Gijs van der Schot
This presentation is the property of its rightful owner.
Sponsored Links
1 / 26

Gijs van der Schot Simone Wanningen PowerPoint PPT Presentation


  • 79 Views
  • Uploaded on
  • Presentation posted in: General

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

Download Presentation

Gijs van der Schot Simone Wanningen

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Gijs van der schot simone wanningen

Gijs van der Schot

Simone Wanningen


Bacteriophages

Bacteriophages


Bacteriophages1

Bacteriophages


Bacteriophages2

Bacteriophages


Host cell lysis

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


Gene e from x174

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


Gijs van der schot simone wanningen

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


Mray and e

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


Mray and e1

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


Mray catalyzes formation of lipid i

MraY catalyzes formation of Lipid I

Phytol Phosphate


Mechanism inhibition mray i

Mechanism Inhibition MraY (I)

  • Mutations in MraY lead to E-resistance

  • MraY from Bacillus suptilis is resistant (BSMraY)


Mechanism inhibition mray ii

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


In this article study

In this article/study:

  • First purifiction of full-length E-protein

  • Characterization of the ability of E-protein to inhibit MraY


Overproduction of e 6his

Overproduction of E6his

  • Induction E allele lethal


Overproduction of e 6his1

Overproduction of E6his

  • Induction E allele and BsMraY overcomes lethality


Purification of e 6his

Purification of E6his

  • Yield of extracted protein: 54uM, 84% pure


Quantification of e 6his in vivo

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


Fluorescent analysis of mray

Fluorescent analysis of MraY

Substrates used:

  • UDP-MurNAc-pentapeptide-DNS

  • Phytol-P

  • Fluorescent labeled product:

    • Phytol-P-P-MurNAc-pentapeptide-DNS


  • Michaelis menten kinetics

    Michaelis-Menten kinetics

    V0 = Initial reaction rate

    VMax = Maximum rate

    KM = Michaels constant

    [S] = substrate concentration


    Determination of km values

    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


    E mediated inhibition of mray i

    E-mediated inhibition of MraY (I)

    • E inhibits MraY specifically when both are present in same membrane


    Reversible inhibitors

    Reversible Inhibitors


    E mediated inhibition of mray ii

    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


    Sensitivity of mray mutant alleles

    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


    Conclusions

    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


    New model inhibition by direct binding

    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


  • Login