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Drug Entry & Exit

Drug Entry & Exit. Impact on Antibiotic Resistance. Gram-negative outer membrane structure. Entry of antibiotics to the periplasmic space through porins . Efflux pumps in both Gram-negative and Gram-positive bacteria. Related antibiotic resistance.

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Drug Entry & Exit

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  1. Drug Entry & Exit Impact on Antibiotic Resistance

  2. Gram-negative outer membrane structure. Entry of antibiotics to the periplasmic space through porins. • Efflux pumps in both Gram-negative and Gram-positive bacteria. • Related antibiotic resistance.

  3. Escherichia coliGram-negative membrane structure PM = plasma membrane PG = peptidoglycan layer OM = outer membrane

  4. Schematic representation of the E.coli envelope adapted from Raetz and Withfield with minor modifications (Raetz and Whitfield, 2002). Abbreviations: LPS, lipopolysaccharide; MDO, membrane-derived oligosaccharides; Kdo, 3-deoxy-d-manno-oct-2-ulosonic acid; PPEtn, phosphoethanolamine http://edoc.hu-berlin.de/dissertationen/kubelt-janek-2004-04-16/HTML/chapter1.html

  5. Porins of Gram-Negative Bacteria • In Escherichia coli, the porins formed under normal laboratory conditions allow the passage of hydrophilic molecules provided their molecular weight is no more than about 600 Daltons. These non-specific channels allow access of low molecular weight nutrients also.

  6. Porins of Gram-Negative Bacteria • The porins reside in the membrane as trimers, each trimer probably forming a single pore. The trans-membrane structure involves b-strands interacting with the lipid interior of the membrane, whilst also forming the hydrophilic channel. Uptake of molecules through the non-specific channels is by simple diffusion.

  7. Porins of Gram-Negative Bacteria • The numbers of different porins and their exclusion properties vary from one bacterium to another. A particular bacterium may have a mix of non-specific and specific porins. • e.g. Pseudomonas aeruginosa has a general non-specific porin (OprF) and a porin relatively specific for gluconate, basic amino acids and peptides, etc.(OprD) which have been implicated in antibiotic resistance. It also has several other specific porins, e.g. OprB, specific for monosaccharide.

  8. Porins of Gram-Negative Bacteria • Alteration in the production or properties of a porin may lead to antibiotic resistance. Any chromosomal mutation responsible must still allow sufficient uptake of nutrients to occur for growth. • Possible types of mutations: a) change of protein sequence to alter character of channel; b) a change in the regulation of gene expression (leading to e.g. a 'new' porin); c) a reduction in porin synthesis, resulting in fewer channels per cell (perhaps as a result of b) above).

  9. Porins of Gram-Negative Bacteria • Qualitative changes in outer membrane permeability of Gram-negative bacteria result in a decrease in sensitivity towards an antibiotic - often towards several antibiotics belonging to different structural classes. .. 'Less sensitive' ……........ 'Resistant' • The severity of resistance may depend on how close the MIC for the 'normal', parental strain is to the concentration of the antibiotic achievable at the site of the infection.

  10. Porins of Gram-Negative Bacteria • Neisseria gonorrhoeae mutants have been found which exhibit low-level resistance to penicillin G and tetracycline. The principal OMP* disappears from SDS-PAGE* of the outer membrane and a new band appears. • Serratiamarcescens mutants have been found which exhibit an increase in MIC's to a range of cephalosporins and aminoglycosides. This is associated with a change in pattern of the SDS-PAGE outer membrane proteins. • * OMP = outer membrane protein • * SDS = sodium dodecyl sulphate • * PAGE = polyacrylamide gel electrophoresis

  11. Porins of Gram-Negative Bacteria • lmipenem resistance in Pseudomonas aeruginosa has been reported in which one OMP has disappeared from the usual SDS-PAGE OMP pattern for this bacterium. OprD presumably recognises imipenem as similar to its ‘natural’ substrates for passage.

  12. Porin mutations, together with other permeability changes, may cause substantial changes in antibiotic sensitivity in a strain.

  13. Kaczmarek FM, Dib-Hajj F, Shang WC, et al.High-level carbapenem resistance in a Klebsiella pneumoniae clinical isolate is due to the combination of bla(ACT-1) beta-lactamase production, porin OmpK35/36 insertional inactivation, and down-regulation of the phosphate transport porin PhoE ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 50 (10): 3396-3406 OCT 2006

  14. Shafer WM, Folster JPTowards an understanding of chromosomally mediated penicillin resistance in Neisseria gonorrhoeae: Evidence for a porin-efflux pump collaboration JOURNAL OF BACTERIOLOGY 188 (7): 2297-2299 APR 2006

  15. Efflux Pumps • Multidrug resistance pumps operate in many different cell types. • For instance, the human P-glycoprotein pump confers drug resistance to certain tumour cells by pumping out chemotherapeutic agents. • A pump may expel a broad range of structurally-unrelated substances, but not cellular metabolites!

  16. Is hydrophobicity a characteristic for recognition by a pump? ciprofloxacin chloramphenicol tetracycline

  17. Benzyl penicillin (penicillin G)

  18. MULTIDRUG RESISTANCE(EFFLUX) PUMPS (MDRs) • MDRs WERE FIRST DISCOVERED IN MAMMALIAN CELLS IN THE MID-1980s. THEY INVOLVE PROTEINS CODED FOR BY GENES.

  19. MULTIDRUG RESISTANCE(EFFLUX) PUMPS (MDRs) • MDRs SEEM TO BE ABLE TO DISTINGUISH BETWEEN HYDROPHOBIC TOXINS (e.g. ANTIBIOTICS) AND HYDROPHILIC CELLULAR COMPONENTS (e.g. NUTRIENTS).

  20. MULTIDRUG RESISTANCE(EFFLUX) PUMPS (MDRs) MAMMALIAN MDR PUMPS USE ATP AS THE SOURCE OF ENERGY FOR DRUG EXTRUSION FROM THE CELL. BUT A WIDE RANGE OF OTHER SUBSTANCES CAN ALSO BE EXPORTED . MANY MICROBIAL MDRs ARE ALSO NOW KNOWN.

  21. MULTIDRUG RESISTANCE(EFFLUX) PUMPS (MDRs) A FEW USE ATP, e.g.Plasmodium falciparum, Candida albicans, and Lactococcuslactis (A RARE BACTERIAL EXAMPLE). KNOWN AS ABC TYPE (ATP-BINDING CASSETTE). OTHER MICROBIAL PUMPS USE THE ENERGY OF THE PROTON MOTIVE FORCE (pmf).

  22. MULTIDRUG RESISTANCE(EFFLUX) PUMPS (MDRs) THESE pmf-TYPE PUMPS FALL INTO 3 WELL-DEFINED FAMILIES:

  23. 1. MAJOR FACILITATOR SUPERFAMILY (MFS) THIS TYPE OF PUMP IS FOUND IN A WIDE RANGE OF BACTERIA - e.g. E. coli, B. subtilis, Staph. aureus, Haemophilusinfluenzae, Mycobacterium smegmatis. GENES HOMOLOGOUS TO THOSE FOUND IN Mycobacterium smegmatis HAVE ALSO BEEN FOUND IN Mycobacterium tuberculosis ANDMycobacterium avium.

  24. RESISTANCE TO A WIDE RANGE OF ANTIBIOTICS CAN BE CONFERRED, e.g. CHLORAMPHENICOL, QUINOLONES, TETRACYCLINE, RIFAMPICIN. VARIOUS GENES ARE INVOLVED IN DIFFERENT SPECIES. SOME ARE CARRIED ON PLASMIDS AND SOME ARE INTRINSIC.

  25. 2. SMALL MULTIDRUG RESISTANCE (SMR) FOUND IN A MORE RESTRICTED RANGE OF BACTERIA THAN MFS, e.g. Klebsiella pneumoniae, E. coli, Staph. aureus. AGAIN, SOME ARE CARRIED ON PLASMIDS AND SOME ARE INTRINSIC.

  26. 3. RESISTANCE NODULATION DIVISION (RND) FOUND ONLY IN GRAM-NEGATIVE BACTERIA, e.g. E. coli, Pseudomonas aeruginosa, Neisseria gonorrhoeae.

  27. MDR PUMPS AND OTHER MECHANISMS HELP EXPLAIN THE HIGH INTRINSIC RESISTANCE OF SOME BACTERIA, e.g. Pseudomonas aeruginosa.

  28. THIS IS THOUGHT TO BE DUE TO A VERY TIGHT OUTER MEMBRANE BARRIER AND A PUMP THAT TRANSPORTS ERYTHROMYCIN, CHLORAMPHENICOL, TETRACYCLINE, QUINOLONES AND OTHER ANTIBIOTICS ACROSS THIS BARRIER AND OUT OF THE CELL.

  29. MDR PUMPS ALSO EXPLAIN THE RESISTANCE OF CLINICAL ISOLATES OF Neisseria gonorrhoeae TO HYDROPHOBIC AGENTS SUCH AS ERYTHROMYCIN AND NEOMYCIN.

  30. THE END

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