Metabolomic Analysis of Bacterial Cell Wall Intermediates Using LC-MS/MS: Peptidoglycan Pathway Response to Different Antibiotics
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The bacterial cell wall is made up of peptidoglycan (PG) and its synthesis is of interest as a target for several antibiotics and for new inhibitor development. Peptidoglycan biosynthesis is a complex process which involves a series of UDP-linked and amine-linked cytoplasmic intermediates assembled together by the Mur (A-G) enzymes¹⁻⁴. Quantification of these intermediates is essential for the studies of current agents, and for the development of new agents targeting the PG pathway. Part I of my dissertation describes a novel LC-MS/MS assay for the determination of in vivo levels of cytoplasmic PG-linked intermediates in S. aureus. Quantitative analysis of UDP-linked intermediates is challenging, in part due to the lack of commercial available intermediates for use as standards. Therefore, we preparatively purified these intermediates from antibiotic treated bacterial cultures by HPLC, and used them to develop LC-MS/MS methods for their detection, and as analytical standards for their quantification. Levels of UDP-linked intermediates were quantitatively determined using ion pairing LC-MS/MS in negative mode⁵, and amine-linked intermediates were quantitatively determined stereospecifically as their Marfey's reagent derivatives in positive mode ⁶,⁷. The developed method was then used to study the in vivo (in bacteria in culture) changes in cytoplasmic peptidoglycan intermediates in the absence and presence of different antibiotics⁸. Three experiments were performed: 1) Exposure to 4x-MIC (acute) levels of antibiotics. 2) Exposure to sub-MIC levels of antibiotics. And 3) a time course of the effect of vancomycin. Our LC-MS/MS results indicate that PG metabolite pool accumulations were consistent with known targets of these antibiotics and suggests at least three potential regulatory loops (GlmU, MurD and Ddl enzymes) within this pathway. Sub-MIC exposures to antibiotics demonstrated effects even at 1/8x MIC which strongly paralleled acute exposure changes. The effect of non-cell wall targeting inhibitors on PG pathway was also performed and reported in this study. These types of analyses provide a reference point for similar studies in other bacteria to facilitate development of new agents targeting PG pathway. Part II of my thesis describes LC-MS/MS-based resolution of aminobutyric acid isomers (ABA) using linear and Gaussian deconvolution. This approach was then demonstrated for quantification of these species in human serum ⁹. Isomeric molecules present a challenge for analytical resolution and quantification, even with MS-based detection. The eight ABA isomers are of interest for their various biological activities, particularly, γ-aminobutyric acid (GABA) and the D- and L-isomers of β-aminoisobutyric acid (β-AIBA; BAIBA). HPLC was able to separate three Mar-ABA isomers completely, with three isomers (GABA, and D/ L-BAIBA) in one chromatographic cluster, and two isomers (α-AIBA (AAIBA) and D-BABA) in a second cluster. Partially separated cluster components were deconvoluted using Gaussian peak fitting and by linear deconvolution using their MS/MS intensity profiles. This approach can be used for the resolution of any challenging isomeric compounds.
Table of Contents
Introduction and literature review -- Ion-pairing chromatography-tandem mass spectrometry -based quantification of UDP-linked intermediates in the staphylococcus aureus cell wall biosynthesis pathway -- Cytoplasmic peptidoglycan intermediate levels in staphylococcus aureus -- Antibiotic effects on staphylococcus aureus cytoplasmic cell wall intermediate levels; evidence for potential metabolite level regulatory loops -- Comparison study of antibiotics effects insensitive and resistant S. aureus -- Gaussian and linear deconvolution of lc-ms/ms chromatograms of the eight aminobutyric acid isomers -- Summary and conclusions -- Appendix
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Ph.D.