Intestinal luminal putrescine is produced by collective biosynthetic pathways of the commensal microbiome

Abstract

The intestinal microbiome produces various metabolites that may harm or benefit the host. However, the production pathways of these metabolites have not been well characterised. The polyamines putrescine and spermidine required for physiological process are also produced by intestinal microbiome. The production and release of these polyamines by microbiome are poorly understood, though we have confirmed that intestinal bacteria produced putrescine from arginine. In this study, we characterised polyamine synthesis by analysing the collective metabolic functions of the intestinal microbiome. In particular, we analysed polyamines and their intermediates in faecal cultures, as well as the colonic contents of rats injected with isotope-labelled arginine through a colon catheter, using mass spectrometry. Isotope-labelled putrescine was detected in faecal cultures and colonic contents of rats injected with isotope-labelled arginine. Putrescine is produced through multiple pathways, and its extracellular intermediates are exchanged between bacterial species. Additionally, we demonstrated that the collective metabolic pathway depends on a complex exchange of metabolites released into the colonic lumen. This study demonstrates the existence of putrescine biosynthetic pathways based on the collective metabolic functions of the intestinal microbial community. Our findings provide knowledge to manipulate the levels of intestinal microbial products, including polyamines, that may modulate host health.

Keywords: Intestinal microbiome; Polyamines; arginine degradation; metabolomics; stable isotope.

Figure 1.

Polyamines and intermediates in faecal cultures Time course of (a) arginine, citrulline, ornithine, agmatine, putrescine and spermidine, as measured by UPLC, and (b) pH of faecal cultures supplemented with 1 mM arginine (red) or PBS (blue) (c) Ratio of total citrulline, ornithine, and putrescine to the molar concentration of arginine at 0 h; error bars indicate standard error (n = 7); *, p < 0.05; **, p < 0.01 compared with PBS by two-way analysis of variance without replication, followed by post-hoc Bonferroni multiple comparison test (a, b).

Figure 2.

Isotope-labelled putrescine in faecal cultures Time course of (a) putrescine concentrations and (b) ratios of isotope-labelled putrescine in faecal cultures supplemented with isotope-labelled arginine (n = 3), as measured by GC-MS.

Figure 3.

Putrescine collective biosynthetic pathway Proposed putrescine collective biosynthetic pathway via (a) ornithine or (b) agmatine in the intestinal microbiome; ADI, arginine deiminase; ADC, arginine decarboxylase; AgDI, agmatine deiminase; OTC, ornithine carbamoyltransferase; ODC, ornithine decarboxylase; CK, carbamate kinase; PTC, putrescine carbamoyltransferase; NCP, N-carbamoyl putrescine amidase; AdoMetDC, S-adenosylmethionine carboxy-lyase; SPDS, spermidine synthase; AUH, agmatine ureohydrolase; ASS, argininosuccinate synthetase; ASL, argininosuccinate lyase; Carbamoyl-P, carbamoylputrescine; Arg-Suc, argininosuccinate; AdoMet, S-adenosylmethionine; dcAdoMet, S-adenosylmethioninamine; MTA, 5’-methylthioadenosine. Red and blue circles represent ¹³C and ¹⁵N. Red arrows indicate the linear pathway from isotope-labelled arginine (M + 10), black arrows indicate the pathway from isotope-labelled arginine (not M + 10), and the green arrows indicate reverse pathways. Dashed lines show the putative pathway.

Figure 4.

Putrescine production from exogenous intermediates Production of putrescine in faecal cultures supplemented with 1 mM citrulline (Cit), ornithine (Orn), and agmatine (Agm) (n = 7), as measured by UPLC; error bars represent standard error; *, p < 0.05; **, p < 0.01 by Student’s t-test versus control.