Facilitated antibiotic translocation through

E.coli and E. aerogenes porins

K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter

Jacobs University, Bremen

2008

Outline

• Artificial Bilayer measurements

• Antibiotic entry through bacterial porins

� OmpF (Cephalosporins and fluroquinolones )

� OmpC (Cephalosporins and fluroquinolones)

� Omp36 (Beta lactams)

• Effect of temperature on ampicillin passage

through OmpF

• Prospects- new structure development

Motivation

Antibiotic

Target

Main resistance mechanisms to drugs

Yim 2007, modified

Gram negative bacteria

Bacterial Porins

• Specific and non-specific channels

• E.coli 3 major porins (OmpF, OmpC and PhoE)

• OmpF plays important role in antibiotic translocation

OmpF 2OMPF OmpC 2J1N

Artificial lipid bilayer measurements

• Electrophysiology

� Ion channels, bacterial porins, toxins etc

• Current measurement- Ion flow

• BLM technique allows to see interactions between antibiotic and porin as time resolved fluctuations of ion current

• Substrate interacting with Pore

can produce long residence times

conductance

time

conductance

time

Artificial lipid bilayer measurements

Antibiotic interacts with porin resulting

in transient blockage of ionic current

Cephalosporins pathway through OmpF

• Target: the bacterial cell wall

• Fourth generation

Cefpirome +/- Cefepime +/-

Time resolved ion current blockages through OmpF

0

50

100

150

200

1 2 3 4 5

OmpF no antibiotic

time,s

current, pA

0 1 2 3 4 5

0

50

100

150

200

time,s

current, pA

OmpF- 10mM cefepime

0 1 2 3 4 50

50

100

150

200

time,s

current,pA

OmpF -5mM cefepime

0 1 2 3 4 5

0

50

100

150

200

time,s

current,pA

OmpF 10mM cefpirome

Conditions: 1M KCl, pH6, antibiotic added at trans side, V = + 50 mV

Binding kinetics OmpF- cefepime

0 5 10

0

10

20

30

40

cis side +50mV

cis side -50mV

trans side +50mV

trans side -50mV

IM KCl pH6, 10mM Cefepime

Cefepime (mM)

Number of events

Single exponential fitting of blockage time histogram

Dwell Time (ms)0 0.5 1 1.5

Count (N

)

0

500

1000

Two quantities are measured

1)Number of blockages

2)Average time of blockage

10mM cefepime

Tau= 0.12± 0.02ms

At 10mM cefepime Average residence time, Tau= 0.12± 0.02ms

Association rate constant Kon=1868 M-1s-1

Dissociation rate constant Koff=9523.8s-1

Antibiotic translocation is facilitated by binding site in the

channel

Binding kinetics OmpF- cefepime

Microbiological assays

Lipid bilayer measurements correlates with microbiological assays

mean lysis

diameter (mm)

Bredin et al, 2002; Vidal et al, 2005

Pathway of fluroquinolones through OmpF

• Fluroquinolones- target DNA

• Enrofloxacin

• zwitterionic

0.2 nS

0.4 sec

20 msec

Conditions: 150 mM KCl, pH6, antibiotic added at cis side, V = - 50 mV

2 mM

enrofloxacin

An example of strong interaction-E.coli OmpF and

Enrofloxacin

Τau -50 mV= 2.98±0.56 msec

Τau 50 mV= 1.82±0.4 msec

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

0

300

600

900

1200

50 mV

- 50 mV

C[Enro], mM

ν, ev/sec

Binding Kinetics

Nestorovich et al, 2002

Conditions: 150 mM KCl, pH6 Conditions: 1 M KCl, pH6

≈10 ev/sec at 3 mM, V=50 mV

≈20 ev/sec at 3 mM, V=-50 mV

≈65 ev/sec at 3 mM, V=50 mV

≈720 ev/sec at 3 mM, V=-50 mV

Enrofloxacin blocking events Ampicillin blocking events

Antibiotic translocation through OmpC

Trimer forms smallest functional unit

OmpC

� 60% identity in amino acid sequence to OmpF

�Key residues conserved, different positions

(Basle et al. 2006)

�More acidic residues and cation selective compared to OmpF

� Liposome swelling assays (Nikaido and Rosenburg 1985)

PorinSingle channel

Conductance(nS)

1M KCL pH 6

Critical voltage for

Channel closure(mV)

Ionic selectivity

PK/Cl

2-4

7-30

4

2.5

100-150

200-250

Cefepime - Fourth generation +/-

Ceftriaxone - Third generation -2

Norfloxacin – Fluroquinolone +/-

Antibiotic translocation through OmpC

OmpF

OmpC

Time resolved ion current blockages through OmpC

OmpC no antibiotic

1s

50pA

OmpC 10mM Cefepime

1s

50pA

OMPC 10mM CEFTRIOXONE

1s

50pA

OmpC 2mM NORFLOXACIN

1s

50pA

Conditions: 1M KCl, pH6, antibiotic added at cis side, V = + 100 mV

Power Spectrum OmpC- Norfloxacin

Frequency (Hz)100 1000 10000

Amplitude (pA² / Hz)

1e-4

0.001

0.01

0.1

0mM norfloxacin

1mM norfloxacin

2mM norfloxacin

3mM norfloxacinTau = 0.2 ±0.02ms

Determination of binding parameters

Determination of binding parameters

Residence time

Cefepime Tau 0.1 ± 0.02ms

Ceftriaxone Tau 0.14 ± 0.02ms

Norfloxacin Tau 0.2 ±0.02

Ampicilin no binding event

Strength antibiotic interaction with OmpC

Norfloxacin > ceftriaxone > cefepime > ampicillin

Antibiotic translocation through Omp36

• E.aerogenes possesses three known porins

• Omp35 (E.coli OmpF, OmpK35 homolog)

• Omp36 (E.coli OmpC, OmpK36 homolog)

• Omp37

Antibiotics

• Ertapenem, cefepime, ceftazidime and ampicillin

0 2 4 6 8 100

50

100

150

200

250

cis side -50 mV

cis side +50 mV

trans side -50 mV

trans side +50mV

Ertapenem concentration, (mM)

Number of events per second

Binding kinetics Omp36

0 5 10 15 20 25

0

5

10

15

20

25

30

trans-side;

V= -50 mV

cis-side;

V= -50 mV

trans-side;

V= +50 mV

cis-side;

V= +50 mV

ν, s-1

cefepime, mM

Number of events per second

Binding kinetics of Omp36

Frequency (Hz)100 1000 10000

Am

plit

ude (pA

² / H

z)

0.001

0.01

10mM ertapenem

25mM cefepime

No antibiotic

-150 -75 0 75 150

0.05

0.10

0.15

0.20

0.25

cis ertapenem

trans ertapenem

cis and trans ertapenem

Applied voltage, mVAverage residence tim

e, ms

Bilayer measurements correlated with antibiotic activities in bacteria

(Chloë E. James)

Tau ertapenem 0.14±0.02ms

Tau cefepime 0.1±0.02ms

Temperature effect on antibiotic

translocation through OmpF

• OmpF highly stable.

• New parameter

• OmpF conductance strongly depends on temperature

• Ampicillin and Norfloxacin (Catalin)

• Temperature (5-55 0 C)

2 5 m s

2 5 p At i m e , m s

current, pA

t i m e , m s

current,pA

t i m e , m s

current,pA

50c

150c

250c

Ion current blockages at different temperatures

Open current

Closed pore current

10mM ampicillin,1M KCl pH 6,+50mV

t i m e , m s

current pA 350c

t i m e , m s

current,pA

450c

550c

2 5 m s

2 5 p A t i m e , m s

current,pA

Determination of binding parameters

0 10 20 30 40 50 60

0

2

4

6

8

10

12

Temperature (c)

Number of events per second

10mM Ampicillin cis side

IM KCl pH 6 +50mV

Dependence of Ampicillin-OmpF binding events on temperature

0 10 20 30 40 50 60

0.0

0.2

0.4

0.6

0.8

1.0

1.2

cis side +50mV

trans side +50mV

10mM ampicillin pH 6

Dependence of residence time on temperature

Temperature C

Residence tim

e,m

s

Chemical rate constants (kon and koff) were determined

Depends on temperature

Prospects

���� We have been able to measure time-resolved single- molecule events of antibiotic entry into the pore, and obtain detailed kinetic information.

� High resolution conductance measurements correlated with antibiotic activities in bacteria (MIC assays)

� Experimental results from lipid bilayer measurementsand microbiological assays compared with moleculardynamics simulations .

� Miniaturation (Nanion)

� Development of new antibiotics with high translocation

efficiency in future.

Nestorovich et al, 2002

Acknowledgements

Group of Prof. Winterhalter

Prof. Jean-Marie Pagès

Chloë E. James

Group of Prof. Ceccarelli

Group of Prof.Paula Gameiro

Dr. Malcolm Page (Basilea)