1. Gene Network Model and Quorum Sensing in Pseudomonas Aeruginosa
ELE 580B- Cellular and Biochemical Computing
Project Presentation
Hidekazu OKI & Canturk ISCI

2. Project Workplan Quorum Sensing mechanisms in P.A.
Gene network for P.A.
Biochemical Reactions
Possible Simulation Techniques
Simulation and results

3. Pseudomonas Aeruginosa
Lethal, opportunistic, Gram negative human pathogen
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
Communal behaviour
Lethal  60% death in PA outbreaks in burn cases 38% of ventilation associated deaths 50 % of AIDS deaths [all from 1.5 p1] Lives in soil, water, vegetation Opportunistic  Needs a break in defense (trauma, surgery, burns, AIDS, etc.) why: 1) not very successful overcoming initial physical barriers I.e. skin 2) When enters a host, not succesful in avoiding host defense mechanisms (in healthy man)[5, p.68]
Some of Proteases produced by PA. Protein elastin major part of lung tissue (extraction and contraction) and blood vessels LasA Elastase makes Elastin vulnarable to degradation by LasB and Alkaline Protease
ExotoxinA: Most toxic product, inhibits protein synthesis in host cell  cell dies, local tissue damage,
Senses cell population density, secretes virulence factors when density high enough cells behave cooperatively as a population

4. Binding Occurs only at high AI concentrations
Quorum Sensing
<Discovered: Vibrio Fischeri  lux system>
Generic Quorum Sensing Mechanism:
(xxx-HSL)
Binding Occurs only at high AI concentrations
marine bacteria around 1970s
xxxI: AutoInducer Synthase Gene  XxxI AutoInducer Synthase Protein  xxx-HSL (Homoserine Lactone) AI xxxR: Transcriptional Activator (R) Protein gene  Transcriptional Activator (R) Protein (not active w/o AI) Low population density  AI diffuses out of cell & gets diluted High population density  Intracellular AI concentration gets high enough  R-Protein/AI complex forms  Transcription of target genes
Gram Negative bacteria use small HSL molecules – AutoInducers – for cell-cell signaling

5. Quorum Sensing Signaling Molecules
Gram ‘-’  HSL ring & Fatty acid side chain
Different side chains  Different AIs
Gram ‘+’  Oligo Peptides
Gram Negative bacteria use small HSL molecules – AutoInducers – for cell-cell signaling
All have the same HSL ring, different length of side chains | different groups on side chains  different AIs

6. Quorum Sensing in PA 2 Hierarchical xxxI-xxxR systems: Las System:
2) rhl system
Las System:
lasI  LasI  3-oxo-C12-HSL (PAI1)
lasR LasR
LasR/3-oxo-C12-HSL lasA, lasB, aprA, toxA, etc. & lasI rhlR – hierarchy!!
Las system:
lasI: AutoInducer Synthase Gene  LasI AutoInducer Synthase Protein  3-oxo-C12-HSL (N-3-oxododecanoyl-L-homoserine lactone) = PAI1(Pseudomonas AutoInducer 1) lasR: Transcriptional Activator (R) Protein gene  LasR: Transcriptional Activator (R) Protein (not active w/o AI) LasR/Pai1 complex  lasA, lasB, aprA ( alkaline phosphatase ), toxA (exeotoxinA ) also  lasI: positive feedback  more PAI1: sudden burst of PAI1 production after threshold: x1000 in gene expression also  rhlR: initiates secondary quorum system > hierarchy

7. Quorum Sensing in PA Rhl System: PQS AutoInducer:
rhlI  RhlI  C4-HSL (PAI2)
rhlR RhlR
RhlR/C4-HSL rhlAB operon, lasA, lasB, aprA & other genes & rhlI
PQS AutoInducer:
Additional link between las-rhl
LasR  PQS  lasB & rhlI
Rhl system:
rhlI: AutoInducer Synthase Gene  RhlI AutoInducer Synthase Protein  C4-HSL (N-butyryl-homoserine lactone) = PAI2(Pseudomonas AutoInducer 2) rhlR: Transcriptional Activator (R) Protein gene  RhlR: Transcriptional Activator (R) Protein (not active w/o AI) RhlR/Pai2 complex  rhlAB operon: encodes rhamnosyltransferase which enables the production of rhamnolipids by catalyzing
the transfer of rhamnosyl from …… to …….
also  lasA, lasB, aprA ( alkaline phosphatase ) also  rhlI: positive feedback  more PAI2, dunno how strong the effect of complex is PQS Autoinducer:
recently discovered
Pseudomonas Quinolone Signal (2-heptyl-3-hydroxy-4-quinolone) , NOT AN HSL!! Also controls expression of lasB PQS expression requires LasR PQS induces rhlI xcription

8. Quorum Sensing in PA Informal Description: PAI2 PAI1 PQS
lasI
lasR
LasI
PAI1
LasR
PQS
rhlI
rhlR
RhlI
PAI2
RhlR
PAI1 can bind to RhlR and block it!
Only quorum sensing mechanisms described
LasR/Pai1  virulence genes & lasI as well
PQS  lasB as well
RhlR/Pai2  virulence genes, some other genes & rhlI as well

9. PA Gene Network
Is it important to make a detailed circuit like the  circuit?
All the promoters, repressors, activators, specified explicitly
We care about i/p-o/p and cause-effect relations
Our Model:
I/p  Gene  O/p Protein ( Secondary o/p)
Details of i/p strength hidden in affinities of chemical reactions

10. PA Gene Network All mentioned genes + las system inputs
alkaline phosphatase
exotoxinA
LasR/{3-oxo-C12-HSL}
{3-oxo-C12-HSL}
Vfr
lasR
lasI
LasR
LasI
lasA
lasB
LasA
LasB
aprA
toxA
xcpP
xcpR ?
GacA +
RsaL -
RhlR/{C4-HSL}
{C4-HSL}
rhlR
rhlI
RhlR
RhlI
RhlR/{3-oxo-C12-HSL}
 Next Slide
PA Gene Network
All mentioned genes
+ las system inputs
+ additional downstream genes
LasR/PAI1  excitatory on rhlR
PAI1  inhibitory on RhlR
PQS
Las system I/ps  RsaL, etc.
Additional downstream genes  xcpR, xcpP
Strong positive feedback for lasI
Strength of positive feedback for rhlI not explained +

11. PA Gene Network All mentioned genes + additional downstream genes RhlR
alkaline phosphatase
RhlR/{C4-HSL}
RhlR
lasA
rhlAB
LasA
RhlAB
aprA
lasB ?
rpoS
 s
LasB
pyocyanin
lecA
cyanide
cytoxic lectin
All mentioned genes
+ additional downstream genes

12. PA Biochemical Reactions
Reactions that describe the core of the quorum sensing mechanism
1) LasR/PAI1 complex:
R1: Concentration of LasR
A1: Concentration of PAI1
C1: Concentration of Lasr/PAI1

13. PA Biochemical Reactions
2) RhlR/PAI2 complex:
R2: Concentration of RhlR
A2: Concentration of PAI2
C2: Concentration of RhlR/PAI2
3) RhlR/PAI1 complex:
C3: Concentration of RhlR/PAI1

14. PA Biochemical Reactions
4) LasR:
bR1: Degradation rate of LasR
VR1: Maximum production rate of LasR
KR1: Affinity between C1 and lasR promoter!
R10: LasR basal production rate

15. PA Biochemical Reactions
5) RhlR:
bR2: Degradation rate of RhlR
VR2: Maximum production rate of RhlR
KR2: Affinity between C1 and rhlR promoter
R20: RhlR basal production rate

16. PA Biochemical Reactions
6) RsaL:
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

17. PA Biochemical Reactions
7) PAI1:
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

18. PA Biochemical Reactions
8) PAI2:
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

19. Simulation Methodology
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.

20. Simulation Results (1)
Low concentration of extra-cellular PAI1 causes cell to remain in inactive state.
LasR/PAI1 complex concentration is low

21. Simulation Results (2)
Increasing extra-cellular concentration of PAI1 beyond 2.0 causes the system to eventually reach active state.

22. Simulation Results (3)
Final Steady-State concentrations vary sharply depending on the extra-cellular PAI1 concentration:
(KR1 = 4, KA1 = 0.4) (KR1= 5.0, KA1 = 0.6 )

23. 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.
Gram Positive: …are characterised by having as part of their cell wall structure eptidoglycan as well as polysaccharides and/or teichoic acids.
Back
Lethal  60% death in PA outbreaks in burn cases 38% of ventilation associated deaths 50 % of AIDS deaths [all from 1.5 p1] Lives in soil, water, vegetation Opportunistic  Needs a break in defense (trauma, surgery, burns, AIDS, etc.)
Back

24. References David's Paper (lecture 11) --> about quorum and PA
--> slides come from this web in the lecture 11 pres
M. Miller and B Bassler, “Quorum Sensing in Bacteria”, Annual Review of
Microbiology, 55: , > Rweiss reading list paper
--> 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