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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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slide1
Gijs van der Schot

Simone Wanningen

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
slide7
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

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A

E

E

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E

E

E

K

K

K

K

K

K

A

A

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A

A

A

A

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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

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A

E

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E

K

K

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A

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Lipid I

M

M

MraY

MurG

Lipid II

M

M

G

G

UDP

M

UDP

MraY and E

Lipid II

out

in

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
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
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