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. 2016 Oct;5(5):802-818.
doi: 10.1002/mbo3.371. Epub 2016 Jun 28.

FolC2-mediated folate metabolism contributes to suppression of inflammation by probiotic Lactobacillus reuteri

Carissa M Thomas  1 , Delphine M A Saulnier  2   3 , Jennifer K Spinler  2   3 , Peera Hemarajata  4 , Chunxu Gao  2   3 , Sara E Jones  1 , Ashley Grimm  2   3 , Miriam A Balderas  2   3 , Matthew D Burstein  5 , Christina Morra  1   3 , Daniel Roeth  6 , Markus Kalkum  6 , James Versalovic  7   8
Affiliations

FolC2-mediated folate metabolism contributes to suppression of inflammation by probiotic Lactobacillus reuteri

Carissa M Thomas et al. Microbiologyopen. 2016 Oct.

Abstract

Bacterial-derived compounds from the intestinal microbiome modulate host mucosal immunity. Identification and mechanistic studies of these compounds provide insights into host-microbial mutualism. Specific Lactobacillus reuteri strains suppress production of the proinflammatory cytokine, tumor necrosis factor (TNF), and are protective in a mouse model of colitis. Human-derived L. reuteri strain ATCC PTA 6475 suppresses intestinal inflammation and produces 5,10-methenyltetrahydrofolic acid polyglutamates. Insertional mutagenesis identified the bifunctional dihydrofolate synthase/folylpolyglutamate synthase type 2 (folC2) gene as essential for 5,10-methenyltetrahydrofolic acid polyglutamate biosynthesis, as well as for suppression of TNF production by activated human monocytes, and for the anti-inflammatory effect of L. reuteri 6475 in a trinitrobenzene sulfonic acid-induced mouse model of acute colitis. In contrast, folC encodes the enzyme responsible for folate polyglutamylation but does not impact TNF suppression by L. reuteri. Comparative transcriptomics between wild-type and mutant L. reuteri strains revealed additional genes involved in immunomodulation, including previously identified hdc genes involved in histidine to histamine conversion. The folC2 mutant yielded diminished hdc gene cluster expression and diminished histamine production, suggesting a link between folate and histadine/histamine metabolism. The identification of genes and gene networks regulating production of bacterial-derived immunoregulatory molecules may lead to improved anti-inflammatory strategies for digestive diseases.

Keywords: folC2; Colitis; Lactobacillus reuteri.; folate; histamine; immunomodulation.

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Figures

Figure 1
Figure 1
MALDI‐MS detection of 5,10‐CH = THF polyglutamates in TFA‐treated L. reuteri cell pellets. Peaks indicating the presence of 5,10‐CH = THF polyglutamate (arrows, MGlun) are marked for strain (A) 6475 and (B) 55730. The subscripted number (n) after MGlu indicates the number of glutamate residues present in the polyglutamate tail, not counting the intrinsic glutamate of folic acid. (C) MALDI ion trap MS/MS fragmentation analysis of MGlu5 at m/z 1101.4 confirms the presence of a folate compound, consistent with the lightest MS/MS fragment ion at m/z values 327.1. (D) Inactivation of folC2 in 6475 inhibited the production of 5,10‐CH = THF compounds as monitored by MALDI MS. (E) The 6475::folC mutant produced 5,10‐CH = THF (structure depicted), but no 5,10‐CH = THF polyglutamates of any chain length. The unidentified ion at m/z 932.4 was consistently observed after TFA treatment of all L. reuteri cell pellets. All spectra were scaled to comparable maximum intensity levels. TFA, trifluoroacetic acid; 5,10‐CH = THF, 5,10‐methenyltetrahydrofolic acid.
Figure 2
Figure 2
The folC2 gene contributed to the TNF‐suppressive ability of L. reuteri 6475. TFA‐treated cell pellets (normalized to cell pellet weight of 1 g) from various L. reuteri strains were tested for the ability to inhibit TNF by TLR2‐activated THP‐1 cells. THP‐1 cells were treated with 100 ng/mL PCK (TLR2 agonist) in the presence of L. reuteri for 3.5 h, and TNF production was monitored by ELISA. Wild‐type 6475 significantly inhibited TNF compared to medium control. The 6475::folC2 mutant yielded reduced capability to inhibit TNF production compared to wild‐type 6475. There was no significant difference between 6475 and 6475::folC strains. Data were analyzed with one‐way analysis of variance with Bonferroni's multiple comparison test correction, mean ± SD, n = 3, *P < 0.05 compared to medium control # P < 0.05 compared to 6475.TLR2, Toll‐like receptor 2; TNF, tumor necrosis factor.
Figure 3
Figure 3
Comparative transcriptomics analysis revealed gene set encoding potential immunomodulins. (A) Flowchart of the comparative transcriptomic analysis employed in this study. N = 3 for each wild‐type and mutant L. reuteri strain that was included in the comparative analysis. (B) GSEA showed significant enrichment of strain 6475 genes up‐regulated in stationary phase in the down‐regulated genes of 6475::folC2. (C) The yellow circle indicates genes significantly down‐regulated in 6475::folC2 that were up‐regulated in wild‐type 6475 (stationary phase). The blue circle indicates genes not down‐regulated in 6475::folC that were up‐regulated in wild‐type 6475 (stationary phase). The red circle indicates genes that were included in the GSEA “core enrichment.” The overlap between these three gene sets revealed a final gene set of interest including 125 genes potentially involved in immunomodulin production by L. reuteri 6475. GSEA, A Gene Set Enrichment Analysis
Figure 4
Figure 4
Inactivation of the folC2 gene resulted in repression of the hdc gene cluster. (A) Quantitative real‐time PCR yielded evidence of decreased expression of hdcA and hdcP genes in the L. reuteri 6475::folC2 mutant compared to wild‐type L. reuteri 6475. The gene narI, which was suggested by the comparative transcriptomics analysis to be down‐regulated in the folC2 mutant, was also repressed in 6475::folC2. Expression ratios of each gene (folC2 mutant vs. wild‐type) were calculated, and results represent the mean ± SD, n = 3, **P < 0.01, *P < 0.05 compared to the theoretical mean of 1.0. (B) Quantitative real‐time PCR demonstrated increased expression of all three hdc genes, hdcA, hdcB, and hdcP, when L. reuteri 6475 was grown in medium supplemented with l‐histidine compared to unsupplemented medium. Expression ratios of each gene (l‐histidine‐supplemented vs. unsupplemented) were calculated. Results represent the mean ± SD, n = 3, ***P < 0.005, **P < 0.01, *P < 0.05 compared to the theoretical mean of 1.0. (C) Quantitative real‐time PCR demonstrated no significant changes in expression of all three hdc genes when L. reuteri 6475::folC2 was grown in medium supplemented with l‐histidine compared to unsupplemented medium. (D). Inactivation of the folC2 gene resulted in decreased histamine production. Quantification of secreted L. reuteri‐derived histamine by a histamine‐specific ELISA demonstrated decreased histamine production in the folC2 mutant compared to wild‐type L. reuteri 6475 even when grown in l‐histidine‐supplemented medium. Data were analyzed by two‐way ANOVA. Results represent the mean ± SD, n = 3, P < 0.0001 compared to wild‐type 6475.
Figure 5
Figure 5
The folC2 gene was essential for colitis attenuation and anti‐inflammatory activity of L. reuteri 6475 in vivo. (A) Supernatant from L. reuteri 6475 significantly decreased weight loss in mice challenged with intrarectal TNBS, whereas 6475::folC2 did not have such effects. Differences in weight loss are shown as percent weight loss 48 h after induction of TNBS colitis. (B) Supernatant from L. reuteri 6475 significantly decreased colonic macroscopic injury in mice challenged with TNBS, whereas 6475::folC2 did not have such effects. Differences in colonic macroscopic injury are shown as Wallace score. Data presented as mean ± SEM. (C) Supernatant from L. reuteri 6475 significantly decreased plasma SAA concentrations in mice challenged with TNBS, whereas 6475::folC2 did not have such effects. Plasma levels of SAA, an indicator of inflammation, were measured by ELISA. N = 19, 50, 47, and 26 for colitis‐negative, colitis‐positive, ATCC 6475, and 6475::folC2, respectively. Statistical analyses were performed using GraphPad Prism (GraphPad Inc., La Jolla,CA). Data were presented using box and whisker plots showing the median and 5th and 95th percentiles. Statistical significance was assessed by nonparametric Kruskal–Wallis test. Differences between experimental groups are reported as mean fold difference ± SEM, ***P < 0.001, **P < 0.01, *P < 0.05.SAA, Serum amyloid A; TNBS, trinitrobenzene sulfonic acid.
Figure 6
Figure 6
Folate synthesis appears to be linked to histamine production in L. reuteri 6475. (A) Folate synthesis in wild‐type L. reuteri 6475 with FolC2 necessary for production of dihydrofolate and FolC responsible for addition of a polyglutamate tail to tetrahydrofolate. Folate synthesis contributes to histamine production and the anti‐inflammatory effect of L. reuteri. (B) Inhibition of folate synthesis in L. reuteri 6475::folC2 leads to reduced histamine production and loss of anti‐inflammatory activity. (C) Inhibition of folate polyglutamylation in L. reuteri 6475::folC does not impact histamine production and L. reuteri anti‐inflammatory activity is preserved. Hdc, histidine decarboxylase.
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