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Marie Curie Meeting 9 th 11 th April 2008 Mission: 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


  1. Marie Curie Meeting 9 th �11 th April 2008 Mission: 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. Chloë E. James and Jean�Marie Pagès

  2. 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 ������������ ������������� ���������� ������������������ ��������������� ���������������� �� !"�#�$�%

  3. ���������������� ���������������������� Pathogenic; commensal; lab tool Environmental; opportunistic; MDR nosocomial infections Well characterized model system MDR within 5 days antibiotherapy ( Regulation; Crystal structure; Susceptibility and porin synthesis Liposome / Bilayer studies; Modelling ) restored after treatment ���� �������������� ��� ��� ��������� �������� ������������������������� ��� ����������������� ������� ���� (quiescent?) ��� ��� (quiescent?)

  4. �������������������������������������� ������������������� ���������������� MDR ������������ ���������������� ��������� ������� • ↓ Expression of Omp36 • Antibiotic stress induces rapid ↓ in OmpF • Switch from Omp35 to Omp36 • Antibiotic flux is faster through OmpF than OmpC (smaller channel and electrostatic diffs) • L3 loop mutations → structural • Mutation in Omp36 L3 loop or electrostatic change; alter flux

  5. 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

  6. Project 1 – Omp36 Transport ��� insert (1 kB) ���� " . ���� ! . ��� HI ���� III ��� # Clone Omp �� & from �� ��������� ATCC and express on the OM of porin�free �� ���� Purify Omp36 by FPLC using a selective detergent protocol with octyl�POE and AKTA Pharmacia apparatus ���� $% ���� � . (Mahendran – Jacobs University) Assess the capability of Omp36 to increase susceptibility to various Insert purified porins into lipid bilayer model antibiotics using MIC assays Analyse channel�drug interactions by observing fluctuations in ion currents through Omp36 in the presence of antibiotics � g/ml 0,125 0,5 2 4 8 16 32 1

  7. Expression of cloned ��� genes �������� BL21 DE3 � ��� (pColdIV+ ����� +/� Induction: 1mM IPTG 24 h @ 15 o C � 36 36 A � � A Total cell + � + � + � � � � + + + + + � � � + + + + + 55 kDa 55 kDa 34 kDa 34 kDa OM 36 A � 36 A Antibodies: Omp36 = α f4 (12 aa peptide from L3 loop) 55 kDa 55 kDa (1:10,000) 34 kDa 34 kDa OmpA = α OmpA from 30 kDa �������� (1:1000)

  8. ���� ���������������������������� FPLC ion exchange chromatography: Solubilisation conditions: 1% POE; 1M NaCl; 37 o C; 1 h; x3 Purification conditions: 0.6 % POE; NaCl gradient -��������������34�5��,���������������+6 -��������������34�5��,���������������+6 -��������������34�5��,���������������+6 ,&���,,��,#���,� ,&���,,��,#���,� ,&���,,��,#���,� ,&����,,����,#���,� ,&����,,����,#���,� ,&����,,����,#���,� ,&���,,����,#����,� ,&���,,����,#����,� ,&���,,����,#����,� &'()����� &'()����� ���*����� ���*����� �++��+���� �++��+���� ,2��+� � �� �, ,2��+� � �� �, ,2��+� � �� �, α �0��������� α �0��������� α �0��������� ����������- -� ����������- -� ����������- -� -��*����������� -��*����������� -��*����������� �./� �./� �./� 1���������� 1���������� 1���������� - -��./� - -��./� - -��./� Purified porin stable for at least 1 week at 4, �20 and RT o C

  9. Β�lactam interaction with Omp36 Bi�layer Experiments (work by K. Mahendran (Jacobs University) 7������������ #4�$��������� ,&�$���������� #4�. #4�� #4�$����������� #4�$������8����� Ion current fluctuation due to transient channel blockages by antibiotic molecules • 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

  10. Antibiotic Sensitivity conferred by Omp36 MIC Assays MIC values ( � g ml –1 ) for porin null ������� expressing Omp36 Grow to OD 600 0.4 or OmpA (repeated 3�5 times) Vector IPTG ERT FEP � CTC FEP + CTC +/� 1 mM IPTG, 1 h pColdIV only +/� 4 4 2�4 Dilute to OD 600 0.001 pColdIV ��� �� � 4 4 2 pColdIV ��� �� + 0.5 4 0.5 Incubate with dilution series of drug +/� IPTG pColdIV ��� � + 4 8 4 (0.5 mM) and CTC CTC = Clavulanic acid, Tazobactam, Cloxacillin (4 � g ml �1 ) • Omp36 expression causes � 8�fold increase in sensitivity to ertapenem � 4�fold increase in sensitivity to cefepime (+ inhibitors) . • No Omp36 increase in sensitivity to cefotaxime observed

  11. 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 β �lactamase degredation and target affinities means that influx rates of different β �lactams 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

  12. Project 2 – OMP regulation in response to stress ���'� 18 kDa enterobacterial OMP 8 stranded antiparallel β�barrel. Over�expression in ������������ : Decreased Omp36 expression Decreased susceptibility to β�lactams Increased OmpX and decreased porins in clinical isolates OmpX expression is increased in response to external stress conditions Work by M. Dupont

  13. Project Strategy Does OmpX play a role in porin regulation in response to external stresses? ���� 1. Construct ���� transcriptional and * ATG * Transcriptional start site translational lacZ fusions 3. Expose cultures to sub�inhibitory external pFus2K pFus2K stress conditions and measure β �galactosidase activity at 30 min time intervals Transcriptional Translational fusion fusion ↓ β �gal activity reflects ↓ ���� 2. Transform expression in constructs into ��� t = 60 90 120 response to stress ���� parent and Add substrate to compared to normal ����� or ����� sample and growth conditions mutants measure OD

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