Microbial species involved in production of 1,2-sn-diacylglycerol and effects of phosphatidylcholine on human fecal microbiota

Abstract

1,2-sn-Diacylglycerols (DAGs) are activators of protein kinase C (PKC), which is involved in the regulation of colonic mucosal proliferation. Extracellular DAG has been shown to stimulate the growth of cancer cell lines in vitro and may therefore play an important role in tumor promotion. DAG has been detected in human fecal extracts and is thought to be of microbial origin. Hitherto, no attempts have been made to identify the predominant fecal bacterial species involved in its production. We therefore used anaerobic batch culture systems to determine whether fecal bacteria could utilize phosphatidylcholine (0.5% [wt/vol]) to produce DAG. Production was found to be dependent upon the presence of the substrate and was enhanced in the presence of high concentrations of deoxycholate (5 and 10 mM) in the growth medium. Moreover, its production increased with the pH, and large inter- and intraindividual variations were observed between cultures seeded with inocula from different individuals. Clostridia and Escherichia coli multiplied in the fermentation systems, indicating their involvement in phosphatidylcholine metabolism. On the other hand, there was a significant decrease in the number of Bifidobacterium spp. in the presence of phosphatidylcholine. Pure-culture experiments showed that 10 of the 12 strains yielding the highest DAG levels (>50 nmol/ml) were isolated from batch culture enrichments run at pH 8.5. We found that the strains capable of producing large amounts of DAG were predominantly Clostridium bifermentans (8 of 12), followed by Escherichia coli (2 of 12). Interestingly, one DAG-producing strain was Bifidobacterium infantis, which is often considered a beneficial gut microorganism. Our results have provided further evidence that fecal bacteria can produce DAG and that specific bacterial groups are involved in this process. Future strategies to reduce DAG formation in the gut should target these species.

FIG. 1.

Production of 1,2-sn-DAG by fecal bacteria in batch culture fermentations run at pHs 6.8, 7.5, and 8.5. Fecal samples were collected from six individuals on two separate occasions and were incubated with 0.5% (wt/vol) phosphatidylcholine and 135 ml of basal nutrient medium in batch culture systems at 37°C. Control fermentors contained no phosphatidylcholine. Samples were obtained at time zero and after 48 h of incubation, and lipids were extracted prior to the measurement of DAG by use of a commercially available kit. The data presented are mean values of initial DAG levels (T0) and levels after 48 h of fermentation with and without phosphatidylcholine.

FIG. 2.

1,2-sn-DAG production by fecal bacteria in batch culture fermentations, measured at the end of the phosphatidylcholine run. Fecal samples were collected from six individuals (numbers 1 to 6) on two separate occasions (a and b) and were incubated with 0.5% (wt/vol) phosphatidylcholine and 135 ml of basal nutrient medium in batch culture systems at 37°C. DAG production was measured after 48 h of fermentation. •, pH 8.5; ▪, pH 7.5; ▴, pH 6.8.

FIG. 3.

Mean bacterial proportions in all samples over the experimental period for pHs 6.8, 7.5, and 8.5, as determined by fluorescence in situ hybridization. Population proportions are expressed as percentages of total bacteria. The inocula were human feces (1% [wt/vol]), and the incubations were carried out in batch cultures for 48 h with phosphatidylcholine as the main substrate at a concentration of 0.5% (wt/vol). Control fermentors did not contain phosphatidylcholine. a, significantly different from the initial sample (P < 0.05); b, significantly different from the initial sample (P < 0.01); c, significantly different from the T48 control (P < 0.05); d, significantly different from the T48 control (P < 0.01); e, significantly different from the T48 sample at pH 7.5 (P < 0.05); f, significantly different from the T48 sample at pH 8.5 (P < 0.05).

FIG. 4.

Production of 1,2-sn-DAG in the presence of DCA by fecal bacteria in batch culture fermentations run at pHs 6.8 (▴), 7.5 (▪), and 8.5 (•). Fecal samples were incubated with DCA, 0.5% phosphatidylcholine, and 135 ml of basal nutrient medium at 37°C. Samples were obtained at 4, 8, 10, 24, and 48 h, and lipids were extracted prior to the measurement of DAG by use of a commercially available kit. The data presented are mean values from duplicate runs. DCA was used at a concentration of 1.2 (a), 2 (b), 5 (c), or 10 (d) mM.