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

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

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

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

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

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

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

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

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

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 +

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

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

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

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

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

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

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

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

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.

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

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

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 )

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

References 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