Towards modeling epigenetic phase variation of virulence factors Marjan van der Woude Centre for...
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Transcript of Towards modeling epigenetic phase variation of virulence factors Marjan van der Woude Centre for...
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
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
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.
restreakrestreak
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