Vitamin B 2 Production by Vaginal Lactobacilli Promotes Symbiosis
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Abstract
The human vaginal microbiome, particularly with lactobacilli as the main inhabitants, plays a key role in maintaining women’s health. While lactic acid-mediated pathogen exclusion is well known, broader metabolic functions of vaginal lactobacilli remain underexplored. In this study, we analyzed the vaginal microbiome and metabolome of 258 healthy women from the Isala program. Using targeted metabolomics analysis, we detected a high prevalence with strong interpersonal differences of most B-vitamins, their precursors, and vitamin A in the vaginal microenvironment. Riboflavin (B 2 ) and biotin (B 7 ) showed strong associations with Lactobacillus crispatus and Limosilactobacillus sp . Comparative genomics, phenotypic assays, and in vivo metatranscriptomic data (VIRGO2) collectively confirmed riboflavin biosynthesis by these taxa. Using a riboflavin overproducing Lim. reuteri as a functional model, we showed that microbially derived riboflavin and its pathway intermediates are transported across the vaginal epithelium and modulate host redox balance, cytokine production, and activation of mucosal-associated invariant T (MAIT) cells via induction of MR1 (Major histocompatibility complex, class I-related protein receptor), revealing a potential immunometabolic interface between the vaginal microbiota and its host.
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Great question, thank you for raising it. We do not exclude a potential contribution from the gut. There are two reasons for this: (1) riboflavin derived from diet or gut microbial production is absorbed in the intestine and transported systemically via riboflavin-binding proteins across the body, and (2) riboflavin intermediates have been shown to cross the gut epithelium rapidly in mice; for example, certain 5-OP-RU reaches the thymus within 1 hour after oral gavage and drive MAIT-cell development (Legoux et al. Science 2019). However, because we did not collect fecal or blood samples in the Isala study, we cannot yet directly assess the extent of systemic or gut-derived contributions. We hope to address this more explicitly in our follow-up study. At the same time, it is important to consider the reverse question: why would these …
Great question, thank you for raising it. We do not exclude a potential contribution from the gut. There are two reasons for this: (1) riboflavin derived from diet or gut microbial production is absorbed in the intestine and transported systemically via riboflavin-binding proteins across the body, and (2) riboflavin intermediates have been shown to cross the gut epithelium rapidly in mice; for example, certain 5-OP-RU reaches the thymus within 1 hour after oral gavage and drive MAIT-cell development (Legoux et al. Science 2019). However, because we did not collect fecal or blood samples in the Isala study, we cannot yet directly assess the extent of systemic or gut-derived contributions. We hope to address this more explicitly in our follow-up study. At the same time, it is important to consider the reverse question: why would these compounds measured in vaginals swabs not originate form local production? Beyond the gut, other human microbiomes, including the skin, oral cavity, and respiratory tract, harbor riboflavin-producing microbes and show responsiveness to riboflavin-MAIT cell signaling. It is therefore plausible that the vaginal microbiome does as well. Indeed, multiple vaginal species possess the genomic capacity for riboflavin biosynthesis; Candida albicans and Prevotella are good examples. In this work, we have als indicated that most vaginal lactobacilli isolates have a predicted genomic biosynthetic capacity, and can actively express these genes in vivo. When we mapped the VIRGO metatranscriptome data onto the corresponding metagenomes, we observed that in Community State Type I, so profiles dominated by L. crispatus, have higher rib transcripts, and they map back to L. criasptus and L. jensenii metagenomes. We also observed that in the more diverse Community State Type IV, where overall riboflavin-related transcription is lower, the transcripts that are present predominantly originate from Prevotella spp. (data not shown in this version of the manuscript). This further supports the idea that local microbial production within the vagina is likely contributing, even if systemic sources may also play a role. And then lastly, you adress in your question whether vitamin intake has an effect on vaginal lactobacilli levels. We are not sure, there were no significant associations in the original Isala cohort of 3345 women (Lebeer et al. Nature Microbiology. (2023). We have adressed the current state of the art in our recent review Dricot et al. npj Biofilms and Microbiomes (2024), which you might be interested in.
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Thank you very much for your thoughtful question and for your interest in our work. In general, vaginal Lactobacillusspecies tend to increase during pregnancy, largely as a result of rising estrogen levels. Estrogen stimulates vaginal epithelial cells to accumulate and release glycogen, which can be metabolized by the resident microbiota. Lactobacilli are particularly efficient at utilizing glycogen, and this typically results in their increased abundance during pregnancy. This shift is considered beneficial, as higher Lactobacillus dominance has been associated with a reduced risk of adverse outcomes such as preterm birth, low birth weight, and other pregnancy complications. I completely agree that longitudinal metabolomic and metagenomic profiling throughout pregnancy would be highly informative for understanding how host–microbe …
Thank you very much for your thoughtful question and for your interest in our work. In general, vaginal Lactobacillusspecies tend to increase during pregnancy, largely as a result of rising estrogen levels. Estrogen stimulates vaginal epithelial cells to accumulate and release glycogen, which can be metabolized by the resident microbiota. Lactobacilli are particularly efficient at utilizing glycogen, and this typically results in their increased abundance during pregnancy. This shift is considered beneficial, as higher Lactobacillus dominance has been associated with a reduced risk of adverse outcomes such as preterm birth, low birth weight, and other pregnancy complications. I completely agree that longitudinal metabolomic and metagenomic profiling throughout pregnancy would be highly informative for understanding how host–microbe metabolic exchange, more specifically of vitamins, evolves in this context. One practical challenge, however, at least in Belgium, is that pregnant individuals are routinely prescribed (multi)vitamin supplementation, given the increased nutritional requirements to support fetal development. In our ongoing clinical trial, we have observed that supplementation with stremely high doses of riboflavin (well above typical dietary intake) can reach the vaginal lumen. This might introduce a confounder when attempting to track endogenous vaginal vitamin levels during pregnancy. In our preprint here, we did not observe these associations with diet or supplementation, while a strong association with L. crispatus was seen. Of course, we would never ask pregnant participants to discontinue these supplements, as doing so would be unethical; their benefits for maternal and fetal health are well established. Nonetheless, this does highlight an important consideration for study design and interpretation in pregnancy-focused microbiome research.
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Riboflavin plays an important role in women’s health due to increased physiological demands linked to menstruation, fertility, pregnancy, and pelvic tissue integrity and is implicated in neurological and psychiatric conditions such as postpartum depression, and migraine
Do Lactobacillus species become reduced in the vaginal microbiome during pregnancy? It would be interesting to follow up on this work to understand how the host-microbe exchange of metabolites changes in pregnancy-shifted microbiomes.
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Our observation that exposure to riboflavin-overproducing lactobacilli significantly enhances 5,6-diMe-Ben, all trans retinal, coenzyme Q10, PABA, β-NAD and reduced glutathione in vaginal epithelial cells suggests a direct contribution of Lactobacillaceae to vaginal host redox homeostasis.
Do you suspect that these compounds originate exclusively from the lactobacilli in the vagina, or is it possible that some contribution is from microbial activity in the gut? Because there was no correlation between vitamin intake and the presence of vaginal lactobacilli, perhaps it's unlikely that the gut microbiome is contributing.
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