Transcript Molecular Basis of Vancomycin Resistance

Molecular Basis of Vancomycin
Resistance-Basic Science Paper
Yashar Kalani
Biochemistry 230
History and Background
• Vancomycin 1.5kD
glycopeptide.
• Introduced in
hospitals ~40 years
ago when strains of
bacteria exhibited
penicillin resistance.
• Vancomycin
resistant enterococci
(VRE) appeared in
1987.
www.omedon.co.uk/ vrsa/vancomycin/
How Does Vancomycin Work?
• Vancomycin binds to
the D-alanyl-D-alanine
residues of the
peptidoglycan
monomers.
• Cross links are not
created, so the
peptidoglycan chains
only form a weak cell
wall. Intense osmotic
pressure ruptures the
cell.
www.omedon.co.uk/ vrsa/vancomycin/
• Vancomycin
binds with high
affinity to the
terminal D-AlaD-Ala through
five hydrogen
bonds.
www.omedon.co.uk/ vrsa/vancomycin/
How Do Bacteria Become Resistant?
2 factors lead to vancomycin resistance:
1) A thickening of the PG layer;
2) Modification of the PG termini from DAla--D-Ala to D-Ala--D-lactate.
www.omedon.co.uk/ vrsa/vancomycin/
And …
• Two genes were shown to provide a high
level of vancomycin resistance. These
genes, (Van A and Van H) code for proteins
which ultimately modify the cell-wall
biosynthetic pathway.
Role of Van H
Hypothesis:
“This raises the possibility that VanH synthesizes a D-a-hydroxycarboxylic
acid, which may be an alternative substrate for VanA. If VanA is able to
condense a D-hydroxy acid instead of a D-amino acid with D-alanine, binding
of the subsequently resulting peptidoglycan precursor to vancomycin would be
directly affected, since one of the hydrogen bonds formed between vancomycin
and N-acetyl-D-Ala-D-Ala is with the NH of the terminal D-alanine residue
(Barna & Williams, 1984).”
X
No decrease in absorbance was observed with L-lactate
dehydrogenase, but a decrease in absorbance of 0.023 was
observed with D-lactate dehydrogenase.
Determination of N-terminal Sequences of L-specific Dehydrogenases
Specificity of VanH favors smaller side chains, best being pyruvate
and 2-keto-butyrate.
2-Hydroxy Acids as Substrates for VanA
VanA has a preference for longer side chains!
Lanes 6-8 show bands
corresponding to D-Ala-D-Hbut,
D-Ala-D-Hval and D-Ala-D-Hcap
consistent with presence of Van A
in BM4147
Lanes 9-11 show no evidence of
synthesis of D-alanyl esters by
BM4147-extract, but D-Ala-DAla is produced.
Very little D-Ala-D-Ala
synthesis is observed with
BM4147.
This suggests that the chromosomal D-Ala-D-Ala
ligase in BM4147 is inhibited.
Substrate Specificity of E. coli D-Ala-D-Ala Adding Enzyme
D-Ala-D-Hbut has a much higher Km than D-Ala-D-Lac
but has a comparable catalytic efficiency
Both turn over quickly; D-Ala-D-Ala amide
bond is not critical for substrate recognition!
Vancomycin Binding Experiments
Peptide with D-Ala termini bind well; those with non-D-Ala don’t bind!
Substitution of larger side chains in C-terminal positions gives rise to
>3000-fold reduction in binding and substitution of amide NH for O gives
>1000-fold reduction in binding.
Functional Link Between
VanH and VanA
D-hydroxy acid products of VanH are
substrates for VanA.
Change from a dipeptide to
depsipeptide removes the amide NH
of terminal D-alanine with interacts
with Vancomycin.
Conclusion
So How Does It All Fit Together?
There are actually 5 genes that
result in resistance.
•“In the absence of vancomycin
the VanH,A,X genes are
transcriptionally inactive. The
presence of vancomycin is somehow
detected by VanS (a sensor kinase)
and the transcription of VanH,A,X
initiated by VanR.”
Why Has Resistance Occurred?
• Most VRSA and MRSA cases are isolated in
hospital wards.
• In these environments natural selection breeds
antibiotic resistant microbes. The susceptible
bacteria are killed by antibiotic agents leaving
behind a select few resistant bacteria along with a
larger pool of nutrients to grow on.