Transcript Towards modeling epigenetic phase variation of virulence factors

Towards modeling epigenetic
phase variation of virulence
factors
Marjan van der Woude
DEPARTMENT OF BIOLOGY
Centre for Immunology and Infection
Expression of
Virulence factors
Infectious dose
Bottlenecks
Haraga 2008
Phase variation: Heritable yet reversible gene expression
Cell division
QuickTime™ and a
TIFF (U ncompressed) decompressor
are needed to see this picture.
QuickTi me™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
restreak
restreak
lacZ reporter
1 in 10-104 cells switch per generation
Cells in a clonal population may never have identical
phenotype
Qui ckTime™ and a
TIFF (LZW) decompressor
are needed to see thi s pi cture.
2.
1.
1. Variable level of response within population
2. Phase variation
Results in heterogeneous clonal population with cells
expressing (ON) and not expressing gene(OFF).
Why study population heterogeneity?
Interesting biology we may be missing:
Host- pathogen /commensal interactions, interaction
with (abiotic) environment, biofilms, resistance
Wider implications:
Combating Infectious Disease
Diagnostics, Epidemiology, Vaccine development
Biological significance of phase variation?
-
Evade the immune system
- ?Alters host pathogen
interactions?
PV of adhesins:
- ?Facilitates bacterial
dispersal?
(from biofilms or colonized host tissue)
Phase variation: Heritable yet reversible gene expression
Cell division
QuickTime™ and a
TIFF (U ncompressed) decompressor
are needed to see this picture.
QuickTi me™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
restreak
restreak
lacZ reporter
1 in 10-104 cells switch per generation
Reporter fusions to analyze PV:
gfp: Green Fluorescent Protein
+1
RNA polymerase
gfp
Also for Flow cytometry
Alternatives:
lux
lacZ
Native protein
Single cells: overlay of phase contrast (all cells)
and fluorescent image (ON cells, GFP+)
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?
Phase variation controlled by DNA methylation
(epigenetic)
OxyR
OFF
-35
-10
Protein CDS
GATCs
promoter
ON
Dam
Protein CDS
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.
OFF
UM
ON
METH
HM
• 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 GATC
WTK12
GATC mutants
OFF
ON
Altered switch
frequency
x
x
(NA locked Off)
x
(NA locked Off)
WTRS218
x
x
x
x
x
Altered switch
frequency
Microbial Challenge #2
Getting the data:
Acquiring relevant numerical data
(low concentration proteins and enzyme)
Microbial Challenge #3
Reduce complexity w/o oversimplifying
(include DNA replication, growth?)
OxyR and Dam-dependent PV:
variation on a theme
E. coli agn family
ON
OFF
Salmonella enterica sp. gtr
ON
OFF
Sarah Broadbent
“Molecular Rules” Dam-dependent PV?
ON
OFF
agn family
gtr family
Agn- outer membrane protein family in E. coli
Gtr- LPS modification operons in Salmonella
Both with evidence of past horizontal (phage) transfer
Expression of gtr can affect Salmonellae serotyping
>2500 serovars
Genus
Species
Subspecies
>98% of human clinical
isolates
Enterica (I)
Bongori (V)
Salmonella
Salamae (II)
Arizonae (IIIa)
Strain
Serotypes
Typhimurium
Typhi
Choleraesuis
Paratyphi
Enteriditis
…
…
…
Enterica
Diarizonae (IIIb)
…
Houtenae (IV)
…
Indica (VI)
…
LT2
14028
DT104
SL1344
TR7095
…
Serotypes (Kauffmann-White
scheme)
-Based on immunoreactivity of
two surface antigens
i) O Antigen (LPS)
ii) H Antigen (Flagellar)
gtr operons modify the O-antigen
Enteritidus PT4_II
Gallinarum_II
Dublin_III
Typhi CT18_II
(Pseudo gtrB)
Typhi TY2_I
Paratyphi A_I
∆gtrA
Typhimurium D23580_BTP1 ∆gtrB
Cholerasuis_II
Infantis_II
Cholerasuis_III
Infantis_I
Cholerasuis_I
Hadar_1
gtrC
Phage ST104
Phage ST64T
Typhimurium DT104_III
Group 1
Paratyphi A_III *
Phage P22
Hadar_II
Typhimurium SL1344_II
Typhimurium D23580_II
Typhimurium DT10_I
Typhimurium DT2_I
Typhimurium LT2_II
Choleraesuis_IV *
Infantis_III
Typhi CT18_I
Typhi TY2_II
Paratyphi A_II *
Enteritidus PT4_I
Gallinarum_I
Typhimurium DT2_II
Typhimurium LT2_I
Typhimurium SL1344_I
Typhimurium DT104_II
Paratyphi B
Typhimurium D23580_I
0.1
L C + increasing #O repeats
Group 2
oafA OAc:O5
Group 4
gtrABC Lt2_I Glc:O122
1
4
6
gtr P22 Glc:O1
1
LPS gel
Gal
Rha
Abe
Man
O4, O12
Lipid-core
S. Typhimurium
LT2 O-antigen
Group 3
SPI16-like
?
∆oafA
∆Lt2_I
∆Lt2_II
WT
ptac
Lt2_1
Which gtr cluster conveys which O-serotype?
Model for gtr phase variation;Dam and OxyR
OxyR
ON
+1
CH3-35
CH3
RNApol
OxyR A
OxyR B
CH3
OFF
-10
CH3
gtrA
OxyR C
OxyR
+1
OxyR A
-35
gtrA
OxyR B
Broadbent et al 2010
-10
OxyR C
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
Predict PV rates /
regulation based
on DNA sequence
and paramters?
OxyR half b.s.motif : ATAG/T.T…A.CTAT
Salmonella LPS modification project
BIOCHEMISTRY
Relate genes to chemical
modification
ROLE OF MODIFICATION
Host-Pathogen interactions
MOLECULAR
-Genome sequencing
SEROTYPING
-Improve ? Complete,
Molecular diagnostics
EXPRESSION
-Phase variation /Regulated?
Can we predict Dam-dependent PV from DNA
sequence? Any methylation dependent PV?
Bacterial species
Affected moiety /phenotype
Campylobacter jejuni
Escherichia coli
*
Salmonella enterica Tm
Haemophilu influenzae
Helicobacter pylori
*
*
Neisseria meningitidis
*
Proteus mirabilis
*
Streptococcus pneumoniae
OxyR and Dam
LOS modif ication
Fimbr iae (type 1, CS18)
Fimbr iae (Pap, S, F1845, Clp)
Outer membrane protein
Fimbr iae (Pef)
DNAmodification
modi fication
LPS
fimbr iae LKP
LOS modif ication
DNA R/M
LPS modif ication
Flagella
Membrane lipid composition
Outer membrane proteins
Haemo globin receptors
Capsule
Fimbr iae (MR/P)
Capsule
Metabolism
DNA R/M
Class(es) of regulated
gene/operon
enzyme
structural, regulatory
structural, regulatory
structural
sructural, regulatory
enzyme
structural
enzyme
enzyme
enzymes
structural
enzyme
structural
structural
enzyme
structural, regulatory
structural
enzyme
enzyme
Molecular
Mechanism
SSM
Recomb
DNA methyl
DNA methyl
DNA methyl
SSM methyl
DNA
SSM
SSM
SSM
SSM
SSM
SSM
SSM
SSM
SSM
Recomb
Recomb
SSM
SSM
Lrp and Dam
from van der Woude and Baumler, 2004
“Molecular Rules” Dam-dependent PV?
ON
OFF
agn family
gtr family
Pap family
Lrp, needs PapI
Microbial Challenge #4
Testing 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?
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Renata Kaminska
Sarah Broadbent
Mark Davies
Matt Lakins
previous lab members
Support from
With previous support from NSF
DEPARTMENT OF BIOLOGY
Centre for Immunology and Infection