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Mission:

Marie Curie Meeting 9 th -11 th April 2008. Mission:. To gain further understanding of the molecular aspects of antibiotic transport through porins and the emergence of drug resistance through porin mutation and regulation mechanisms. Chlo ë E. James and Jean-Marie Pag è s.

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Mission:

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  1. Marie Curie Meeting 9th-11th April 2008 Mission: To gain further understanding of the molecular aspects of antibiotic transport through porins and the emergence of drug resistance through 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 Enterobacteriaceae) • Tightly regulated • Rapid response to environment • Antibiotic Resistance •  level / type of porins expressed • Express mutated porin • Modulation of porin activity

  3. Escherichia coli Enterobacter aerogenes Pathogenic; commensal; lab tool Environmental; opportunistic; MDR nosocomial infections Well characterized model system (Regulation; Crystal structure; Liposome / Bilayer studies; Modelling) MDR within 5 days antibiotherapy Susceptibility and porin synthesis restored after treatment OmpF low osmolarity OmpC high osmolarity OmpN (quiescent?) Omp 35 dominant in vitro Omp 36 dominant in vivo Omp 37 (quiescent?)

  4. Resistance Strategies associated with porin modification: Escherichia coli MDR E. aerogenes • Clinical studies •  Expression of Omp36 • Switch from Omp35 to Omp36 • Mutation in Omp36 L3 loop • in vitro studies • Antibiotic stress induces rapid  in OmpF • Antibiotic flux is faster through OmpF than OmpC (smaller channel and electrostatic diffs) • L3 loop mutations  structural or electrostatic change; alter flux

  5. Projects 1) Role of E. aerogenes 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. omp insert (1 kB) BamHI HindIII PstI 0,125 2 4 g/ml 0,5 8 16 32 1 Project 1 – Omp36 Transport Step 2. Step 1. Clone Omp36 & A from E. aerogenes ATCC and express on the OM of porin-free E. coli Purify Omp36 by FPLC using a selective detergent protocol with octyl-POE and AKTA Pharmacia apparatus Step 4. Step 3. (Mahendran – Jacobs University) Assess the capability of Omp36 to increase susceptibility to various antibiotics using MIC assays Insert purified porins into lipid bilayer model Analyse channel-drug interactions by observing fluctuations in ion currents through Omp36 in the presence of antibiotics

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

  8. FPLC Ion-exchange chromatography: 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

  9. Β-lactam interaction with Omp36 Bi-layer Experiments (work by K. Mahendran (Jacobs University) No antibiotic 25mM cefepime 10mM ertapenem 25pA 25ms 25mM ampicillin 25mM ceftazidime 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 E. coli expressing Omp36 or OmpA (repeated 3-5 times) 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 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 E. coli sensitivity to ertapenem and cefepime and electrostatic data shows their strong interaction with the channel • Hypothesis:ERT translocates Omp36 more frequently than FEP due to stronger affinity with an interaction site inside the channel • Varying sensitivities to -lactamase degredation and target affinities means that influx rates of different -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 et al. submitted to EMBO Reports

  12. Project 2 – OMP regulation in response to stress OmpX:18 kDa enterobacterial OMP 8 stranded antiparallel β-barrel. Over-expression in E. aerogenes: 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. ompF * ATG * Transcriptional start site Project Strategy Does OmpX play a role in porin regulation in response to external stresses? 1. Construct ompF transcriptional and translational lacZ fusions 3. Expose cultures to sub-inhibitory external stress conditions and measure -galactosidase activity at 30 min time intervals pFus2K pFus2K Transcriptional fusion Translational fusion -gal activity reflects  ompF expression in response to stress compared to normal growth conditions 2.Transform constructs into E. coli parent and marA- or ompX- mutants t = 60 90 120 Add substrate to sample and measure OD

  14. % Decrease in ompF expression in response to 90 min stress Transcription Transcriptional regulation is independent of OmpX Down-regulation of OmpF transcription is partially dependent on MarA Translation n = 10 P < 0.05 SD 2-20 % Translational regulation is independent of OmpX Down-regulation of OmpF translation is dependent on MarA (micF action) Novobiocin may induce a Mar independent regulation pathway

  15. Conclusions from Project 2 • OmpF expression is decreased 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 translation (micF action) • An alternate regulatory pathway may be induced by novobiocin • No detected role for ompX in regulation of ompF expression at transcriptional and translational levels • Dupont M, James C E et al., 2007 AAC (9) 3190-8 • 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 et al., 2007 PLoS one 2(4): e365

  16. F4 Project 3 – Effect of OmpF L3 loop mutations on rate of antibiotic influx 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 ompF mutants

  17. ompF BamHI HindIII 0,125 2 4 g/ml 0,5 8 16 32 1 Methodology Assess the effect of OmpF mutations on the rate of antibiotic activity. 1. Clone ompF and D113A; D121A mutant genes into expression vector and express in porin-null strain BL21omp 3. Expose cultures to -lactams (2x MIC)and record decrease in cfu at 30 min time intervals. 2.Determine the MIC for various -lactams on BL21omp expressing WT and mutated OmpF * D113A WT D121A MIC x2

  18. 0.01 0.01 0.001 0.001 0.0001 0.0001 4th generation cephalosporin MW 514.58 4th generation cephalosporin MW 480.56 • PIR and FEP have a similar kill rate • 113A and 121A residues influence flux of FEP and PIR through ompF equally

  19. 0.01 0.01 0.001 0.001 0.0001 0.0001 4th generation cephalosporin MW 480.56 Carbapenem MW 475.5 • ERT kill rate is much faster (influx rate, stability + target affinity) • 113A and 121A residues influence flux of ERT through OmpF much more than cephalosporins

  20. Conclusions from Project 3 • Mutation at residues D113A and D121A results in faster translocation of -lactams through OmpF • Ertapenem action is much faster than cephalosporins due to more rapid transport through OmpF,increased target binding affinity and increased stability against degredation. The 113A mutation has a more dramatic effect. • Can molecular dynamics modeling of -lactam 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

  21. 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 andAntibiotic susceptibility studies (Marseille) • Testing new molecules (Basel) • Molecular Dynamics • Mathematical predictions of different porin – drug interactions based on sequence info (Cagliari)

  22. The Big Picture • Exploring dialogue between antibiotic and channel • will aid design of smart drugs that efficiently travel through porins for rapid delivery to target sites. • Exploring dialogue between bacteria and antibiotic therapy • will tell us how bacteria respond to antibiotic therapy: • changes in porin profiles? • mutations leading to porin constriction to deny them access? • An understanding of the regulation of membrane permeability will provide insight into new global targets for drug design

  23. Merci! Jean-Marie Pagès Jean-Michel Bolla Myrielle Dupont Alexander Molitor Laurent Mamelli Anne Favard Mathias Winterhalter Mahendran K

  24. vector control No IPTG control OmpA control Strategy for Omp Expression and Purification Porin null DE3 BL21 omp pColdIV(omp36) ompF- ompC- ompA- lamB- IPTG induce at 15 oC 24 h Omp36 expression Extract Outer Membrane Fraction MIC assays +/- -lactamase inhibitors Purification using octyl-POE solubilization and FPLC with AKTA Pharmacia apparatus

  25. Solubilisation of Omp36: Solubilisation of Omp36: Varying POE% does not improve solubilisation 1M NaCl improves solubilisation of Omp36 T S I S I OM - + - + - + S soluble I insoluble 55 kDa 55 kDa Omp36 34 kDa 34 kDa Omp36 + heat 95°C - no heat OM +1M NaCl 1 % POE 3 % POE 1 % POE HiTrap Desalting solubilised protein: 37 °C improves solubilisation of Omp36 S I S I S I S I 1) Protein eluted in fractions 1-3 55 kDa 2) Salt began to elute in fraction 3 34 kDa Omp36 1 % POE, 1M NaCl 1 37 °C (1) (2) RT (3) 2

  26. FPLC Ion-exchange chromatography: FPLC ion exchange chromatography:

  27. Extra data

  28. ompF BamHI HindIII 0,125 2 4 g/ml 0,5 8 16 32 1 Methodology 4. Expose strains to 2x MIC -lactams and plate dilutions at time 30 min intervals 1. Clone ompF and D113A; D121A mutant genes into expression vector and express in porin-null strain BL21omp * t = 60 t = 0 t = 30 1:103 3. Expose BL21omp expressing WT or mutated OmpF to 2x MIC -lactams andUse Miles Misra plating at time intervals to determine dilution factors for analysis of killing rate of -lactams MIC x2 2.Determine the MIC for various -lactams on BL21omp expressing WT and mutated OmpF neat 1:10 1:105 1:102 1:104 1:103

  29. 4th generation cephalosporin: MW 514.58 3rd generation cephalosporin: MW 546.58 • CAZ and FEP have a similar kill rate • 113A and 121A residues influence flux of FEP through ompF more than CAZ

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