The folylpolyglutamate synthase gene in probiotic Lactobacillus reuteri ATCC PTA 6475 contributes to its anti-inflammatory activity.

Dysregulated intestinal microbiota has been described in patients with inflammatory bowel disease (IBD). Supplementation with probiotic Lactobacillus species yielded promising results in ameliorating colitis in IBD patients and rodent models. We have previously shown that the culture supernatant from human-derived probiotic strain Lactobacillus reuteri ATCC PTA 6475 suppresses tumor necrosis factor (TNF) production by activated human and mouse monocytes. The aims of our current study are: 1) to confirm the anti-inflammatory activity of the culture supernatant from L. reuteri 6475 in a murine colitis model and 2) to identify probiotic factors responsible for the anti-inflammatory activity. Based on mass spectrometry, we hypothesized that folylpolyglutamates associated with the bacterial cell surface and supernatant of L. reuteri 6475 contributes its anti-inflammatory activity. Targeted insertional mutagenesis was used to inactivate fpgs1, one of the two folylpolyglutamate synthase genes in L. reuteri 6475. The fpgs1 mutant and wild type L. reuteri 6475 were subsequently studied using mass spectrometry, TNF suppression assay (in vitro) and colitis-attenuation assay (in vivo). The cell surface associated factors of the wild type L. reuteri 6475 contained folylpolyglutamates and could suppress TNF production from activated monocytes. In comparison, the cell surface associated factors of the fpgs1 mutant lacked folylpolyglutamates and showed a significant reduction of TNF suppressive activity. Likewise, the low molecular weight (< 3kD) (LMV) supernatant fraction of the wild type L. reuteri 6475 suppressed TNF production from activated monocytes, while that of the fpgs1 mutant yielded diminished TNF suppressive activity. To test for anti-inflammatory activities of the probiotic supernatants in vivo, the supernatants were injected intraperitoneally into colitic mice generated using enemas of trinitrobenzene sulfonic acid (TNBS). The concentrated LMV supernatant fraction from the wild type L. reuteri 6475 reduced colitis scores and systemic inflammation in colitic mice, while the fpgs1 mutant did not to prevent colitis. To confirm that the fpgs1 gene contributes to the anti-inflammatory activity of L. reuteri 6475, we complemented the fpgs1 mutant with a full length fpgs1 gene. Complementation partially restored the mutant's TNF suppressive activity in monocytes and ability to suppress colitis in mice. In summary, our results showed that the LMV supernatant fraction of L. reuteri 6475 attenuated murine colitis, and the fpgs1 gene in L. reuteri 6475 contributed to the anti-inflammatory phenotype of this probiotic strain. Bioactive metabolites produced by human-derived probiotics may facilitate selection of probiotics and enhancement of their functionalities for health promotion and disease prevention.