Translocation StudiesF.Vidal-Aroca, J.Dreier and M.Page

Background

• Deteriorating situation regarding treatment of Gram-negative infections

– Growing concern over:• Multi-resistant Acinetobacter• Multi-resistant Pseudomonas aeruginosa• Carbapenem-resistant Klebsiella pneumoniae• ESBL-producing K. pneumoniae and E. coli

– Warnings for:• Carbapenem-resistant Enterobacter• GNNFs like Burkholderia, Stenotrophomonas….

Interests & Expectationsat Basilea

• Insight into the permeation of β-lactam antibioicsin clinical isolates

• Permeation route(s) of novel compounds

• Role of porins in resistance

• Role of efflux systems in resistance

RationaleFocus on the role of porins and efflux systems in antibiotic resistance of

Gram-negative bacteria

antibiotic

Uptake

Efflux

Antibiotic-resistant Cells

Permeability of BridgedMonobactams

Bridged monobactams arepotent β-lactamase inhibitors

When R2 = H, activity againstβ-lactamase in situ can bemodulated by R1

When R2 = OMe or larger, allactivity against β-lactamase in situ is lost, irrespective ofR1

NSO3HO

HH

O

R1R2

Permeability of BridgedMonobactams

R2IC50 β-lactamase

in vitro(µM)

MIC against cells

in situ

Effect on OMPF Conductance(from Mathias)

RO 47-7303 OCH3 0.06 >64* No interaction

RO 47-0243 CH.CONH2 0.05 >64* Blocking

RO 46-9392 Cl 0.1 >64* No interaction

RO 46-8377 S.tetrazole 0.009 >64* Blocking

RO 44-4454 H 0.1 2* No effect

Carbenicillin - - 4 No interaction

Penicillin G - - 16 Some blocking

* In combination with penicillin

Permeability of BridgedMonobactams

• Is the lack of correlation due to OmpC?• Knock-out strain with only OmpF

• Need more data points to look for trends• Select more penicillins with a spread of MIC values• Select more bridged monobactams

• Are the conditions appropriate? •low pH, high salt vs neutral pH, isotonic

Gene Disruption

genomic DNATarget geneA B B C1 2 3 4

PCR 1/2 & 3/4 cloning vector

gene replacement vector (TcR, SacB)

antibiotic resistant & sucrose sensitive1st crossing-over:

antibiotic sensitive & sucrose resistant2nd crossing-over:

Knock-out mutant (or reversal to wt)

Complementation

Complementation experiments are needed to show a functional linkbetween observed phenotype(s) and given K.O. mutation(s).

OmpC Knock-out (1)1 2 31 2 3 4 5 6

1 2 3 4 5 1 2 3 4 5 OmpCup OmpCdw(1) OmpC up & down

fragment amplification

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 M

20 21 22 23 24 M12345678

910111213141516

1718192021222324

(2) Preparation of the fragments into TopoCloning

vector

OmpC Knock-out (2)(3) Digestion of the Gene

replacement vector (pex18Gm) and the OmpC up

& down fragments

(4) Confirmation for the first fragment cloned into

pEX18Gm

(5) Waiting for the second fragment cloned into the

plasmid from step 4

(6) Gene replacement: Double homologous recombination

Piddock L.J.V. Clin Microbiol Rev, Apr. 2006, p. 382–402

Efflux Pumps

Resistance Mechanism1- Enzymatic inactivation

2- Alternative metabolic pathways

3- Reduced uptake

4- Alteration of the target site

5- Membrane bound efflux pumps

Pseudomonas aeruginosa

Ubiquitous environmental bacterium

One of the top causes of opportunistic human infections

bacteraemia in burn victimsurinary-tract infectionshospital-acquired pneumoniaAIDS populationpredominant cause of morbidity and mortality of cystic fibrosis patients

Intrinsic resistance to antibiotics and disinfectants

Efflux Systems in P. aeruginosa

7 of 12 RND-pump encoding operons in P. aeruginosa have been characterized:

System substratesMexAB/ OprM * β-lactams, BLI, chloramphenicol, novobiocin, macrolides, quinolones,

sulfonamides, tetracyclines, trimethoprim, thiolactomycin, detergents, triclosan,…MexXY/ OprM* aminoglycosides, β-lactams, erythromycin, fluoroquinolones, tetracyclines,…

MexCD/ OprJ* β-lactams, quinolones, chloramphenicol, novobiocin, sulfonamides, tetracyclines, trimethoprim triclosan,…

MexEF/ OprN* fluoroquinolones, tetracycline, chloramphenicol, trimethoprim, triclosan…

MexJK/ OprM ciprofloxacin, erythromycin, tetracycline, triclosan

Mex(G)HI/ OpmD vanadium, norfloxacin,…

MexVW/ OprM chloramphenicol, erythromycin, fluoroquinolones, tetracycline,…

* major contributors to MDR

Other clinically relevant bacteria with RND efflux systems:

A. baumannii, B. cepacia, B. pseudomallei, S. maltophilia, N. gonorrhoeae, N. meningitidis, S. marcescens, E. coli, S. enterica, E. aerogenes, K. pneumoniae, K. oxytoca, C. jejuni, P. mirabilis, H. influenzae.

Knock-out Mutants

http://www.pseudomonas.com

1. Make single and multiple knock-out mutants of all major RND pumps in P. aeruginosa.

2. Measure susceptibility to all available antibiotics in comparison to the parent strain.

Expression Analysis byReal-time PCR:

Measure the expression levels of the 4 main efflux pumps in:

• P. aeruginosa PAO1 wt and K.O. mutants• P. aeruginosa PAO1 wt and K.O. mutants as a response to antibiotics• clinical isolates• clinical isolates as a response to antibiotics

Results of Translocation Studies

E. coli and P. aeruginosa Knock-out Mutants

Strains and Plasmids AcquiredNº Strains Properties or genotype1 Pseudomonas aeruginosa PAO1 wild type2 E.coli S17-1 lambdaPir thi pro hsd R rec A RP4-2-TcR::Mu-KmR::Tn7 λpir (TpR & SmR100)3 E.coli HB101 (RK2013) Smr, recA thi pro leu hsdRM+ (Mobilizing strain for triparental mating KmR (HELPER))4 E.coli HB101 (RK600) Smr, recA thi pro leu hsdRM+(Mobilizing strain for triparental mating CmR)/RK600 Cmr ori-ColE1 RK2-mob+RK2-tra+ 5 E.coli S-17-1 λpir / pDM47 E.coli JM109 /pMMB66EH8 E.coli JM109 /pMMB66HE9 E.coli JM109 /pVI533EH10 E.coli JM109 /pVI533HE11 E.coli JM109 /pVLT3112 P.putida Paw94 /pVLT31 Benzoate-1,2-dioxygenase-negative KT2440 mutant / pVLT3113 E.coli /pKNG10114 E.coli TOP10 (SmR100) F- mcrA ?(mrr-hsdRMS-mcrBC) Φ80lacZ?M15 ?lacΧ74 recA1 araD139 ?(araleu) 7697 galU galK rpsL (StrR) endA1 nupG15 E.coli JM109 recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi ∆(lac-proAB) F' [traD36 proAB+ lacIq lacZ∆M15]16 E.coli W3110 prototrophic derivative of K-12 F- IN(rrnD-rrnE)1 lambda-17 E.coli TransforMax EC100D pir-116 F- mcrA ∆(mrr-hsdRMS-mcrBC) φ80dlacZ∆M15 ∆lacX74 recA1 endA1 araD139 ∆(ara, leu)7697 galU galK λ- rpsL nupG pir-116(DHFR).SmR100

18 E.coli S17-1 lambdaPir bertoni's lab19 E.coli EC100D pir-116/pEX18Gm20 E.coli S17-1 lambdaPir/pEX18Gm21 E.coli HPS1/pEX18Gm22 E.coli HPS1/pEX18Tc23 E.coli JM109/pluxCDABE

Plasmids Properties1 pDM4 Suicide vector carrying the sac BR genes for sucrose sensitivity (Cmr)2 pEX18Gm GmR; oriT+ sac B+, gene replacement vector with MCS from pUC183 pMMB66EH Apr, broad-host-range lacIq Ptac expression vectors4 pMMB66HE Apr, broad-host-range lacIq Ptac expression vectors5 pVLT31 Tcr, broad-host-range lacIq Ptac expression vector6 pVI533EH Apr, broad-host-range araC-PBAD expression vector7 pVI533HE Apr, broad-host-range araC-PBAD expression vector8 pKNG101 Suicide vector carrying the sac BR genes for sucrose sensitivity (Smr)9 pEX18Tc TcR; oriT+ sac B+, gene replacement vector with MCS from pUC18

Isolation of Genomic DNA fromE. coli and P. aeruginosa

E.coli

P.aeru

ginosa

Marker

Set-up of PCR for Analysis of KO constructs

(A) Primer design (B) Fragment amplification

KO primers 24 pairs

1. S17-1 lambda pir/ pEX18Gm2. EC100D pir116/ pEX18Gm3. HPS1/ pEX18Gm4. HPS1/ pEX18Tc

Electroporation and Conjugation:Two Methods to be Improved

(A) Electroporation

(B) Conjugation

1 2

3 4

PAO1

PAO1+vector + Triton X-100

PAO1+vector -Triton X-100

42C 37C

Growth of P. aeruginosa in Minimal Medium

3

5

1

4

6

2

a bc

1 2 3 4

1 MM + gentamycin a. EC100pir116 /pEX18Gmb. HPS1/pEX18Gmc. S17 lambdapir/pEX18Gm

2 MM + carbenicillin E.coli JM109

3 MM + carbenicillin S17-1 lpir from Bertoni’s lab

4 MM S17-1 lpir from Bertoni’s lab

5 MM PAO1/pVI533EH

6 MM + carbenicillin PAO1/pVI533EH

Results of Translocation Studies

Expression Analysis by Real-time PCR

Real-time PCR: Optimization of Conditions

Primer design

RT-PCR primers 16 pairs

RNA extractionCheck the primers 12 3

1 2 3 4 51 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Progress

Task StatusK.O. mutants:

Design of primers

Work out method ongoing

Transformation and cross-over in PAO1 and E.coli ongoing

Organize tools (bacterial strains, plasmids, primers)Work out selective growth conditionsPlasmid preparations & quality controlSet up of suicide selection systemSet up of transformation system (electroporation, conjugation)

Cloning of homology fragments into pGEX ongoing

RT-PCR:Primers for RT-PCR

Acknowledgements

ItalyBertoni’s Lab

UKCamara’s Lab

USASchweizer’s Lab

BasileaCaspers’ Lab

BasileaShapiro’s Lab

Thank you

for your attention!!!

Reverse Genetics(1) Amplification of up and down stream sequences of the target gene

Target geneA B B C1 2 3 4

Genomic DNA

PCR 1/2 & 3/4

pCR®2.1-TOPO

Antibiotic Resistances

A B C

(2) Preparation of the Gene replacement vector

pCR®2.1-TOPO

Antibiotic Resistances

A B C

Gene replacement vector

sacBTcR

(3) Gene replacement: Double homologous recombination

X

Gene replacement vector

sacBTcR

Genomic DNA

1st crossing-over: MERODIPLOID

Antibiotic resistant & Sucrose sensitive

X X2nd crossing-over

Knock-out Mutant Wild type

Antibiotic sensitive & Sucrose resistant