Short-Chain Fatty Acids Modulate Metabolic Pathways and Membrane Lipids in Prevotella bryantii B14

Abstract

Short-chain fatty acids (SCFAs) are bacterial products that are known to be used as energy sources in eukaryotic hosts, whereas their role in the metabolism of intestinal microbes is rarely explored. In the present study, acetic, propionic, butyric, isobutyric, valeric, and isovaleric acid, respectively, were added to a newly defined medium containing Prevotella bryantii B₁4 cells. After 8 h and 24 h, optical density, pH and SCFA concentrations were measured. Long-chain fatty acid (LCFA) profiles of the bacterial cells were analyzed via gas chromatography-time of flight-mass spectrometry (GC-ToF MS) and proteins were quantified using a mass spectrometry-based, label-free approach. Cultures supplemented with single SCFAs revealed different growth behavior. Structural features of the respective SCFAs were identified in the LCFA profiles, which suggests incorporation into the bacterial membranes. The proteomes of cultures supplemented with acetic and valeric acid differed by an increased abundance of outer membrane proteins. The proteome of the isovaleric acid supplementation showed an increase of proteins in the amino acid metabolism. Our findings indicate a possible interaction between SCFAs, the lipid membrane composition, the abundance of outer membrane proteins, and a modulation of branched chain amino acid biosynthesis by isovaleric acid.

Keywords: Prevotella bryantii B14; branched-chain amino acids proteome; lipid membrane; long-chain fatty acids; outer membrane proteins; short-chain fatty acids.

Figure 1

Assignment and comparison of pathways and cellular functions in P. bryantii B₁4 exposed to various short-chain fatty acids based on relative protein abundances. Principle component analysis was done based on S17 Bray Curtis similarity out of the resemblance matrix. Elevated main protein functions causing the separation of the different proteomes are indicated close to the clusters. Clusters illustrate a similarity of either 88% (dotted line) or 92% (solid line). Supplementations with single SCFAs are displayed by following symbols: Acet (○), Prop (▲), But (◻), iBut (■), iVal (◇), Val (◆). PTM: post translational modifications. The clustering of supplementation conditions was significant by a p-value < 0.05 (Monte Carlo correction).

Figure 2

Routes of acetate formation and acetate utilization in response to SCFA exposure of P. bryantii B₁4. The abundance of the mentioned enzymes (Uniprot ID) is represented by relative abundance (n = 3) for every treatment. Protein abundance of Acet cultivation is set to one. Above the reaction arrows are the EC numbers of the enzymes displayed. The color code is given in the lower left corner. (A) Acetyl-phosphate path with bidirectional reactions, where production of acetate is more likely due to ATP formation. (B) Reaction via the acetyl-CoA synthetase for SCFA assimilation, explicitly seen for iVal.

Figure 3

Influence of SCFAs on the relative abundance of enzymes required for the conversion of pyruvate to branched-chain amino acids in P. bryantii B₁4. This pathway is based on the KEGG pathway (ko00290). Grey boxes indicate the mean relative abundance (n = 3) of the enzymes (including EC numbers close to the arrows) and their detected subunits or copies, as shown by their Uniprot IDs. The color code on the right side displays fold change with respect to the Acet condition, which is set to one. Dotted arrow illustrates an undetected enzyme.

Figure 4

Influence of SCFAs on long-chain fatty acid (LCFA) profiles in P. bryantii B₁4 cell membranes. (A) LCFA profiles after 8 h (n = 1) and (B) after 24 h of incubation (n = 1) are presented as relative abundances of LCFA using AUC values. The nomenclature of the LCFA is defined by the maximal chain length and number of carbon atoms, given by the capital C, followed by the number of carbon double bonds after the colon. The position of methylation (Me) is described by the number in front while the type of branching is given in brackets and was implemented with respective patterns. Linear LCFAs (without pattern), iso-branched (diagonal upward stripes), anteiso-branched (dotted pattern).

Figure 5

Growth of P. bryantii B₁4 in minimal medium (M2-B) supplemented with glucose as the only energy supporting carbon source. Following parameters are given for a duplicate (A: empty, B: filled symbols) of a two-liter batch culture: optical density at 600 nm (circles), glucose concentration in millimolar (squares) and pH as a color gradient from pH 7 (green) to pH 5.2 (red). The interpolation curves for optical density (dotted line) and glucose (solid line) represent the trend for each duplicate.