이지효 (한국기초과학지원연구원)

황금숙 (한국기초)

이지효 (한국기초과학지원연구원)

이주은 (한국기초과학지원연구원)

황금숙 (한국기초)

Acinetobacter baumannii is a representative multidrug-resistant pathogen that can cause infections and sepsis. To discover new antimicrobial targets, it is important to elucidate the functions and mechanisms of bacterial metabolic enzymes. In particular, central carbon metabolism is essential for bacterial survival and proliferation, making it a key target for understanding the roles of metabolic enzymes. In this study, we established and applied a semi-targeted LC-MS/MS method to evaluate changes in central carbon metabolism in specific metabolic enzyme-deficient strains of A. baumannii.

Central carbon metabolism includes glycolysis, the pentose phosphate pathway (PPP), and the tricarboxylic acid (TCA) cycle; therefore, a comprehensive analytical method is required to cover these interconnected pathways. In particular, phosphate-containing metabolites can interact with metal surfaces in LC systems and columns, leading to peak tailing, reduced sensitivity, and poor reproducibility. To overcome these limitations and maximize metabolite coverage, LC conditions were optimized using a metal interaction-reduced system LC system coupled with a zwitterionic HILIC column. Using this approach, a total of 23 central carbon metabolites, including glycolysis, PPP, and TCA cycle, were analyzed by MRM-based semi-targeted LC-MS/MS.

Analysis of standard mixtures demonstrated good analytical performance, with all 14 metabolites showing reproducibility (CV% < 30) and most metabolites achieving limits of quantification (LOQs) at the nM level with S/N ratios greater than 10. In A. baumannii bacterial extracts, a total of 19 central carbon metabolites were stably detected. In metabolic enzyme-deficient strain A, glucose 6-phosphate was increased compared with wild-type (WT) and metabolic enzyme-deficient strain B, while several TCA cycle intermediates, including citric acid and isocitric acid, showed decreasing trends. These results suggest that metabolic enzymes A and B play important roles in regulating central carbon metabolism in A. baumannii. and demonstrate that semi-targeted LC-MS/MS method can effectively characterize bacterial metabolic responses.

The semi-targeted LC-MS/MS method established in this study enables comprehensive analysis of central carbon metabolites in A. baumannii. This approach is expected to be useful for antimicrobial target discovery, the screening of antibiotic candidates, and the functional characterization of bacterial metabolic enzymes.