Marie Curie Meeting 9 th 11 th April 2008 Mission: To gain further - - PowerPoint PPT Presentation

To gain further understanding of the molecular To gain further understanding of the molecular aspects of antibiotic transport through porins aspects of antibiotic transport through porins and the emergence of drug resistance through and the emergence of drug resistance through porin mutation and regulation mechanisms. porin mutation and regulation mechanisms. Mission: Chloë E. James and JeanMarie Pagès Marie Curie Meeting 9th11th April 2008

Role of Porins in Antibiotic Susceptibility of Gram Negative Bacteria

Porins

Influence membrane permeability High level of expression (in ) Tightly regulated Rapid response to environment

Antibiotic Resistance

↓ level / type of porins expressed Express mutated porin Modulation of porin activity

• !"#$%

• Environmental; opportunistic;

MDR nosocomial infections MDR within 5 days antibiotherapy Susceptibility and porin synthesis restored after treatment

• (quiescent?)
• Well characterized model system

(Regulation; Crystal structure;

Liposome / Bilayer studies; Modelling)

Pathogenic; commensal; lab tool

• (quiescent?)

• Antibiotic stress induces rapid

↓ in OmpF

• Antibiotic flux is faster through

OmpF than OmpC (smaller channel and electrostatic diffs)

• L3 loop mutations → structural
• r electrostatic change; alter flux
• ↓ Expression of Omp36
• Switch from Omp35 to Omp36
• Mutation in Omp36 L3 loop

MDR

Projects

1) Role of porin Omp36 in antibiotic transport 2) Regulation of OmpF expression in response to antibiotic treatment: a role for OmpX? 3) Effect of L3 loop mutations in OmpF on rate of antibiotic influx

Project 1 – Omp36 Transport

Clone Omp & from ATCC and express on the OM of porinfree

!. ".

Purify Omp36 by FPLC using a selective detergent protocol with octylPOE and AKTA Pharmacia apparatus

. (Mahendran – Jacobs University)

Insert purified porins into lipid bilayer model Analyse channeldrug interactions by observing fluctuations in ion currents through Omp36 in the presence of antibiotics

#

insert (1 kB)

III HI

0,125 g/ml 0,5 1 2 4 8 16 32

Assess the capability of Omp36 to increase susceptibility to various antibiotics using MIC assays

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Expression of cloned genes

BL21 DE3 (pColdIV++/ Induction: 1mM IPTG 24 h @ 15 oC

55 kDa 34 kDa

Antibodies: Omp36 = αf4 (12 aa peptide from L3 loop) (1:10,000) OmpA = αOmpA from (1:1000) + + + + +

36 A + + +

55 kDa 34 kDa

36 A

55 kDa 34 kDa 55 kDa 34 kDa 30 kDa

36 A

+ + + + +

36 A

• Total cell

OM

&'() * +++ ,&,,,#, ,&,,,#, ,&,,,#,

• -

./ α0 1

• *
• -./

,2+,

• 345,+6

&'() * +++ ,&,,,#, ,&,,,#, ,&,,,#,

• -

./ α0 1

• *
• -./

,2+,

• 345,+6

,&,,,#, ,&,,,#, ,&,,,#,

• -

./ α0 1

• *
• -./

,2+,

• 345,+6

FPLC ion exchange chromatography:

Solubilisation conditions: 1% POE; 1M NaCl; 37 oC; 1 h; x3 Purification conditions: 0.6 % POE; NaCl gradient Purified porin stable for at least 1 week at 4, 20 and RT oC

7 ,&$ #4$ #4$8 #4$

Bilayer Experiments

Βlactam interaction with Omp36

• Ertapenem and cefepime cause fluctuations in ion currents through purified

Omp36 channels reconstituted into planar lipid bilayers

• Ertapenem molecules interact with the channel more strongly than cefepime
• No interaction between Omp36 and ampicillin or cefotaxime observed

Ion current fluctuation due to transient channel blockages by antibiotic molecules (work by K. Mahendran (Jacobs University)

Antibiotic Sensitivity conferred by Omp36

MIC Assays

• Omp36 expression causes 8fold increase in sensitivity to ertapenem

4fold increase in sensitivity to cefepime (+ inhibitors) .

• No Omp36 increase in sensitivity to cefotaxime observed

Grow to OD600 0.4 +/ 1 mM IPTG, 1 h Dilute to OD600 0.001 Incubate with dilution series of drug +/ IPTG (0.5 mM) and CTC

MIC values (g ml –1) for porin null expressing Omp36

• r OmpA (repeated 35 times)

4 8 4 + pColdIV 0.5 4 0.5 + pColdIV 2 4 4

• pColdIV

24 4 4 +/ pColdIV only FEP + CTC FEP CTC ERT IPTG Vector CTC = Clavulanic acid, Tazobactam, Cloxacillin (4 g ml 1)

Conclusions from Project 1

• Omp36 expression increases sensitivity to ertapenem

and cefepime and electrostatic data shows their strong interaction with the channel

• & ERT translocates Omp36 more frequently than

FEP due to stronger affinity with an interaction site inside the channel

• Varying sensitivities to βlactamase degredation and target

affinities means that influx rates of different βlactams cannot be directly compared by MIC assays alone

• Quantification of flux through purified porins in lipid bilayers,

together with MIC assays provides crucial information about the molecular dialogue between channel and drug. James C E, Mahendran K R submitted to EMBO Reports

Project 2 – OMP regulation in response to stress

' 18 kDa enterobacterial OMP

8 stranded antiparallel βbarrel. Overexpression in : Decreased Omp36 expression Decreased susceptibility to βlactams

OmpX expression is increased in response to external stress conditions

Increased OmpX and decreased porins in clinical isolates

Work by M. Dupont

Project Strategy

Does OmpX play a role in porin regulation in response to external stresses?

• ATG

* * Transcriptional start site Transcriptional fusion Translational fusion pFus2K pFus2K

• 1. Construct

transcriptional and translational lacZ fusions

• 2. Transform

constructs into parent and

• r

mutants

• 3. Expose cultures to subinhibitory external

stress conditions and measure βgalactosidase activity at 30 min time intervals

Add substrate to sample and measure OD

↓ βgal activity reflects ↓ expression in response to stress compared to normal growth conditions

t = 60 90 120

& 4 ,& ,4 #& #4 & 4

• .

1 79 7: $,&(, : ./,&&

• &

,& #& & 0& 4& (&

• .

1 79 7: $,&(, : ./,&&

• % Decrease in expression in response

to 90 min stress

( (

T regulation is independent of OmpX Downregulation of OmpF transcription is dependent on MarA T regulation is independent of OmpX Downregulation of OmpF translation is on MarA ( action) Novobiocin may induce a Mar independent regulation pathway n = 10 P < 0.05 SD ±220 %

Conclusions from Project 2

• expression is and OmpX increased very rapidly in

response to antibiotic and chemical stress (within 60 min)

• The regulatory response is:

largely via the Mar system at the level of transcription and ( action)

• An alternate regulatory pathway may be induced by novobiocin
• No detected role for ompX in regulation of expression at

transcriptional and translational levels Dupont M, James C E 2007 9) 31908

• OmpX protein may perform posttranslational regulation of OmpF by

competing with porins for proper export, folding and assembly into the OM forcing protease degradation Viveiros M 2007 PLoS one 2(4): e365

Project 3 – Effect of OmpF L3 loop mutations on rate of antibiotic influx

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Subtle sequence differences in the L3 loop can cause dramatic structural differences to hinder or aid passage through the porin eyelet

)!"! *!! )!! !" +" $" ,!

• OmpF studied due to availability of

mutants

Methodology

• II

I H I

MIC

*

0,125 g/ml 0,5 1 2 4 8 16 32

x2

!% Clone and D113A; D121A mutant genes into expression vector and express in porin null strain BL21 "% Determine the MIC for various βlactams on BL21 expressing WT and mutated OmpF % Expose cultures to βlactams (". MIC) and record decrease in cfu at 30 min time intervals.

WT D113A D121A

• /

0%

• 0.125

0.06 0.5 MIC g ml1 Rate ↑ 90% killed 150 min WT 53 % 70 min 113A 20 % 120 min 121A 0.125 0.06 0.25 MIC g ml1 Rate ↑ 90% killed 165 min WT 58 % 70 min 113A 33 % 110 min 121A

• PIR and FEP have a similar kill rate
• 113A and 121A residues influence flux of FEP and PIR through ompF equally

4th generation cephalosporin MW 514.58 4th generation cephalosporin MW 480.56

• 0.125

0.06 0.5 MIC g ml1 20 % 53 % Rate ↑ 120 min 70 min 150 min 90% killed 121A 113A WT 0.03 0.03 0.06 MIC g ml1 28 % 1 Rate ↑ 65 min 25 min 90 min 90% killed 121A 113A WT

• ERT kill rate is much faster (influx rate, stability + target affinity)
• !! and 121A residues influence flux of ERT through OmpF much more than

cephalosporins

• 4th generation

cephalosporin MW 480.56 Carbapenem MW 475.5

Conclusions from Project 3

• Mutation at residues )!! and D121A results in faster

translocation of βlactams through OmpF

• Ertapenem action is much faster than cephalosporins due to

through OmpF2 / and increased / against degredation. The 113A mutation has a more dramatic effect.

• Can molecular dynamics modeling of βlactam translocation

through WT and mutated OmpF back up this data?

• If so, we may have a powerful tool for
• Identifying the crucial residues involved in drug transport

through porin channels

• Predicting the efficacy of transport of new molecules

through porin channels

What’s Next?

Purified porins Omp36, 35, 37 and OmpF, Omp36 mutants will allow us to study the specific role of each porin in antibiotic resistance. This will elucidate the relationship between specific differences in porin eyelet structure and functional activity Such investigations will be carried out working with other RTN partners:

• Cellular biology and activity
• Insert purified porins into artificial membranes (Bremen)
• Insert purified porins into liposomes (Porto)
• Omp purification and Antibiotic susceptibility studies (Marseille)
• Testing new molecules (Basel)
• Molecular Dynamics
• Mathematical predictions of different porin – drug interactions based
• n sequence info (Cagliari)

• An understanding of the regulation of membrane permeability will provide

insight into new global targets for drug design

The Big Picture

• Exploring dialogue between / and

will aid design of smart drugs that efficiently travel through porins for rapid delivery to target sites.

• Exploring dialogue between / and antibiotic

will tell us how bacteria respond to antibiotic therapy:

• changes in porin profiles?
• mutations leading to porin constriction to deny them access?

Merci!

34567 JeanMichel Bolla Myrielle Dupont Alexander Molitor Laurent Mamelli Anne Favard 58 Mahendran K