Quantitation of wall teichoic acid in Staphylococcus aureus by direct measurement of monomeric units using LC-MS/MS

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• 作者：Olga Berejnaia ; Hao Wang ; Marc Labroli ; Christine Yang ; Charles Gill ; Jianying Xiao ; David Hesk ; Reynalda DeJesus ; Jing Su ; Christopher M. Tan ; Payal R. Sheth ; Michael Kavana ; David G. McLaren ; ^{david.mclaren@merck.com}
• 关键词：Staphylococcus aureus ; Wall teichoic acid ; Tunicamycin ; Tarocins ; Liquid chromatography ; Mass spectrometry ; Methicillin resistance
• 刊名：Analytical Biochemistry
• 出版年：2017
• 出版时间：1 February 2017
• 年：2017
• 卷：518
• 期：Complete
• 页码：9-15
• 全文大小：980 K
• 卷排序：518

文摘

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 EC50s for 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.