antibiotic translocation marsielle meeting... · k.r. mahendran, t. mach, a. bessonov, h. weingart,...
TRANSCRIPT
![Page 1: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/1.jpg)
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
![Page 2: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/2.jpg)
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
![Page 3: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/3.jpg)
Motivation
Antibiotic
Target
Main resistance mechanisms to drugs
Yim 2007, modified
![Page 4: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/4.jpg)
Gram negative bacteria
![Page 5: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/5.jpg)
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
![Page 6: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/6.jpg)
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
![Page 7: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/7.jpg)
conductance
time
conductance
time
Artificial lipid bilayer measurements
Antibiotic interacts with porin resulting
in transient blockage of ionic current
![Page 8: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/8.jpg)
Cephalosporins pathway through OmpF
• Target: the bacterial cell wall
• Fourth generation
Cefpirome +/- Cefepime +/-
![Page 9: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/9.jpg)
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
![Page 10: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/10.jpg)
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
![Page 11: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/11.jpg)
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
![Page 12: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/12.jpg)
Microbiological assays
Lipid bilayer measurements correlates with microbiological assays
mean lysis
diameter (mm)
Bredin et al, 2002; Vidal et al, 2005
![Page 13: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/13.jpg)
Pathway of fluroquinolones through OmpF
• Fluroquinolones- target DNA
• Enrofloxacin
• zwitterionic
![Page 14: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/14.jpg)
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
![Page 15: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/15.jpg)
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
![Page 16: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/16.jpg)
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)
![Page 17: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/17.jpg)
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
![Page 18: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/18.jpg)
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
![Page 19: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/19.jpg)
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
![Page 20: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/20.jpg)
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
![Page 21: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/21.jpg)
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
![Page 22: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/22.jpg)
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
![Page 23: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/23.jpg)
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
![Page 24: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/24.jpg)
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)
![Page 25: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/25.jpg)
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
![Page 26: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/26.jpg)
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
![Page 27: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/27.jpg)
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
![Page 28: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/28.jpg)
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
![Page 29: antibiotic translocation marsielle meeting... · K.R. Mahendran, T. Mach, A. Bessonov, H. Weingart, M. Winterhalter Jacobs University, Bremen 2008. ... Antibiotic translocation is](https://reader034.vdocuments.us/reader034/viewer/2022042023/5e7ba1cb90a1885a894fcd03/html5/thumbnails/29.jpg)
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)