Iron Modulates Butyrate Production by a Child Gut Microbiota In Vitro

Abstract

The aim of this study was to investigate the effect of iron (Fe) availability on butyrate production in the complex bacterial ecosystem of the human gut. Hence, different Fe availabilities were mimicked in an in vitro colonic fermentation model (the polyfermenter intestinal model called PolyFermS) inoculated with immobilized gut microbiota from a child and in batch cultures of the butyrate producer Roseburia intestinalis. Shifts in the microbial community (16S rRNA sequencing and quantitative PCR), metabolic activity (high-performance liquid chromatography), and expression of genes involved in butyrate production were assessed. In the PolyFermS, moderate Fe deficiency resulted in a 1.4-fold increase in butyrate production and a 5-fold increase in butyryl-coenzyme A (CoA):acetate CoA-transferase gene expression, while very strong Fe deficiency significantly decreased butyrate concentrations and butyrate-producing bacteria compared with the results under normal Fe conditions. Batch cultures of R. intestinalis grown in a low-Fe environment preferentially produced lactate and had reduced butyrate and hydrogen production, in parallel with upregulation of the lactate dehydrogenase gene and downregulation of the pyruvate:ferredoxin-oxidoreductase gene. In contrast, under high-Fe conditions, R. intestinalis cultures showed enhanced butyrate and hydrogen production, along with increased expression of the corresponding genes, compared with the results under normal-Fe conditions. Our data reveal the strong regulatory effect of Fe on gut microbiota butyrate producers and on the concentrations of butyrate, which contributes to the maintenance of host gut health.

Importance: Fe deficiency is one of the most common nutritional deficiencies worldwide and can be corrected by Fe supplementation. In this in vitro study, we show that environmental Fe concentrations in a continuous gut fermentation model closely mimicking a child's gut microbiota strongly affect the composition of the gut microbiome and its metabolic activity, particularly butyrate production. The differential expression of genes involved in the butyrate production pathway under different Fe conditions and the enzyme cofactor role of Fe explain the observed modulation of butyrate production. Our data reveal that the level of dietary Fe reaching the colon affects the microbiome, and its essential function of providing the host with beneficial butyrate.

FIG 1

Experimental setup used in this study. Continuous colonic in vitro fermentation model PolyFermS, with a first-stage inoculum reactor containing beads with immobilized child gut microbiota and second-stage control and test reactors operated in parallel and continuously inoculated with effluent from the inoculum reactor at 5% (vol/vol) of the reactor volume. Ninety-five percent fresh medium containing different concentrations of Fe was continuously added during periods 1 to 3. In period 3, the inoculum reactor was stopped and the fecal microbiota beads were divided among the control and test reactors.

FIG 2

Changes in bacterial communities and diversities in reactor effluents during application of medium differing only in Fe availability. 16S rRNA genes were sequenced using Roche 454 pyrosequencing and analyzed with QIIME and SILVA. (a) Relative abundances of 16S rRNA genes annotated on the family level. (b) Principal coordinate analysis (PCoA) of microbial community β-diversity reveals shifts due to the application of medium differing in Fe availability. P1, P2, and P3, periods 1, 2, and 3; CR, control reactor; TR1 and TR2, treatment reactors 1 and 2. High-Fe (217.8 mg Fe liter⁻¹ fermentation medium), low-Fe (200dip; Fe chelated with 200 µM 2,2′-dipyridyl in the fermentation medium), and very-low-Fe (300dip; Fe chelated with 300 µM 2,2′-dipyridyl in the fermentation medium) conditions are indicated.

FIG 3

Butyrate concentrations (measured by HPLC), butyryl-CoA:acetate CoA-transferase (butCoAT) gene copy numbers (measured by qPCR in total DNA extracts from effluents), and butCoAT gene expression levels (measured by qPCR in total RNA extracts from effluents and normalized to 16S rRNA gene expression) in low-Fe 200 µM dip and very-low-Fe 300 µM dip fermentation medium (period 3) and in high-Fe fermentation medium (period 1) were calculated relative to the corresponding data from the control reactor (CR; dotted line). Data are mean results ± SD for the last 3 days of the corresponding fermentation period. Bars marked by an asterisk show values significantly different from the corresponding value for the CR within the same parameter (P < 0.05).

FIG 4

Consumption of glucose and acetate and production of lactate, formate, butyrate, and hydrogen by R. intestinalis under normal-Fe, low-Fe (50 µM dip) and high-Fe conditions after 24 h of incubation. Values are mean results ± SD (n = 3). Columns marked by an asterisk show values significantly different from the corresponding values for normal-Fe conditions (P < 0.05).

FIG 5

Relative expression levels of the Fe²⁺ transporter gene (feoB), pyruvate:ferredoxin-oxidoreductase gene (pfo), lactate dehydrogenase gene (ldh), pyruvate formate lyase-activating enzyme gene (PFL-AE gene), butyryl-CoA:acetate CoA-transferase gene (butCoAT), and hydrogenase gene (hyd) in R. intestinalis grown under normal-Fe, low-Fe (50 µM dip), and high-Fe conditions. Gene induction was calculated relative to gene expression under normal-Fe conditions. Values are mean results ± SD (n = 6). Columns marked by an asterisk show values significantly different from the corresponding values for normal-Fe conditions (P < 0.05).

FIG 6

Schematic view of genes and metabolites in the butyrate production pathway of R. intestinalis grown in low-Fe 50 µM dip (a) and in high-Fe (b) YCFA medium. The pyruvate:ferredoxin-oxidoreductase gene (pfo), lactate dehydrogenase gene (ldh), pyruvate formate lyase-activating enzyme gene (PFL-AE gene), butyryl-CoA:acetate CoA-transferase gene (butCoAT), and hydrogenase gene (hyd) are depicted in gray to show reduced expression or in boldface to show increased induction compared with their expression in normal-Fe YCFA medium. Metabolites are depicted as decreased (gray) or increased (boldface) compared with their levels in normal-Fe YCFA medium. *, the regeneration of reduced ferredoxin to the oxidized form is also processed by a membrane-associated NADH:ferredoxin-oxidoreductase (adapted from references , , and 37).