Interesting biology we may be missing:

Wider implications:

Combating Infectious Disease

Diagnostics, Epidemiology, Vaccine development

Biological significance of phase variation?

PV of adhesins:- ?Facilitates bacterial

dispersal? (from biofilms or colonized host tissue)

- Evade the immune system

- ?Alters host pathogen interactions?

Cell division

Cell divisions C

ell d

ivis

ions

Phase variation: Heritable yet reversible gene expression

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

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1 in 10-104 cells switch per generationlacZ reporter

Single cells: overlay of phase contrast (all cells) and fluorescent image (ON cells, GFP+)

Reporter fusions to analyze PV: gfp: Green Fluorescent Protein

gfp

+1RNA polymerase

Alternatives:luxlacZNative protein

Also for Flow cytometry

Microbial Challenge #1

Getting the data:Analyze and Visualize an infrequent event

Suitability -population, individual cells-lab, in vitro or infection model

Sensitivity (need single copy for PV)

Reporter relation to “native” protein?

promoter

OFF

ON

Protein CDS

Phase variation controlled by DNA methylation(epigenetic)

Dam

OxyR

GATCs

Protein CDS

-10-35

Example: OxyR is a repressor but can only bind if 3 Dam target sequences (GATC) are unmethylated. Once OxyR is bound, Dam can not access GATC.

HM

OFFUMON

METH

• Competition DNA binding protein and processive enzyme• Actual DNA and protein concentration (at site) [Kaminska et al 2010]

• Role passage DNA replication fork(s) [Kaminska et al 2010]

• Other growth related variables

Significance OxyR binding affinity

Role of each GATCG

AT

C m

utan

ts x

(NA locked Off)

x

x

x (NA locked Off)

Altered switch frequency

OFF ONWTK12

x xx x

WTRS218Altered switch frequency

Microbial Challenge #2

Getting the data:Acquiring relevant numerical data

(low concentration proteins and enzyme)

Microbial Challenge #3Reduce complexity w/o oversimplifying

(include DNA replication, growth?)

OxyR and Dam-dependent PV:variation on a theme

agn family

ON OFF

gtr Salmonella enterica sp.

E. coli

Sarah Broadbent

ON

OFF

“Molecular Rules” Dam-dependent PV?

agn familyON OFF

gtr family

Agn- outer membrane protein family in E. coliGtr- LPS modification operons in SalmonellaBoth with evidence of past horizontal (phage) transfer

Expression ofExpression of gtr gtr can affect can affect SalmonellaeSalmonellae serotyping serotyping >2500 serovars>2500 serovars

Salmonella

Bongori (V)

Enterica

Genus Species Subspecies Serotypes

Enterica (I)

Salamae (II)

Arizonae (IIIa)

Diarizonae (IIIb)

Houtenae (IV)

Indica (VI)

TyphimuriumTyphiCholeraesuisParatyphiEnteriditis……

…

…

…

…

LT214028DT104SL1344TR7095…

Serotypes (Kauffmann-White scheme)

-Based on immunoreactivity of two surface antigensi) O Antigen (LPS)ii) H Antigen (Flagellar)

Serotypes (Kauffmann-White scheme)

-Based on immunoreactivity of two surface antigensi) O Antigen (LPS)ii) H Antigen (Flagellar)

Strain

>98% of human clinical isolates

0.1

Enteritidus PT4_II

Gallinarum_IIDublin_III

Typhi CT18_IITyphi TY2_I

Paratyphi A_ITyphimurium D23580_BTP1

Cholerasuis_II

Infantis_IICholerasuis_IIIInfantis_ICholerasuis_I

Hadar_1

Phage ST104Phage ST64TTyphimurium DT104_III

Paratyphi A_III *

Phage P22

Hadar_IITyphimurium SL1344_IITyphimurium D23580_IITyphimurium DT10_ITyphimurium DT2_I

Typhimurium LT2_IICholeraesuis_IV *Infantis_III

Typhi CT18_I

Typhi TY2_IIParatyphi A_II *

Enteritidus PT4_IGallinarum_I

Typhimurium DT2_IITyphimurium LT2_ITyphimurium SL1344_ITyphimurium DT104_IIParatyphi B

Typhimurium D23580_I

Group 3SPI16-like

Group 4

Group 1

Group 2

∆gtrA ∆gtrB

gtrCgtrC

?

gtrABC Lt2_I Glc:O1221

4

oafA OAc:O5

gtr P22 Glc:O1 16

S. Typhimurium LT2 O-antigenS. Typhimurium LT2 O-antigen

(Pseudo gtrB)

Gal Rha Man

Abe

O4, O12L C

+ in

cre

asi

ng

#O

re

pe

ats

∆oafA ∆Lt2_I∆Lt2_II

WTptac Lt2_1

Lipid-core

LPS gel

gtr operons modify the O-antigen

Which Which gtrgtr cluster conveys which O-serotype? cluster conveys which O-serotype?

Model for gtr phase variation;Dam and OxyR

gtrA

OxyR B OxyR C

-10-35

+1

OxyR ARNApol

CH3 CH3

ON

gtrA

OxyR C

-10-35

+1OxyR A

OxyR B

OFF

CH3CH3 OxyR

OxyR

Broadbent et al 2010

gtrABC: modifies the O-antigen and phase varies

0-4 copies of gtr-family operons per Salmonella genome (phage remnants)

Also on phage genomes

If 3 of 4 copies PV then one can have 8 phenotypic variants in a population just from the gtr family!

Combine with PV of possibly as many as 11 adhesins …..

WebLogo of 33 gtr regulatory regions identifies putative important elements

OxyR half b.s.motif : ATAG/T.T…A.CTAT

Predict PV rates / regulation based on DNA sequence and paramters?

Salmonella Salmonella LPS modification projectLPS modification project

SEROTYPING

-Improve ? Complete, Molecular diagnostics

SEROTYPING

-Improve ? Complete, Molecular diagnostics

BIOCHEMISTRYRelate genes to chemical

modification

BIOCHEMISTRYRelate genes to chemical

modification

MOLECULAR-Genome sequencing

MOLECULAR-Genome sequencing

EXPRESSION-Phase variation /Regulated?

EXPRESSION-Phase variation /Regulated?

ROLE OF MODIFICATIONHost-Pathogen interactionsROLE OF MODIFICATIONHost-Pathogen interactions

van der Woude and Baumler, 2004

Can we predict Dam-dependent PV from DNA sequence? Any methylation dependent PV?

Bacterial species Affected moiety /phenotype Class(es) of regulated

gene/operon Molecular

Mechanism

Campylobacter jejuni LOS modification enzyme SSM Escherichia coli Fimbriae (type 1, CS18) structural, regulatory Recomb Fimbriae (Pap, S, F1845, Clp) structural, regulatory DNA methyl Outer membrane protein structural DNA methyl Salmonella enterica Tm Fimbriae (Pef) sructural, regulatory DNA methyl Haemophilu influenzae DNA modification enzyme SSM fimbriae LKP structural SSM LOS modification enzyme SSM Helicobacter pylori DNA R/M enzyme SSM LPS modification enzymes SSM Flagella structural SSM Membrane lipid composition enzyme SSM Neisseria meningitidis Outer membrane proteins structural SSM Haemoglobin receptors structural SSM Capsule enzyme SSM Proteus mirabilis Fimbriae (MR/P) structural, regulatory Recomb Streptococcus pneumoniae Capsule structural Recomb Metabolism enzyme SSM DNA R/M enzyme SSM

OxyR and Dam Lrp and Damfrom

LPS modification DNA methyl*

*

*

*

*

“Molecular Rules” Dam-dependent PV?

Pap familyLrp, needs PapI

agn familyON OFF

gtr family

Microbial Challenge #4Testing relevance

Choosing the strain and conditions that represent a natural situation of

relevance

Microbial Challenge #5

Devising and executing experiments within adhering to those wishes

Challenge(s) #6

What is enough data to make modeling feasible?

How to decide if modeling is a worthwhile endeavor for the system?

If the system is the best for the modeling?

Support from

• Renata Kaminska• Sarah Broadbent • Mark Davies• Matt Lakins• previous lab members

With previous support from NSF

Centre for Immunology and Infection

DEPARTMENT OF BIOLOGY