Untargeted Microbial Exometabolomics and Metabolomics Analysis of Helicobacter pylori J99 and jhp0106 Mutant

Abstract

Untargeted metabolomic profiling provides the opportunity to comprehensively explore metabolites of interest. Herein, we investigated the metabolic pathways associated with Jhp0106, a glycosyltransferase enzyme in Helicobacter pylori. Through untargeted exometabolomic and metabolomic profiling, we identified 9 and 10 features with significant differences in the culture media and pellets of the wild-type (WT) J99 and jhp0106 mutant (Δjhp0106). After tentative identification, several phosphatidylethanolamines (PEs) were identified in the culture medium, the levels of which were significantly higher in WT J99 than in Δjhp0106. Moreover, the reduced lysophosphatidic acid absorption from the culture medium and the reduced intrinsic diacylglycerol levels observed in Δjhp0106 indicate the possibility of reduced PE synthesis in Δjhp0106. The results suggest an association of the PE synthesis pathway with flagellar formation in H. pylori. Further investigations should be conducted to confirm this finding and the roles of the PE synthesis pathway in flagellar formation. This study successfully demonstrates the feasibility of the proposed extraction procedure and untargeted exometabolomic and metabolomic profiling strategies for microbial metabolomics. They may also extend our understanding of metabolic pathways associated with flagellar formation in H. pylori.

Keywords: Helicobacter pylori; exometabolomics; flagellum; jhp0106 mutant; liquid chromatography-mass spectrometry; microbial metabolomics.

Figure 1

Continuous extraction tests of culture media (100 μL), performed with 400 μL of methanol (a), and of H. pylori pellets, performed with 1500 μL of methanol (b).

Figure 2

PLS−DA plot of the features obtained from the culture media of WT J99 and Δjhp0106 H. pylori (a); heat map revealing features with higher VIP scores from the PLS−DA model (b); volcano plot (c) and boxplots (d) of features with a fold change of >2 and a p value of <0.05. The y-axis of (d) represents the normalized peak area after the autoscaling normalization. (The black dots in (d) are the sample values; while the yellow dots are the mean values).

Figure 3

PLS−DA plot of the features obtained from WT J99 and Δjhp0106 H. pylori pellets (a); heat map revealing features with higher VIP scores from the PLS−DA model (b); volcano plot (c) and boxplots (d) of features with a fold change of >2 and a p value of <0.05. The y-axis of (d) represents the normalized peak area after the autoscaling normalization. (The black dots in (d) are the sample values; while the yellow dots are the mean values).

Figure 4

Scheme presenting tentatively identified metabolites in the culture media and within H. pylori (a); the illustrated pathways are associated with the tentatively identified metabolites (b).