Native Microbiome Members of C. elegans Act Synergistically in Biosynthesis of Pyridoxal 5'-Phosphate

Abstract

The roles of the healthy microbiome on the host and the relationships between members of the microbiome remain to be fully characterized. Due to the complexity of the interactions between the mammalian microbiome and its host, the use of model organisms such as the nematode worm Caenorhabditis elegans is a promising strategy to study host-microbiome interactions in vivo, as well as bacterial crosstalk within the host. Previously it was found that native bacterial isolates of the worm, Chryseobacterium sp. CHNTR56 MYb120 and Comamonas sp. 12022 MYb131, possess genomic diversity in the biosynthesis of the active form of vitamin B6, pyridoxal 5'-phosphate (PLP), and contribute to host fitness and lifespan extension. However, the relative contribution of PLP from each isolate, as well as the existence of interbacterial relationships within the worm gut remain to be characterized. In the present work, we investigated the presence and measured the abundance of PLP in the isolates and in the worms grown with the isolates using ultraperformance liquid chromatography tandem-mass spectrometry (UPLC-MS/MS). Our analyses confirmed the presence of PLP in vitro and in vivo. The elevated abundance of PLP in the isolates (which reached statistically significant levels when the two isolates were combined), and within worms grown with the combination of bacterial isolates, compared to control, indicated synergism between the isolates in the production of PLP. Isotope labeling revealed that Comamonas sp. 12022 MYb131 was the main provider of PLP in worms grown with the combination of bacterial isolates. The dominance of this isolate inside the worm was further confirmed by a colonization assay. An untargeted metabolomics analysis of the bacteria showed that the pathways related to cell growth, protein synthesis and lipid synthesis/energy production were regulated in the combination group in comparison with Comamonas sp. 12022 MYb131 alone. Furthermore, glutamine, involved in the de novo synthesis of purine and pyrimidines, was specifically abundant in this group, indicating the potential role of this metabolite in initiating and sustaining bacterial growth. This bacterial crosstalk is suggested to promote the growth of Comamonas sp. 12022 MYb131 in vivo, and synthesis of bacterial metabolites such as PLP in the worm gut.

Keywords: C. elegans; LC-MS/MS; glutamine; metabolomics; microbiome; pyridoxal 5′-phosphate; vitamin B6.

Figure 1

Abundance of PLP (nM) in bacterial isolates. (a) Linear curve of standard PLP with R² score is shown; (b) The abundance of PLP (normalized to 10¹⁰ cells) was found to be the highest in the combined bacterial isolates. *: p < 0.05.

Figure 2

Abundance of PLP (nM) in worms grown with bacterial isolates. (a) Linear curve of standard PLP with R² score is shown; (b) The abundance of PLP (from 2000 worms) was significantly higher in worms grown with the combination of bacterial isolates, while it was similar among worms grown with the isolates individually or E. coli OP50. *: p < 0.05.

Figure 3

A depiction of recovered bacterial colonies from worms grown with both isolates. Majority of colonies (white colonies) belonged to Comamonas sp. 12022 MYb131, at a dilution factor of the worm homogenate at 10⁻⁴ dilution, which further supports the isotope-labeling findings. Orange colony represents Chryseobacterium sp. CHNTR56 MYb120. The images of the colonies were zoomed in on to show the bacterial color properly at the indicated dilution where bacterial colonies can be deciphered.

Figure 4

Principal component analysis (PCA). PCA demonstrates the similarity of the samples (n = 5 per group) based on their metabolite patterns for positive (a) and negative (b) modes. The QC samples are almost identical, indicating the robustness for each injection.

Figure 5

Heatmap of the top 25 significant features identified in positive (a) and negative (b) modes. The relative abundance of the feature (m/z = 147.0764), indicated with asterisk (*), is specifically promoted in the combination group in positive mode.

Figure 6

Abundance of glutamine in bacterial isolates. (a) Linear curve of standard glutamine (m/z = 147.0764) with R² score is shown; (b) The abundance of glutamine (normalized to 10¹⁰ cells) was estimated to be the highest in combination. *: p < 0.05.