GC MS-Based Metabolomic Approach to Understand the Mucin Utilization of Vibrio vulnificus

Abstract

Mucin, a highly glycosylated large glycoprotein, is a major component of mucus layer in the human body and could serve as a source of nutrient to support the growth of enteropathogens. To gain insight into the metabolisms used by human enteropathogen Vibrio vulnificus to utilize mucin, the metabolic profiles of V. vulnificus in the media containing mucin and glucose were compared for in vitro analysis. Also, V. vulnificus infecting to HT29-MTX mucin secreting cell line and medium control were compared for ex vivo analysis. For ex vivo experiment, mucin secreting cell line was constructed and confirmed whether it secretes mucin or not by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and confocal laser scanning microscope (CLSM). The global metabolite profiling was conducted by using gas chromatography-time of flight mass spectrometry (GC-TOF-MS) and in-house programmed database and standards. In the in vitro experiment, principal component analysis revealed clear separations between the intracellular metabolite profiles of V. vulnificus utilizing mucin. When V. vulnificus grown with mucin, both the level of N-Acetyl-D-mannosamine-6-phosphate and N-Acetylglucosamine-6-phosphate a catabolic intermediate of N-acetylneuraminic acid (Neu5Ac) which is a terminal carbohydrate of mucin were increased. The level of amino acids, fatty acids and pyruvate were increased in the mucin utilizing cells both in vitro and ex vivo metabolomics analysis. Taken together, the use of mucin as a nutrient source in V. vulnificus led to the increase abundances of amino acids, fatty acids, sialic acid, and TCA cycle intermediates by activating the catabolic pathways.