Identificationof and porins RTN - - PowerPoint PPT Presentation

Identificationof and porins

RTN MTRmeeting QueTien Tran,Marseille,April 11th 2008

Dealing with infectious diseases

• Emergenceof nosocomialinfections
• Decreasing antibiotic susceptibility among Gramnegative

bacteria (GNB)inclinic – Multidrug resistant (MDR) isolates frequently observed

• Inefficacity of conventional antibacterial agents Intrinsic

resistance

• Requirement of newalternativeefficacious molecules for

therapeutic

• Fullunderstanding aboutresistance mechanisms and their

regulation is difficult buturgently necessary

The bacterial outer membranes

• First barrier of cell defense
• Molecular filter forhydrophilic molecules orpermeability barrier

forhydrophobic substances(especially inenteric bacteria).

• Modificationof the permeability may concern several antibiotic

classes

• Intrinsic resistance of bacteria

Porins

• Poreforming proteins inouter membraneof GNB,

mitochondria,plastids

• Uptake pathway of small molecules (≤600Da)including

antibiotics:βlactams,quinolones…

• Modified porin expression(decreasing ormutation)→ change
• f membranepermeability
• Moststudied porins: OmpF,OmpC &PhoE
• Porins classification:

– General (OmpF,OmpC ,PhoE)orspecific (LamB,ScrY…) – Trimer(OmpF,OmpC,PhoE)ormonomer (OmpG…)

• A)Trimer viewedfromthetop.B)Monomerviewedfromtheside.

C)Residuesattheconstrictionzoneofthemonomerviewed from thetop(Cowan,1992)

OmpF

ConstrictiondomainofOmpF porin

Itisnowrequiredtoextendourexpertise concerningtheporins tootherless studiedGramnegativepathogensthatcause emerginginfectiousdiseasesduetotheir highresistancelevel.

and

• GNB,,tribe
• Straightrods,motile,facultativeanaerobic
• Environment,gastrointestinalflora
• Opportunisticpathogens,urinarytractinfection(bladder

stone)→ Riskgroup2(labaccessibilityinMarseille)

• Hospitalacquiredinfection
• Theyareclosed intermofDNArelatednessbutdistantfrom

notyet bySangerInstitute Genomic annotation Aminoglycosides, fluoroquinolone,C3G, carbapenem Aminoglycosides,quinolone, C3GC4G,carbapenem, azthreonam,ticarcillin clavulanic acid,trimethoprime sulfamethoxazole Antibiotic susceptibility

• Species
• and

• Cutaneous colonisation
• Specieswithisolatesthemostresistanttowards

antibioticsofthefamily

• Susceptibility:C3G

Carbapenem Aminoglycoside Fluoroquinolone

• Antibioticresistance

– Cephalosporinase (AmpC) – ESBL: TEM,SHV,CTXM,VEB… – Permeability:LPSmodification Outermembraneproteins?!

ND 16 2 16 4 0.125 ≤0.06 ≤0.06 1 5860 ND 64 4 >2 >2 2 0.125 ≤0.06 2 99594 ND 32 4 0.25 0.25 0.25 ≤0.06 ≤0.06 2 ATCC29906(PR14) 128 512 64 >512 >256 >256 0,5 1 4 2636 ND 32 64 >512 >256 >256 1 1 4 99645 32 256 64 >512 >256 >256 10.5 4 84 NEA16 128 256 32 256 64 128 ≤0.06 0.25 2 65237 ND 32 16 4 1 0.5 0.25 0.25 2 19539 ≤0.06 32 2 ≤0.06 ≤0.06 ≤0.06 ≤0.06 ≤0.06 2 ATCC29914 SFX CM FOX CAZ CPO FEP MPM EPM IPM

Abbreviations:IPM(imipenem),EPM(ertapenem),MPM(meropenem),FEP(cefepime),CPO(cefpirome), CAZ(ceftazidime),FOX(cefoxitin),CM(chloramphenicol),SFX(sparfloxacine),ND(notdetermined).

Susceptibility toantibiotics byminimalinhibitory concentration (MICinIg/ml)

Analysis of outer membraneproteins

• 1. ATCC29914
• 2. 65237
• 3. 19539
• 4. NEA16
• 5. ATCC29906
• 6. 5860

Negative detection of the antigenic sitelocated inside porin which is reported asloop 3markerof enterobacterial porins

resistance selection with cefepime

1) ATCC 29914(no antibiotictreatment) 2)FEP0.06Ig/ml 3)FEP0.09Ig/ml 4)FEP0.25Ig/ml 5)FEP0.5Ig/ml Theabbreviationmeansthat thevariantwasselectedatthe concentrationofcefepime indicated.

Same response observed with cefoxitin

Resistance selection with imipenem

1) ATCC 29914(no antibiotictreatment), 2)IPM1Ig/ml, 3)IPM2Ig/ml, 4)IPM4Ig/ml, 5)IPM6Ig/ml, 6)IPM8Ig/ml. Theabbreviationindicatesthatthe variantswereselectedatthe concentrationofimipenem mentioned.

Analysis ofouter membraneproteins inresistant variants

• Several proteins involved inresponse toantibiotic bylosing or

decreasing the expression

• Cefepime &cefoxitin variantspresent adecrease ofproduction
• fimmunorelated porins

Resistance bylowering cephalosporin uptake

• Noresistant variantselected with imipenem exhibit a

modificationofimmunorelated porins Porin expressioninresponse toimipenem is different than to cephalosporins (at least with the concentrationstested)

Comparison ofpredicted a.asequences OmpPm & OmpPv with OmpF,OmpC,PhoE

The internal loop 3domain

In,L3loop length is well conserved with important modificationof residues compared to general porins Thismay explain the negative detection of the enterobacterial loop 3markerbyF4antibody

• Semiconservation of amino acid sequences compared

to general porins

• Conservationof typical porin structurewith:

16βstrands 8periplasmic turns 8extracellular loops

• Several porin key residues conserved
• Importantmodificationinthe internal L3loop

porin

Special conformationof the eyelet inside porin ? Different functional and structuralorganization of porin channel ?! Involvement inantibiotic susceptibility ?Role of L3 loop ?

DiscussiononOmpPm

What about porin?

Southern blotting

Templates: genomic DNA Positivecontrols: amplicon Different restrictases used, Positively clear signalobtained with HindIII Probe: Hybridization t°:50°C

• gDNA

gDNA EcoRI HindIII Fragmentabout 3.5Kb

Southern blotting

porin amplificationbyPCR

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PCRfor porin amplification

The products were cloned into pGemTeasy vector forsequencing

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porin sequence compared to homolog

• mpPst

Adaptor PCRasatechniqueof choice toget the whole gene

• ?

?

Adaptor PCR

Adaptor PCRin

Ta=50°C Ta=45°C

Restrictases :NcoI NcoI_adaptor Sequencing:inwaiting

Futureaims

Expression– Regulation of the porins Physicochemical characterization Antibiotic fluxthrough the porins …

Acknowledgement

Prof.MathiasWinterhalter Prof.MatthiasUllrich Dr.Helge Weingart Dr.JeanMariePagès Dr.AnneDavinRégli

Biophysics group Microbiology group Laboratory UMRMD1, FacultésdeMédecineetde Pharmacie,Marseille

Thank you foryour attention!