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Gijs van der Schot Simone Wanningen

Gijs van der Schot Simone Wanningen. Bacteriophages. Bacteriophages. Bacteriophages. Host cell lysis. Large double stranded DNA phages: Employ an invariable holin Make use of endolysin Single stranded nucleic acid bacteriophages: Expression of single gene No muralytic enzyme needed

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Gijs van der Schot Simone Wanningen

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  1. Gijs van der Schot Simone Wanningen

  2. Bacteriophages

  3. Bacteriophages

  4. Bacteriophages

  5. Host cell lysis • Large double stranded DNA phages: • Employ an invariable holin • Make use of endolysin • Single stranded nucleic acid bacteriophages: • Expression of single gene • No muralytic enzyme needed • Example: Gene E from MicroviridaeΦX174

  6. Gene E from ΦX174 • Encodes a membrane protein of 91 residues • α-helical shape • Causes lysis of several Gram-negative hosts • Protein E causes lysis by inhibiting MraY

  7. G M G G A A A A A A E E E E E E K K K K K K A A A A A A A A A A A A Lipid I M M MraY MurG Lipid II M M G G UDP M UDP MraY Lipid II out in

  8. G M G G A A A A A A E E E E E E K K K K K K A A A A A A A A A A A A Lipid I M M MraY MurG Lipid II M M G G UDP M UDP MraY and E Lipid II out in

  9. G M G G A A A A A A E E E E E E K K K K K K A A A A A A A A A A A A Lipid I M M MraY MurG Lipid II M M G G UDP M UDP MraY and E Lipid II out in

  10. MraY catalyzes formation of Lipid I Phytol Phosphate

  11. Mechanism Inhibition MraY (I) • Mutations in MraY lead to E-resistance • MraY from Bacillus suptilis is resistant (BSMraY)

  12. Mechanism Inhibition MraY (II) • Two models explaining Inhibition: • E affects functioning MraY directly • E affects functioning MraY indirectly (i.e. assembly heteromultimeric complex) • Epep fragment contains 37 N-terminal residues: • Lysis of membrane containing overexpressed MraY • No lysis in detergent-solubilized membranes

  13. In this article/study: • First purifiction of full-length E-protein • Characterization of the ability of E-protein to inhibit MraY

  14. Overproduction of E6his • Induction E allele lethal

  15. Overproduction of E6his • Induction E allele and BsMraY overcomes lethality

  16. Purification of E6his • Yield of extracted protein: 54uM, 84% pure

  17. Quantification of E6his in vivo • Previous indirect in vivo approaches: • ~100-300 molecules/cell • ~1000 molecules/cell • This study used purified E6his • ~500 molecules/cell • We think: • ~750 molecules/cell

  18. Fluorescent analysis of MraY Substrates used: • UDP-MurNAc-pentapeptide-DNS • Phytol-P • Fluorescent labeled product: • Phytol-P-P-MurNAc-pentapeptide-DNS

  19. Michaelis-Menten kinetics V0 = Initial reaction rate VMax = Maximum rate KM = Michaels constant [S] = substrate concentration

  20. Determination of Km values Al-Dabbagh et al. (ref 27): C55-P – 0.2 mM UM5 – 0,94 mM E resistance is not due to an altered substrate affinity

  21. E-mediated inhibition of MraY (I) • E inhibits MraY specifically when both are present in same membrane

  22. Reversible Inhibitors

  23. E-mediated inhibition of MraY (II) Km parameters for both substrates unchanged in presence of E Vmax in both substrates decreased in presence of E E is a non-competitive inhibitor of MraY with respect to both lipid and sugar-nucleotide substrates • Ki averages of 0,53 +/- 0,12 uM

  24. Sensitivity of MraY mutant alleles • Ability of E to inhibit the MraY proteins form the 5 mutant alleles • 5 mutants in 3 classes: • MraYG186S and MraYV291M • MraYp170L and MraY∆L172 • MraYF288L • Matches classes of apparent affinities

  25. Conclusions • Overproduction of protein E achieved • Possible to do structural and biophysical characterization of E • E acts as a non-competitive inhibitor with respect to both lipid and sugar-nucleotide substrates of MraY

  26. New model: Inhibition by direct binding • Interaction of one TMD of E and TMD 5 and 9 of MraY • Non-competitive binding results in conformational change

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