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-Ala-D-Ala through five hydrogen bonds.

www.omedon.co.uk/ vrsa/vancomycin/

www.omedon.co.uk/ vrsa/vancomycin/

2 factors lead to vancomycin resistance: 1) A thickening of the PG layer;2) Modification of the PG termini from D-Ala--D-Ala to D-Ala--D-lactate.

How Do Bacteria Become Resistant?

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, bindingof the subsequently resulting peptidoglycan precursor to vancomycin would be directly affected, since one of the hydrogen bonds formed between vancomycinand 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.

VanA has a preference for longer side chains!

2-Hydroxy Acids as Substrates for VanA

Lanes 6-8 show bands corresponding to D-Ala-D-Hbut, D-Ala-D-Hval and D-Ala-D-Hcapconsistent 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-D-Ala is produced.

Very little D-Ala-D-Ala synthesis is observed with BM4147.

This suggests that the chromosomal D-Ala-D-Alaligase in BM4147 is inhibited.

Substrate Specificity of E. coli D-Ala-D-Ala Adding Enzyme

Both turn over quickly; D-Ala-D-Ala amide bond is not critical for substrate recognition!

D-Ala-D-Hbut has a much higher Km than D-Ala-D-Lac but has a comparable catalytic efficiency

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.