facilitated antibiotic translocation through and porins
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Facilitatedantibiotictranslocationthrough and porins K.R.Mahendran,T.Mach,A.Bessonov,H.Weingart,M.Winterhalter JacobsUniversity,Bremen


  1. Facilitated�antibiotic�translocation�through� ������� and� ������������ porins K.R.�Mahendran,�T.�Mach,�A.�Bessonov,�H.�Weingart,��M.�Winterhalter Jacobs�University,�Bremen 2008

  2. 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 • Prospects0 new�structure�development

  3. Motivation Main�resistance�mechanisms�to�drugs Antibiotic Target Yim 2007,�modified

  4. Gram�negative�bacteria

  5. Bacterial�Porins • Specific�and�non0specific�channels • ������� 3�major�porins�(OmpF,�OmpC�and�PhoE) • OmpF�plays�important�role�in�antibiotic�translocation OmpF�2OMPF OmpC��2J1N

  6. Artificial�lipid�bilayer�measurements • Electrophysiology � Ion�channels,�bacterial�porins,�toxins�etc • Current�measurement0 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� ��������������������

  7. Artificial�lipid�bilayer�measurements ����������� ���� ����������� Antibiotic�interacts�with�porin�resulting in�transient�blockage�of�ionic�current ����

  8. Cephalosporins�pathway�through�OmpF • Target:�the�bacterial�cell�wall • Fourth�generation Cefpirome���+/0 Cefepime�+/0

  9. Time�resolved�ion�current�blockages�through�OmpF OmpF��no�antibiotic OmpF�05mM�cefepime 200 200 150 150 current,�pA current,pA 100 100 � 50 50 0 0 0 1 2 3 4 5 1 2 � 3 4 5 time,s time,s OmpF0�10mM�cefepime� OmpF��10mM�cefpirome 200 200 150 150 current,�pA current,pA 100 100 � 50 50 0 0 0 1 2 3 4 5 0 1 2 3 4 5 time,s � time,s � Conditions:�1M�KCl,�pH6,�antibiotic�added�at�trans�side,�V�=�+�50�mV

  10. Binding�kinetics�OmpF0 cefepime Two�quantities�are�measured� 1)Number�of�blockages 2)Average�time�of�blockage �� Single�exponential�fitting�of�blockage�tim e�histogram �cis�side�+50mV �cis�side�050mV 10mM�cefepime �trans�side�+50mV 1000 Number�of�events �� �trans�side�050mV Tau=�0.12± 0.02ms IM�KCl�pH6,�10mM�Cefepime Count�(N) �� � 500 �� 0 � 0 0.5 1 1.5 � � �� �Dwell�Tim e�(m s) Cefepime��(mM) �

  11. Binding�kinetics�OmpF0 cefepime At��10mM�cefepime� Average�residence�time,�Tau=�0.12± 0.02ms Association�rate�constant�Kon=1868�M 01 s 01 Dissociation�rate�constant�Koff=9523.8s01 Antibiotic�translocation�is�facilitated�by�binding�site�in�the� channel

  12. Microbiological�assays mean�lysis� diameter�(mm) Lipid�bilayer�measurements�correlates�with�microbiological�assays Bredin et�al,�2002;�Vidal�et�al,�2005

  13. Pathway�of�fluroquinolones�through�OmpF • Fluroquinolones0 target�DNA • Enrofloxacin • zwitterionic

  14. An�example�of�strong�interaction0 ������ OmpF�and� Enrofloxacin ������� ���� enrofloxacin Τau�050�mV=�2.98±0.56�msec ������ ������� Τau��50�mV=�1.82±0.4�msec Conditions:�150�mM�KCl,�pH6,�antibiotic�added�at�cis�side,�V�=�0 50�mV

  15. Binding�Kinetics Enrofloxacin�blocking�events Ampicillin�blocking�events Conditions:�150�mM�KCl,�pH6 Conditions:�1�M�KCl,�pH6 ν ,� ev/sec 1200 0�50�mV 900 600 300 50�mV 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 ≈10�ev/sec�at�3�mM,�V=50�mV� C[Enro],�mM ≈20�ev/sec�at�3�mM,�V=050�mV ≈65�ev/sec�at�3�mM,�V=50�mV Nestorovich�et�al,�2002 ≈720�ev/sec�at�3�mM,�V=050�mV

  16. Antibiotic�translocation�through�OmpC Trimer�forms�smallest�functional�unit OmpC� � 60%�identity�in�amino�acid�sequence�to�OmpF � Key�residues�conserved,�different�positions� (Basle� ������� 2006) � More�acidic�residues�and�cation selective�compared�to�OmpF � Liposome�swelling�assays�(Nikaido�and�Rosenburg�1985)

  17. Antibiotic�translocation�through�OmpC Single�channel����������� Critical�voltage�for� Ionic�selectivity� Porin Conductance(nS) Channel�closure(mV) P K/Cl 1M�KCL�pH�6 4 1000150 OmpF 204 OmpC 2.5 2000250 7030 Cefepime����0 Fourth�generation�+/0 Ceftriaxone�0 Third�generation��02 Norfloxacin�– Fluroquinolone��+/0

  18. Time�resolved�ion�current�blockages�through�OmpC OmpC�no�antibiotic OmpC�10mM�Cefepime 50pA 50pA 1s 1s OMPC�10mM�CEFTRIOXONE OmpC�2mM�NORFLOXACIN 50pA 50pA 1s 1s Conditions:�1M�KCl,�pH6,�antibiotic�added�at�cis side,�V�=�+�100�mV

  19. Determination�of�binding�parameters Power�Spectrum�OmpC0�Norfloxacin 0.1 ����������������� 3mM�norfloxacin Amplitude�(pA²�/�Hz) 0.01 2mM�norfloxacin 1mM�norfloxacin 0.001 0mM�norfloxacin 1e04 100 1000 10000 Frequency�(Hz)

  20. 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

  21. Antibiotic�translocation�through Omp36 • ������������ possesses�three�known�porins • Omp35�( ������ OmpF,�OmpK35�homolog) • Omp36�( ������� OmpC,�OmpK36�homolog) • Omp37 Antibiotics • Ertapenem,�cefepime ,�ceftazidime�and�ampicillin

  22. Binding�kinetics�Omp36 ν,� s 01 30 250 Number�of�events�per�second trans0side; �cis�side�050�m V Number�of�events�per�second V=�050�mV �cis�side�+50�m V 25 �trans�side�050�m V 200 �trans�side�+50m V trans0side; 20 V=�+50�mV 150 15 � � 100 cis0side; 10 V=�+50�mV 50 5 cis0side; V=�050�mV 0 0 0 2 4 6 8 10 0 5 10 15 20 25 Ertapenem �concentration,�(m M ) � cefepime,�mM

  23. Binding�kinetics�of�Omp36 ���� 10mM�ertapenem �cis�ertapenem trans�ertapenem A m p litu d e �( p A ² �/�H z ) cis�and�trans�ertapenem Average�residence�time,�ms ���� 0.01 ���� 25mM�cefepime � 0.001 ���� No�antibiotic ���� 100 1000 10000 ���� ��� � �� ��� Frequency�(Hz) � Applied�voltage,�m V Tau�ertapenem�0.14±0.02ms Tau�cefepime����0.1±0.02ms Bilayer�measurements�correlated�with�antibiotic�activities�in�bacteria� (Chloë E.�James)�

  24. Temperature�effect�on�antibiotic� translocation�through�OmpF • OmpF�highly�stable. • New�parameter • OmpF�conductance�strongly�depends�on�temperature • Ampicillin�and�Norfloxacin�(Catalin) • Temperature (5055� 0� C)

  25. Ion�current�blockages�at�different�temperatures Open�current current,pA Closed�pore�current 5 0 c t i m e , m s 15 0 c current,pA t i m e , m s 25 0 c current,�pA 2 5 p A 10mM�ampicillin,1M�KCl�pH�6,+50mV t i m e , m s 2 5 m s

  26. 35 0 c current�pA t i m e , m s current,pA 45 0 c t i m e , m s 55 0 c current,pA t i m e , m s 2 5 p A 2 5 m s

  27. Determination�of�binding�parameters Dependence�of�residence�time�on�temperature Dependence�of�Ampicillin0OmpF�binding�events��on�temperature 1.2 12 10mM�Ampicillin�cis�side� ��cis�side�+50mV IM�KCl�pH�6��+50mV �trans�side�+50mV 1.0 10 10mM�ampicillin�pH�6 Residence�time,ms 0.8 Number�of�events�per�second 8 0.6 6 � � 0.4 4 0.2 2 0.0 0 0 10 20 30 40 50 60 0 10 20 30 40 50 60 Temperature�(c) Temperature�C � � Chemical�rate�constants�(kon�and�koff)�were�determined� Depends�on�temperature

  28. Prospects � � � � We�have�been�able�to�measure�time0resolved�single0 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�measurements and�microbiological�assays�compared�with�molecular dynamics�simulations�. � Miniaturation�(Nanion) � Development�of�new�antibiotics�with�high�translocation efficiency�in�future. Nestorovich�et�al,�2002

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