Folic acid prevents C. elegans folate deficiency indirectly via bacterial uptake of a breakdown product: a route that can also increase bacterial toxicity and ageing

Supplementation with the synthetic oxidised folate, folic acid is used to prevent neural tube defects and other symptoms of folate deficiency. However, several unanswered questions remain over folic acid efficacy, safety and interactions with gut microbes. Prevention of a development defect caused by folate deficiency in the nematode worm Caenorhabditis elegans requires > 10 fold higher concentrations of folic acid compared to folinic acid, a reduced folate. Here we show that the major route for folic acid to restore normal development is indirect via the Escherichia coli used to feed C. elegans. This route occurs mainly via the E. coli transporter AbgT, which takes up the folic acid breakdown product para-aminobenzoate-glutamate (PABA-glu). We found that folic acid preparations, including a commercial supplement, contain 0.3- 4.0 % of this breakdown product. Previously, we have shown that inhibiting bacterial folate synthesis increases C. elegans lifespan by removing a life-shortening bacterial activity. Here, we show that folic acid restores bacterial folate synthesis and reverses this lifespan increase. It is still to be determined whether this bacterial route increases host folate levels in humans and if there are situations where increased bacterial folate synthesis has negative health complications.


INTRODUCTION
The folate cycle is a set of essential biosynthetic reactions known as one carbon metabolism (Ducker and Rabinowitz, 2017). Folates are a family of molecules with a central aromatic core derived from para-amino benzoic acid (PABA), a pterin ring that can be modified and chain of one or more glutamates (Green and Matthews, 2007). At each step of the folate cycle, an enzyme mediates a modification of the pterin ring of the bound folate that allows the transfer of a chemical group containing one carbon atom (methyl, formyl etc.) to or from the compound being synthesised (Ducker and Rabinowitz, 2017). Because of this cofactor role for folate molecules, there are recycled and only required in very small amounts. Animals cannot synthesise folates and must acquire them from their diet or gut microbes. A symptom of human folate deficiency is neural tube defects during embryonic development (Ducker and Rabinowitz, 2017). It has been found the rate of these defects can be lowered by preconception supplementation with folic acid, an oxidised form of folate not found in nature. In many countries including the US and Canada, there is mandatory fortification of flour with folic acid and this intervention has successfully decreased the incidence of birth defects (Bailey et al., 2015). There are some concerns that folic acid supplementation may have adverse effects (Kim, 2007;Marean et al., 2011;Pickell et al., 2011;Smith et al., 2008), and there are many unknowns about the efficacy of uptake and biological utilisation of the supplement (Gregory et al., 2005). However, recent reviews of the evidence by experts acting for government public health bodies have concluded that the risks are minimal and have recommended either the supplementation of flour or other food products as a beneficial intervention (Bailey et al., 2015;Boyles et al., 2016;Public_Health_England, 2017). These reviews do not mention the potential role of gut microbes in considering the mechanisms and safety of folic acid supplementation.
Research from our group and others has shown that inhibiting folate synthesis in Escherichia coli, either by treatment with the drug sulfamethoxazole (SMX) or mutation of the PABA synthesis pathway (e. g. a pabA or pabB mutant), extends the lifespan of the nematode Caenorhabditis elegans that feeds on it Virk et al., 2012;Virk et al., 2016). While these interventions decrease the folate levels in both E. coli and C. elegans, there remains sufficient folate available to support normal growth of both (Virk et al., 2012). Numerous lines of evidence suggest that lifespan is increased because inhibiting folate synthesis prevents a life-shortening activity of the bacteria rather than because animal folate levels are decreased (Virk et al., 2016). A mutant in the C. elegans homologue of the human GCPII enzyme (which cleaves glutamates from polyglutamated folates (Halsted et al., 1998)) shows delayed development and infertility on low folate E. coli (Virk et al., 2016). This defect is due to folate deficiency because it can be prevented by adding 1-10 µM folinic acid, a reduced folate. Prevention with folic acid requires much higher concentrations (100 µM) (Virk et al., 2016). This result may reflect the fact that the only characterised C. elegans folate transporter is FOLT-1, a reduced folate carrier that shows specificity for folinic acid (Balamurugan et al., 2007). However, we also discovered that at high concentrations folic acid can partially reverse the lifespan increase caused by inhibiting E. coli folate synthesis. This finding suggests that E. coli can take up folic acid (Virk et al., 2012). E. coli does not possess a folate transporter (Nickerson and Webb, 1956;Webb, 1955) but can take up the folic acid breakdown product PABA-glu through the transporter AbgT and catabolise it to PABA (Carter et al., 2007). PABA can also diffuse across E. coli membranes. PABA from either source can be used to synthesise folate.
In this study, we show that the prevention of delays in C. elegans development by folic acid depends on the E. coli AbgT transporter, demonstrating that bacterial uptake is the major route of folic acid supplementation. Consistent with this finding, we show that folic acid increases E. coli folate levels through the AbgT transporter and we find PABA-glu present in folic acid preparations. We also show that uptake of PABA-glu by AbgT can reverse the lifespan increase caused by inhibiting E. coli folate synthesis. Thus, this pathway can have both positive effects on development and negative consequences for ageing.

The major route of folic acid prevention of a C. elegans developmental defect requires the E. coli AbgT transporter
In order to test whether folic acid requires E. coli to prevent developmental defects in our C. elegans folate deficiency model, we tested whether the E. coli abgT genotype influenced the outcome of supplementation. We first established that the C. elegans gcp-2.1(ok1004) mutant grown on the E. coli pabA mutant on defined media (DM) has the same growth defect as these worms grown on wild type E. coli treated with 128 µg/ml SMX (Figure 1a, Virk et al. 2016). Addition of 2.5 µM folinic acid or higher prevented this growth defect regardless of the abgT genotype ( Figure 1b). In contrast, folic acid was found to increase gcp-2.1 mutant body length on the pabA mutant in a dose-dependent manner, achieving full growth only at 200 µM folic acid ( Figure 1c). On the E. coli abgTpabA double mutant, a much weaker response to folic acid was observed, whereas overexpression of abgT in the E. coli pabA mutant resulted in normal C. elegans growth at lower concentrations of folic acid (Figure 1c). Analysing the experiment by two-way ANOVA, we find that there is a significant interaction effect of abgT genotype (F=102.67, p<0.0001) and folic acid concentration (F=123.55, p<0.0001) on C. elegans gcp-2.1 body length. Mutation of abgT alone did not influence growth of the gcp-2.1 mutant (Supplementary Figure 1a). These results are consistent with folinic acid being taken up directly by the worm (Balamurugan et al., 2007), and that the major route of folic acid uptake requires E. coli and the E. coli AbgT transporter.

Folic acid supports growth of E. coli pabA mutants via abgT-dependent uptake of PABA-glu
The dependence on E. coli abgT for folic acid to rescue the gcp-2.1 developmental delay strongly suggests that PABA-glu is available to E. coli following folic acid supplementation. To test the relative ability of E. coli to take up PABA-glu and folic acid, we added these compounds to pabA, abgT pabA and pabA (abgT OE) E.
coli and assessed growth on DM agar plates in PABA-free conditions under which the pabA mutant showed slower growth than wild type. We found that growth in the presence of folic acid and PABA-glu depended on abgT expression; 10 µM folic acid rescued growth of the pabA mutant, whereas 100 µM was needed to achieve an equivalent rescue in the abgT pabA double mutant (Figure 2a). In the presence of 10 µM folic acid, growth of the pabA strain over-expressing AbgT was greater than of the pabA mutant alone or the wild type. Supplementation by PABA-glu had a similar effect to folic acid but at a 10-fold lower concentration ( Figure 2a). PABA, which can diffuse across biological membranes (Tran and Nichols, 1991),  elegans at L4 stage raised on DM agar plates seeded with WT E. coli (control), pabA mutant or WT E. coli treated with 128 μg/ml SMX. b) body length of gcp-2.1 mutant C. elegans at L4 stage raised on plates seeded with pabA mutant, abgTpabA double mutant or pabA mutant over-expressing abgT with increasing concentrations of folinic acid and c) folic acid: by two-way ANOVA analyses, we find that there is a significant interaction effect of strain type (F=102.67, p<0.0001) and folic acid concentration (F=123.55, p<0.0001) on C. elegans gcp-2.1 body length. Over-expression is conferred by transformation with a high copy number plasmid, pJ128. pabA and abgTpabA strain are transformed with the empty vector, pUC19. Error bars represent standard deviation of C. elegans body length; n ≥40.

Folic acid increases E. coli folate levels in an AbgT-dependent mechanism
It order to verify that E. coli growth following folic acid supplementation is attributable to restored bacterial folate synthesis, we used LC-MS/MS to detect E. coli folate levels under the conditions used in the above experiment. Levels of the most detectable and thus likely the most abundant THF species, 5-methyl THFglu 3 , are presented in Figure 2b. In the absence of folic acid, folate levels in the pabA mutant strains were significantly lower than in WT extracts (

Folic acid preparations contain PABA-glu and PABA
Together, the data presented here indicate that the main route of C. elegans folic acid supplementation is indirect via E. coli uptake of PABA-glu and PABA. We used LC-MS/MS to test for the presence of these breakdown products in folic acid preparations from Schircks (used in all other experiments in this study), Sigma Aldrich and Boots, a UK retailer. We also tested for further folic acid breakdown under the experimental conditions used here by analysing extracts from agar media supplemented with Schircks folic acid and incubated at 25 °C for 4 days. We detected PABA-glu in all three folic acid sources at between 0.3% (Schircks) and 4% (Boots, Figure 3). Under the conditions used for C. elegans experiments PABA-glu increased to 1.18%, suggesting further breakdown. PABA was found at between 0.01% (Schircks) and 0.06% (Boots, Figure 3).

Folic acid shortens C. elegans lifespan via AbgT-dependent uptake of PABA-glu during adulthood
Inhibiting bacterial folate synthesis, without affecting bacterial growth, is known to increase C. elegans lifespan (Virk et al., 2012;Virk et al., 2016). It was therefore hypothesized that factors that increase bacterial folate synthesis, such as folic acid (as shown here), may shorten C. elegans lifespan. Consistent with our previous findings (Virk et al., 2016), we find that C. elegans maintained on any E. coli pabA mutant are long-lived compared to C. elegans fed wild type E. coli ( Figure

DISCUSSION
In this study we have found that a major route of folic acid uptake by C. elegans is via E. coli. This route is dependent on the spontaneous breakdown of folic acid into PABA-glu and salvage of this product by the E.
coli PABA-glu transporter, AbgT. We found that this route led to increases in the levels of several folate species in E. coli following folic acid supplementation. This route can also lead to folic acid shortening C. elegans lifespan when worms are cultured on bacteria with impaired folate synthesis. The increase in folate synthesis caused by folic acid supplementation leads to a bacterial activity/toxicity that is harmful to the worm over the long term ( Figure 5).
Is it possible that this bacterial route for folic acid exists in humans? As far as we know, no other studies have tested folic acid supplements for the presence of PABA-glu or PABA, but several studies have reported issues with the stability and dissolution of commercial folic acid supplements (Hoag et al., 1997;Sculthorpe et al., 2001). In light of these issues, manufacturers have adopted a policy of 'overages' in order to ensure sufficient folic acid is released in the small intestine following ingestion (Andrews et al., 2017). The presence of PABA-glu and PABA in a commercially available folic acid source (Figure 3) combined the instability of folic acid at the low pH conditions of the stomach, makes it likely that PABA-glu and PABA will be available to the gut microbiota following supplementation. PABA has been identified as a human fecal excretory product after ingestion of folic acid (Denko et al.). The abgT gene is widespread in the human gut microbiota and studies in rodents (Rong et al., 1991) and piglets (Asrar and O'Connor, 2005) have demonstrated that infusion of labelled PABA into the cecum results in the incorporation of bacterially synthesized folate in host tissues. Thus the literature suggests that the components for such route exists in humans, but whether this accounts for significant amounts of folic acid is yet to be seen. We could only detect this route in C. elegans because of the poor bioavailability of folic acid in our folate deficiency model. Folic acid is taken up well by humans and leads to increases in serum folate levels, but folic acid is often found in the oxidised form, suggesting that it is not always bioavailable (Bailey et al., 2015;Gregory et al., 2005;Patanwala et al., 2014).
Further studies are required in order to determine whether folic acid supplementation affects the folate status of human gut microbes and whether this in turn impacts host health. Interestingly, there are several diseases associated with an increased abundance of folate-synthesizing gut bacteria, such as inflammatory bowel disease (Shin et al., 2015) and small intestinal bacterial overgrowth (Camilo et al., 1996), but a causal relationship between bacterial folate and disease has not been established. The abgT gene is found in the genomes of several enteric pathogens, including Enterobacter cloacae, N. gonorrhoeae, Salmonella enterica, Shigella boydii and Staphylococcus aureus in addition to E. coli. Whilst there is much consideration about the consequences of folic acid supplementation (Bailey et al., 2015;Boyles et al., 2016;Public_Health_England, 2017), our work here indicates that folic acid supplement instability and bacterial genotype are previously unexplored variables that may impact human health and thus warrant consideration in future reports and studies.

Figure 5. Schematic of the impact of folic acid supplementation on C. elegans via indirect uptake of breakdown products by E. coli.
Folic acid is not taken up well by C. elegans directly. We find that the major uptake of folic acid by C. elegans is dependent on its breakdown into PABA-glu and uptake by the E. coli AbgT transporter. This route increases bacterial folate synthesis in both wild-type and pabA mutant E. coli. Under conditions of folate-deficiency (pabA mutant E. coli), increasing bacterial folate via this route is beneficial for C. elegans development. During C. elegans adulthood, this route has a negative impact on longevity as it promotes a bacterial folate-dependent toxicity.

Culture conditions
Defined media (DM) was prepared as described (Virk et al., 2016), except that 10 nM B12 was added. B12, folic acid and antibiotics are added post-autoclaving for agar plates. DM for liquid culture is filter-sterilised.
0.1 µM PABA added to the liquid DM media used to seed the plates in order to maintain bacterial growth (apart the growth experiments in Figure 2). 30 µl of 3 ml fresh overnight LB culture is used to inoculate 5ml DM (15 ml Falcon tube). 25 µg/ml kanamycin (50 ug/ml ampicillin if necessary) added to both LB and DM pre-incubation. DM liquid cultures are incubated for 18 hr at 37°C, 220 RPM.

C. elegans strains used
SS104 glp-4(bn2), UF208 (wild-type), and UF209 gcp-2.1(ok1004) (Virk et al., 2016). onto the plate and a glass spreader to scrape off the bacterial lawn. The bacterial suspension was pipetted into a 1.5 ml Eppendorf and the volume was recorded (v). Tubes were vortexed vigorously to obtain a homogenised solution. 100 µl was taken and diluted with 900 µl M9 in a cuvette. A spectrophotometer was used to read bacterial growth at 600 nm. Bacterial growth was calculated by multiplying OD 600 by the volume of the sample (v).

E. coli folate extraction
Bacterial lawns were scraped from plates into micro centrifuge tubes using M9 solution and kept on ice.
Volume (v), multiplied by the OD 600 of the solution (diluted 1:5) gives a measure of the amount of material.
Samples were concentrated in chilled microcentrifuge and pellets were snap frozen in liquid nitrogen.
Samples were vortexed vigorously and left on ice for 15 min before centrifugation in a cooled microcentrifuge for 15 min at full speed. Supernatants were used for analysis.

Folate LC-MS/MS analysis
Folates were detected by multiple reaction monitoring (MRM) analysis using a SCIEX QTRAP 6500 instrument. MRM conditions for folic acid, PABA, PABA-Glu, 5-Me-H 4 PteGlu 3 (5-methylTHF-Glu 3 ) and 5/10-CHO-H 4 PteGlu 3 (formyl THF 3 ) were optimised by infusion of standards into the instrument. The optimised conditions for -Glu 3 folates were applied to other higher folates using MRM transitions described by Lu et al., 2007(Lu et al., 2007. Further confirmation of identity for folates of interest was achieved by performing enhanced product ion scans and comparing the fragment spectra with known standards.
The QTRAP 6500 was operated in ESI+ mode and was interfaced with a Shimadzu Nexera UHPLC system.
Samples were separated using a Thermo PA2 C18 column (2.2 µm, 2.1 x 100 mm) with a gradient of 0.1% formic acid in water (mobile phase A) and acetonitrile (mobile phase B). Samples were maintained at 4⁰C and 2 µL aliquots were injected. The column was maintained at 40⁰C with a flow rate of 200 µL/min, starting at 2% B, held for 2 minutes, with a linear gradient to 100% B at 7 minutes, held for 1 minute, before a 7-minute re-equilibration step at 2% B that was necessary for consistent retention times. The column eluate flow to the MS was controlled via the QTRAP switching valve, allowing analysis between 4 and 8 minutes to minimise instrument contamination. Folates were quantified with reference to external standards and matrix effects were assessed by spiking of standards into extracted samples.

Lifespan analysis
Survival analyses were performed as described (Virk et al., 2012). glp-4(bn2) worms were maintained at 15°C and shifted to 25°C at the L3 stage. At the L4/young adult stage, animals were placed on bacteria under the experimental conditions. All lifespan data is in Table S1.