Quantitation of wall teichoic acid in Staphylococcus aureus by direct measurement of monomeric units using LC-MS/MS.
The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has created an urgent need for new therapeutic agents capable of combating this threat. We have previously reported on the discovery of novel inhibitors targeting enzymes involved in the biosynthesis of wall teichoic acid (WTA) and demonstrated that these agents can restore β-lactam efficacy against MRSA. In those previous reports pathway engagement of inhibitors was demonstrated by reduction in WTA levels measured by polyacrylamide gel electrophoresis. To enable a more rigorous analysis of these inhibitors we sought to develop a quantitative method for measuring whole-cell reductions in WTA. Herein we describe a robust methodology for hydrolyzing polymeric WTA to the monomeric component ribitol-N-acetylglucosamine coupled with measurement by LC-MS/MS. Critical elements of the protocol were found to include the time and temperature of hydrofluoric acid-mediated hydrolysis of polymeric WTA and optimization of these parameters is fully described. Most significantly, the assay enabled accurate and reproducible measurement of depletion EC50sfor tunicamycin and representatives from the novel class of TarO inhibitors, the tarocins. The method described can readily be adapted to quantifying levels of WTA in tissue homogenates from a murine model of infection, highlighting the applicability for both in vitro and in vivo characterizations.
Merck Research Laboratories, 2000 Galloping Hill Road, Kenilworth, NJ 07033, United States.
Olga
O
Berejnaia
Merck Research Laboratories, 2000 Galloping Hill Road, Kenilworth, NJ 07033, United States.
Hao
H
Wang
Merck Research Laboratories, 2000 Galloping Hill Road, Kenilworth, NJ 07033, United States.
Marc
M
Labroli
Merck Research Laboratories, 2000 Galloping Hill Road, Kenilworth, NJ 07033, United States.
Christine
C
Yang
Merck Research Laboratories, 2000 Galloping Hill Road, Kenilworth, NJ 07033, United States.
Charles
C
Gill
Merck Research Laboratories, 2000 Galloping Hill Road, Kenilworth, NJ 07033, United States.
Jianying
J
Xiao
Merck Research Laboratories, 2000 Galloping Hill Road, Kenilworth, NJ 07033, United States.
David
D
Hesk
Merck Research Laboratories, 2000 Galloping Hill Road, Kenilworth, NJ 07033, United States.
Reynalda
R
DeJesus
Merck Research Laboratories, 2000 Galloping Hill Road, Kenilworth, NJ 07033, United States.
Jing
J
Su
Merck Research Laboratories, 2000 Galloping Hill Road, Kenilworth, NJ 07033, United States.
Christopher M
CM
Tan
Merck Research Laboratories, 2000 Galloping Hill Road, Kenilworth, NJ 07033, United States.
Payal R
PR
Sheth
Merck Research Laboratories, 2000 Galloping Hill Road, Kenilworth, NJ 07033, United States.
Michael
M
Kavana
Merck Research Laboratories, 2000 Galloping Hill Road, Kenilworth, NJ 07033, United States. Electronic address: david.mclaren@merck.com.
David G
DG
McLaren
D002853:Chromatography, Liquid / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods*; D055624:Methicillin-Resistant Staphylococcus aureus / Q000737:chemistry / Q000378:metabolism*; D013682:Teichoic Acids / Q000737:chemistry / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology
D013682:Teichoic Acids; D014415:Tunicamycin
Liquid chromatography; Mass spectrometry; Methicillin resistance; Staphylococcus aureus; Tarocins; Tunicamycin; Wall teichoic acid