Transcript Gene Network Model and Quorum Sensing in Pseudomonas

Gene Network Model
and
Quorum Sensing
in
Pseudomonas Aeruginosa
ELE 580B- Cellular and Biochemical Computing
Project Presentation
Hidekazu OKI & Canturk ISCI
Project Workplan
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Quorum Sensing mechanisms in P.A.
Gene network for P.A.
Biochemical Reactions
Possible Simulation Techniques
Simulation and results
Pseudomonas Aeruginosa
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Lethal, opportunistic,
Gram negative human
pathogen
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LasB elastase, LasA Elastase, Alkaline Protease 
degrade Elastin (lung & blood vessels)
ExotoxinA  inhibit protein synthesis
Uses cell-cell signaling – quorum sensing – to
overcome host defense
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Communal behaviour
Quorum Sensing
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<Discovered: Vibrio Fischeri  lux system>
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Generic Quorum Sensing Mechanism:
(xxx-HSL)
Binding Occurs
only at high AI
concentrations
Quorum Sensing
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Signaling Molecules
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Gram ‘-’  HSL ring &
Fatty acid side chain
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Different side chains  Different AIs
Gram ‘+’  Oligo Peptides
Quorum Sensing in PA
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2 Hierarchical xxxI-xxxR systems:
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1) las system
2) rhl system
Las System:
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lasI  LasI  3-oxo-C12-HSL (PAI1)
lasR LasR
LasR/3-oxo-C12-HSL
lasA, lasB, aprA, toxA, etc. & lasI
rhlR – hierarchy!!
Quorum Sensing in PA
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Rhl System:
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rhlI  RhlI
 C4-HSL (PAI2)
rhlR RhlR
RhlR/C4-HSL
rhlAB operon, lasA, lasB, aprA & other
genes & rhlI
PQS AutoInducer:
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Additional link between las-rhl
LasR  PQS  lasB & rhlI
Quorum Sensing in PA
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Informal Description:
PAI1 can
bind to RhlR
and block it!
PAI2
PAI1
RhlR
PQS
RhlR
RhlI
LasI
LasR
LasR
lasI
lasR
rhlI
RhlR
rhlR
PA Gene Network
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Is it important to make a detailed circuit like
the  circuit?
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All the promoters,
repressors, activators,
specified explicitly
We care about i/p-o/p and cause-effect relations
Our Model:
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I/p  Gene  O/p Protein ( Secondary o/p)
Details of i/p strength hidden in affinities of
chemical reactions
PA Gene
Network
lasI
LasI
RsaL
-
LasR/{3-oxo-C12-HSL}
lasR
Vfr
{3-oxo-C12-HSL}
LasR
LasR
+
lasB
LasB
GacA
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All mentioned genes
+ las system inputs
+ additional
downstream genes
LasR/PAI1 
excitatory on rhlR
PAI1 
inhibitory on RhlR
+
lasA
LasA
PQS
toxA
exotoxinA
aprA
alkaline phosphatase
xcpR
?
xcpP
?
rhlI
RhlI
{C4-HSL}
RhlR/{C4-HSL}
rhlR
RhlR
RhlR
{3-oxo-C12-HSL}
RhlR/{3-oxo-C12-HSL}
RhlR
 Next Slide
PA Gene Network
RhlR/{C4-HSL}
RhlR
rhlAB
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All mentioned genes
+ additional
downstream genes
RhlAB
lasA
LasA
lasB
LasB
aprA
alkaline phosphatase
rpoS
s
?
pyocyanin
lecA
cytoxic lectin
?
cyanide
PA Biochemical Reactions
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Reactions that describe the core of the
quorum sensing mechanism
1) LasR/PAI1 complex:
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R1: Concentration of LasR
A1: Concentration of PAI1
C1: Concentration of Lasr/PAI1
PA Biochemical Reactions
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2) RhlR/PAI2 complex:
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R2: Concentration of RhlR
A2: Concentration of PAI2
C2: Concentration of RhlR/PAI2
3) RhlR/PAI1 complex:
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C3: Concentration of RhlR/PAI1
PA Biochemical Reactions
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4) LasR:
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bR1: Degradation rate of LasR
VR1: Maximum production rate of LasR
KR1: Affinity between C1 and lasR promoter!
R10: LasR basal production rate
PA Biochemical Reactions
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5) RhlR:
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bR2: Degradation rate of RhlR
VR2: Maximum production rate of RhlR
KR2: Affinity between C1 and rhlR promoter
R20: RhlR basal production rate
PA Biochemical Reactions
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6) RsaL:
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S : RsaL concentration
bS: Degradation rate of RsaL
VS: Maximum production rate of RsaL
KS: Affinity between C1 and rsaL promoter
S0: RsaL basal production rate
PA Biochemical Reactions
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7) PAI1:
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bA1: Degradation rate of PAI1
VA1: Maximum production rate of PAI1
KA1: Affinity between C1 and lasI promoter
KS1: Affinity between RsaL and lasI promoter
A10: PAI1 basal production rate
A1ex: Extracellular PAI1
PA Biochemical Reactions
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8) PAI2:
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bA2: Degradation rate of PAI2
VA2: Maximum production rate of PAI2
KA2: Affinity between C2 and rhlI promoter
A20: PAI2 basal production rate
A2ex: Extracellular PAI2
Simulation Methodology
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Deterministic, Single-Cell model
Numerical Integration of Ordinary
Differential Equations.
C Program simulator.
Time step = 0.01 hours.
Total simulated time varied from 100
hours to 10,000 hours.
Simulation Results (1)
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Low concentration of extra-cellular PAI1
causes cell to remain in inactive state.
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LasR/PAI1 complex concentration is low
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
LasR
RhlR
PAI1
PAI2
LasR_PAI1
0
50
100
Time (hours)
150
RhlR_PAI2
RhlR_PAI1
P.A. Quorum Sensing (PAI_ex = 1.5)
Concentration (umolar)
Concentration (u-molar)
P.A. Quorum Sensing (PAI1_ex = 1.0)
1
LasR
RhlR
0.8
0.6
PAI1
PAI2
0.4
0.2
LasR_PAI1
RhlR_PAI2
0
0
50
100
Time (hours)
150
RhlR_PAI1
Simulation Results (2)
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Increasing extra-cellular concentration of
PAI1 beyond 2.0 causes the system to
eventually reach active state.
P.A. Quorum Sensing
(PAI1_ex = 2.5)
8
4
LasR
RhlR
PAI1
2
PAI2
6
0
0
1000
2000
Time (hours)
3000
LasR_PAI1
RhlR_PAI2
RhlR_PAI1
Concentration (u-molar)
Concentrations (umolar)
P.A. Quorum Sensing
(PAI1_ex = 2.0)
7
LasR
6
RhlR
5
PAI1
4
PAI2
3
LasR_PAI1
2
RhlR_PAI2
1
RhlR_PAI1
0
0
200
400
Time (hours)
600
Simulation Results (3)
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Final Steady-State concentrations vary sharply
depending on the extra-cellular PAI1 concentration:
8
6
4
2
0
-2 0
1
2
3
PAI1_ex concentration
(u-molar)
(KR1 = 4, KA1 = 0.4)
P.A. Quorum Sensing
(Steady State)
LasR
RhlR
PAI1
PAI2
LasR_PAI1
RhlR_PAI2
RhlR_PAI1
Concentration (u-molar)
Concentration of
other species
(u-molar)
P.A. Quorum Sensing
(Steady State)
9
8
7
6
5
4
3
2
1
0
-1 0
LasR
RhlR
PAI1
PAI2
LasR_PAI1
RhlR_PAI2
RhlR_PAI1
1
2
3
4
5
Extra-cellular PAI1 concentration (u-molar)
(KR1= 5.0, KA1 = 0.6 )
Index of Terms
Gram negative: …cell wall of Gram-negative bacteria is a thinner
structure with distinct layers. There is an outer layer which is more
like a cytoplasmic membrane in composition with the typical
trilaminar structure.
Ba
ck
Gram Positive: …are characterised by having as part of their cell wall
structure eptidoglycan as well as polysaccharides and/or teichoic
acids.
Ba
ck
References
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David's Paper (lecture 11) --> about quorum and PA
http://www.cdc.gov/ncidod/eid/vol4no4/vandelden.htm --> slides come from
this web in the lecture 11 pres
M. Miller and B Bassler, “Quorum Sensing in Bacteria”, Annual Review of
Microbiology, 55: 165--199, 2001 --> Rweiss reading list paper
http://info.bio.cmu.edu/Courses/03441/TermPapers/99TermPapers/Quorum/ -->
WEB page about PA and quorum
L. Passador and B. Iglewski, "Quorum Sensing and Virulence Gene Regulation in
Pseudomonas Aeruginosa", Virulence mechanisms of bacterial pathogens, 1995
lecture 7 slides --> the lambda cct and the determinstic vs stochastic simulation
models
Fagerlind, Magnus. “The role of regulators on the expression of quorum-sensing
signals in Pseudomonas aeruginosa” A thesis of 20p in molecular computational
biology for the degree of Bachelor of Science at the University of Skovde. Oct,
2001
Albus, Anne M., etal. “Vfr Controls Quorum Sensing in Pseudomonas
Aeruginosa” Journal of Bacteriology, June 1997, p 3928-3935