Presentation on theme: "Towards modeling epigenetic phase variation of virulence factors"— Presentation transcript:
1 Towards modeling epigenetic phase variation of virulence factors Intro: there is a reason I am before Mustafa!! Towards, vs fait accompliMarjan van der WoudeCentre for Immunology and InfectionDEPARTMENT OF BIOLOGY
2 Expression of Virulence factors Haraga 2008Infectious doseBottlenecks
3 Phase variation: Heritable yet reversible gene expression Cell divisionCell divisionsSwitch frequencyrestreak1 in cells switch per generationlacZ reporter
4 Cells in a clonal population may never have identical phenotype 2.In absence of and despite of varying environmental conditions1.Variable level of response within populationPhase variationResults in heterogeneous clonal population with cells expressing (ON) and not expressing gene(OFF).
5 Why study population heterogeneity? Interesting biology we may be missing:Host- pathogen /commensal interactions, interaction with (abiotic) environment, biofilms, resistanceLosick labMention salmonellla mechanisms unknown! And coli…..If it is really that important should every pathogen have it or is this a reflection of the nature of interactions- which host, how long (vibrio short, etc)End argue that this is a fascinating but also critical towards understanding the behaviour of populations in natural environments- modeling diseaseWider implications:Combating Infectious DiseaseDiagnostics, Epidemiology, Vaccine development
6 Biological significance of phase variation? Evade the immune system?Alters host pathogen interactions?OFF phase: loss of antigen leads to loss function - functional redundancy?Baumler !A future event which is possible but can not predicted with certaintyBacillus subtilis (soil) (4.2 Mb genome)swrA gene (SSM) - swarming behaviorKearns DB, et al 2004 Mol Microbiol. 52:PV of adhesins:?Facilitates bacterial dispersal?(from biofilms or colonized host tissue)
7 Phase variation: Heritable yet reversible gene expression Cell divisionCell divisionsSwitch frequencyrestreak1 in cells switch per generationlacZ reporter
8 Reporter fusions to analyze PV: gfp: Green Fluorescent Protein Also for Flow cytometryAlternatives:luxlacZNative proteinSingle cells: overlay of phase contrast (all cells) and fluorescent image (ON cells, GFP+)
9 Analyze and Visualize an infrequent event Microbial Challenge #1Getting the data:Analyze and Visualize an infrequent eventLab vs during infection!Suitability-population, individual cells-lab, in vitro or infection modelSensitivity (need single copy for PV)Reporter relation to “native” protein?
10 Phase variation controlled by DNA methylation (epigenetic)OxyROFF-35-10GATCsProtein CDSpromoterONProtein CDSDamExample: 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.
11 Competition DNA binding protein and processive enzyme OFFUMONMETHStochastic elements: competition, concentration of proteins (local and cellular)HMCompetition DNA binding protein and processive enzymeActual DNA and protein concentration (at site) [Kaminska et al 2010]Role passage DNA replication fork(s) [Kaminska et al 2010]Other growth related variables
12 Significance OxyR binding affinity Role of each GATCOFFONWTK12GATC mutantsx(NA locked Off)Altered switch frequencyGATC-I mutant On to OFF especially altered: increased almost 10 foldEffect DESPITE sufficient OxyRxWTRS218Altered switch frequency
13 Microbial Challenge #2 Microbial Challenge #3 Getting the data: Acquiring relevant numerical data(low concentration proteins and enzyme)Lab vs during infection!Relevant: in cell vs in vitro!Microbial Challenge #3Reduce complexity w/o oversimplifying(include DNA replication, growth?)
14 OxyR and Dam-dependent PV: variation on a theme E. coliagn familyOFFONSalmonella enterica sp.gtrONOFFSarah Broadbent
15 “Molecular Rules” Dam-dependent PV? ONOFFagn familyDam processivity- inter and intra? LocationRNA polymeraseDNA replicationgtr familyAgn- outer membrane protein family in E. coliGtr- LPS modification operons in SalmonellaBoth with evidence of past horizontal (phage) transfer
16 Expression of gtr can affect Salmonellae serotyping >2500 serovars StrainLT214028DT104SL1344TR7095…Genus Species Subspecies Serotypes>98% of human clinical isolatesTyphimuriumTyphiCholeraesuisParatyphiEnteriditis…Enterica (I)Salamae (II)Arizonae (IIIa)Diarizonae (IIIb)Houtenae (IV)Indica (VI)Serotyping main way that outbreaks are characterized!Bongori (V)…SalmonellaSerotypes (Kauffmann-White scheme)Based on immunoreactivity of two surface antigensi) O Antigen (LPS)ii) H Antigen (Flagellar)…Enterica………
18 Model for gtr phase variation;Dam and OxyR gtrAOxyR BOxyR C-10-35+1OxyR ARNApolCH3CH3CH3CH3gtrAOxyR C-10-35+1OxyR AOxyR BOFFOxyRBroadbent et al 2010
19 modifies the O-antigen and phase varies gtrABC:modifies the O-antigen and phase varies0-4 copies of gtr-family operons per Salmonella genome (phage remnants)Also on phage genomesIf 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 …..Rmeind: agn with 2 GATC’s: altered switch rates!(Neisseria PV over 210 variants theoretically possible!)
20 Predict PV rates / regulation based on DNA sequence and paramters? WebLogo of 33 gtr regulatory regions identifies putative important elementsPredict PV rates / regulation based on DNA sequence and paramters?Spacing closest two gatcs same as agn gatcII and gatcIII-mutate either of those in agn and loose phase variationOxyR half b.s.motif : ATAG/T.T…A.CTAT
21 Salmonella LPS modification project BIOCHEMISTRYRelate genes to chemical modificationROLE OF MODIFICATIONHost-Pathogen interactionsFrom molecular stochastic events to how it effects host -pathogen stohcastic eventsMOLECULAR-Genome sequencingSEROTYPING-Improve ? Complete, Molecular diagnosticsEXPRESSION-Phase variation /Regulated?
22 Can we predict Dam-dependent PV from DNA sequence Can we predict Dam-dependent PV from DNA sequence? Any methylation dependent PV?**LPS modification DNA methylEnd : signficance virulence (and funding…???)***OxyR and DamLrp and Damfromvan der Woude and Baumler, 2004
23 “Molecular Rules” Dam-dependent PV? ONOFFagn familyDam processivity- inter and intra? LocationRNA polymeraseDNA replicationgtr familyPap familyLrp, needs PapI
24 Devising and executing experiments within adhering to those wishes Microbial Challenge #4Testing relevanceChoosing the strain and conditions that represent a natural situation of relevanceLab vs during infection!Relevant: in cell vs in vitro!Microbial Challenge #5Devising and executing experiments within adhering to those wishes
25 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?Lab vs during infection!Relevant: in cell vs in vitro!
26 With previous support from NSF Renata KaminskaSarah BroadbentMark DaviesMatt Lakinsprevious lab membersSupport fromWith previous support from NSFCentre for Immunology and InfectionDEPARTMENT OF BIOLOGY