Metabolomic Analysis of Vibrio vulnificus Biofilm and Validation of Control Target

Abstract

Biofilms are assemblages of microbial cells enclosed in self-produced extracellular polymeric matrix (EPM). Biofilm formation provides Vibrio vulnificus with resistances to antimicrobial agents and a variety of stresses in the environment. In order to gain insight into the cellular mechanisms in mature biofilms, the global metabolite profiling of V. vulnificus biofilms and planktonic cells was analyzed by using gas chromatography-time of flight mass spectrometry (GC-TOF-MS) and multivariate statistical analysis. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) showed significant differences in metabolic profiles between biofilms and planktonic cells. The levels of some amino acids, monosaccharides and nucleotides were elevated in biofilms compared to the planktonic counterparts. These metabolites appear to function as building blocks for structural components of extracellular matrix. Metabolites involved in the anaerobic energy production, such as glycolysis and pyruvate fermentation, were increased in biofilms. These suggest that V. vulnificus may obtain energy by using glycolysis and pyruvate fermentation in biofilms. The amounts of product of putrescine biosynthetic pathway in bioflms were also greater than in the planktonic cells. These results suggested that putrescine may have a positive effect on formation of V. vulnificus biofilms. Furthermore, the amounts and expression levels of the metabolites and genes, respectively, involved in N-acetylglucosamine (GlcNAc) utilization were highly increased in V. vulnificus biofilms. The combined results suggest that, when V. vulnificus forms mature biofilms and encounters conditions of low-nutrient, GlcNAc-specific phosphotransferase system (PTS) may transfer GlcNAc which is derived from extracellular environments. To find out whether GlcNAc-specific PTS affects biofilm forming ability of V. vulnificus, biofilm forming activities of the wild type and GlcNAc-specific phosphotransferase (ptsG) mutant were compared. Under the condition of low carbon source, biofilm forming activity was reduced in the ptsG mutant compared to the wild type and recovered in the complemented strain. Composition analysis of exopolysaccharide (EPS) using GC-TOF-MS revealed that GlcNAc is one of the constituent of EPS. In these aspects, when bacterial cells in V. vulnificus biofilms are faced with adverse conditions, such as insufficient nutrient, PtsG appears to transport GlcNAc, as a carbon source, which stems from extracellular matrix. Consequently, V. vulnificus in biofilms exhibits a distinct mode of growth characterized by matrix components syntheses, anaerobic energy production, polyamine metabolism and N-acetylglucosamine utilization.