Optical coherence tomography-guided Brillouin microscopy highlights regional tissue stiffness differences during anterior neural tube closure in the Mthfd1l murine mutant.

Neurulation is a highly synchronized biomechanical process leading to the formation of the brain and spinal cord, and its failure leads to neural tube defects (NTDs). Although we are rapidly learning the genetic mechanisms underlying NTDs, the biomechanical aspects are largely unknown. To understand the correlation between NTDs and tissue stiffness during neural tube closure (NTC), we imaged an NTD murine model using optical coherence tomography (OCT), Brillouin microscopy and confocal fluorescence microscopy. Here, we associate structural information from OCT with local stiffness from the Brillouin signal of embryos undergoing neurulation. The stiffness of neuroepithelial tissues in Mthfd1l null embryos was significantly lower than that of wild-type embryos. Additionally, exogenous formate supplementation improved tissue stiffness and gross embryonic morphology in nullizygous and heterozygous embryos. Our results demonstrate the significance of proper tissue stiffness in normal NTC and pave the way for future studies on the mechanobiology of normal and abnormal embryonic development.

Formate-tetrahydrofolate ligase: supplying the cytosolic one-carbon network in roots with one-carbon units originating from glycolate.

The metabolism of tetrahydrofolate (H4PteGlun)-bound one-carbon (C1) units (C1 metabolism) is multifaceted and required for plant growth, but it is unclear what of many possible synthesis pathways provide C1 units in specific organelles and tissues. One possible source of C1 units is via formate-tetrahydrofolate ligase, which catalyzes the reversible ATP-driven production of 10-formyltetrahydrofolate (10-formyl-H4PteGlun) from formate and tetrahydrofolate (H4PteGlun). Here, we report biochemical and functional characterization of the enzyme from Arabidopsis thaliana (AtFTHFL). We show that the recombinant AtFTHFL has lower Km and kcat values with pentaglutamyl tetrahydrofolate (H4PteGlu5) as compared to monoglutamyl tetrahydrofolate (H4PteGlu1), resulting in virtually identical catalytic efficiencies for the two substrates. Stable transformation of Arabidopsis plants with the EGFP-tagged AtFTHFL, followed with fluorescence microscopy, demonstrated cytosolic signal. Two independent T-DNA insertion lines with impaired AtFTHFL function had shorter roots compared to the wild type plants, demonstrating the importance of this enzyme for root growth. Overexpressing AtFTHFL led to the accumulation of H4PteGlun + 5,10-methylene-H4PteGlun and serine, accompanied with the depletion of formate and glycolate, in roots of the transgenic Arabidopsis plants. This metabolic adjustment supports the hypothesis that AtFTHFL feeds the cytosolic C1 network in roots with C1 units originating from glycolate, and that these units are then used mainly for biosynthesis of serine, and not as much for the biosynthesis of 5-methyl-H4PteGlun, methionine, and S-adenosylmethionine. This finding has implications for any future attempts to engineer one-carbon unit-requiring products through manipulation of the one-carbon metabolic network in non-photosynthetic organs.

Orthogonal genome-wide screens of bat cells identify MTHFD1 as a target of broad antiviral therapy.

Bats are responsible for the zoonotic transmission of several major viral diseases, including those leading to the 2003 SARS outbreak and likely the ongoing COVID-19 pandemic. While comparative genomics studies have revealed characteristic adaptations of the bat innate immune system, functional genomic studies are urgently needed to provide a foundation for the molecular dissection of the viral tolerance in bats. Here we report the establishment of genome-wide RNA interference (RNAi) and CRISPR libraries for the screening of the model megabat, Pteropus alecto. We used the complementary RNAi and CRISPR libraries to interrogate P. alecto cells for infection with two different viruses: mumps virus and influenza A virus, respectively. Independent screening results converged on the endocytosis pathway and the protein secretory pathway as required for both viral infections. Additionally, we revealed a general dependence of the C1-tetrahydrofolate synthase gene, MTHFD1, for viral replication in bat cells and human cells. The MTHFD1 inhibitor, carolacton, potently blocked replication of several RNA viruses, including SARS-CoV-2. We also discovered that bats have lower expression levels of MTHFD1 than humans. Our studies provide a resource for systematic inquiry into the genetic underpinnings of bat biology and a potential target for developing broad-spectrum antiviral therapy.

Common Variants in One-Carbon Metabolism Genes (MTHFR, MTR, MTHFD1) and Depression in Gynecologic Cancers.

We investigated the association between methylenetetrahydrofolate reductase (gene MTHFR 677C>T, rs1801133), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR 2756A>G, rs1805087), and methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1 (gene MTHFD1 1958G>A, rs2236225)-well-studied functional variants involved in one-carbon metabolism-and gynecologic cancer risk, and the interaction between these polymorphisms and depression. A total of 200 gynecologic cancer cases and 240 healthy controls were recruited to participate in this study. Three single-nucleotide variants (SNVs) (rs1801133, rs1805087, rs2236225) were genotyped using the PCR-restriction fragment length polymorphism method. Depression was assessed in all patients using the Hamilton Depression Scale. Depression was statistically significantly more frequent in women with gynecologic cancers (69.5% vs. 34.2% in controls, p < 0.001). MTHFD1 rs2236225 was associated with an increased risk of gynecologic cancers (in dominant OR = 1.53, p = 0.033, and in log-additive models OR = 1.37, p = 0.024). Moreover, an association was found between depression risk and MTHFR rs1801133 genotypes in the controls but not in women with gynecologic cancers (in codominant model CC vs. TT: OR = 3.39, 95%: 1.49-7.74, p = 0.011). Cancers of the female reproductive system are associated with the occurrence of depression, and ovarian cancer may be associated with the rs2236225 variant of the MTHFD1 gene. In addition, in healthy aging women in the Polish population, the rs1801133 variant of the MTHFR gene is associated with depression.

Oncogenic Ras expression increases cellular formate production.

One-carbon units, critical intermediates for cell growth, may be produced by a variety of means, one of which is via the production of formate. Excessive formate accumulation, known as formate overflow and a characteristic of oxidative cancer, has been observed in cancer cells. However, the basis for this high rate of formate production is unknown. We examined the effect of elevated expression of oncogenic Ras (RasV12), on formate production in NIH-3T3 cells (mouse fibroblasts) cultured with either labelled 13C-serine or 13C-glycine. Formate accumulation by the fibroblasts transformed by RasV12 was increased two-threefold over those by vector control (Babe) cells. The production of formate exceeded the rate of utilization in both cell types. 13C-formate was produced almost exclusively from the #3 carbon of 13C-serine. Virtually no labelled formate was produced from either the #2 carbon of serine or the #2 carbon of glycine. The increased formate production by RasV12 cells was associated with increased mRNA abundances for enzymes of formate production in both the mitochondria and the cytosol. Thus, we find the oncogenic RasV12 significantly increases formate overflow and may be one way for tumor cells to produce one-carbon units required for enhanced proliferation of these cells and/or for other processes which have not been identified.

Melatonin modulates metabolic remodeling in HNSCC by suppressing MTHFD1L-formate axis.

Metabolic remodeling is now widely recognized as a hallmark of cancer, yet its role in head and neck squamous cell carcinoma (HNSCC) remains largely unknown. In this study, metabolomic analysis of melatonin-treated HNSCC cell lines revealed that exogenous melatonin inhibited many important metabolic pathways including folate cycle in HNSCC cells. Methylenetetrahydrofolate dehydrogenase 1 like (MTHFD1L), a metabolic enzyme of the folate cycle regulating the production of formate, was identified as a downstream target of melatonin. MTHFD1L was found to be markedly upregulated in HNSCC, and MTHFD1L overexpression was significantly associated with unfavorable clinical outcome of HNSCC patients. In addition, MTHFD1L promoted HNSCC progression in vitro and in vivo and reversed the oncostatic effects of exogenous melatonin. More importantly, the malignant phenotypes suppressed by knockdown of MTHFD1L or exogenous melatonin could be partially rescued by formate. Furthermore, we found that melatonin inhibited the expression of MTHFD1L in HNSCC cells through the downregulation of cyclic AMP-responsive element-binding protein 1 (CREB1) phosphorylation. Lastly, this novel regulatory axis of melatonin-p-CREB1-MTHFD1L-formate was also verified in HNSCC tissues. Collectively, our findings have demonstrated that MTHFD1L-formate axis promotes HNSCC progression and melatonin inhibits HNSCC progression through CREB1-mediated downregulation of MTHFD1L and formate. These findings have revealed new metabolic mechanisms in HNSCC and may provide novel insights on the therapeutic intervention of HNSCC.

Metabotype analysis of Mthfd1l-null mouse embryos using desorption electrospray ionization mass spectrometry imaging.

Mammalian folate-dependent one-carbon (1C) metabolism provides the building blocks essential during development via amino acid interconversion, methyl-donor production, regeneration of redox factors, and de novo purine and thymidylate synthesis. Folate supplementation prevents many neural tube defects (NTDs) that occur during the embryonic process of neurulation. The mechanism by which folate functions during neurulation is not well understood, and not all NTDs are preventable by folate supplementation. Mthfd1l is a mitochondrial 1C metabolism enzyme that produces formate, a 1C donor that fuels biosynthesis and the methyl cycle in the cytoplasm. Homozygous deletion of the Mthfd1l gene in mice (Mthfd1lz/z) causes embryonic lethality, developmental delay, and folate-resistant NTDs. These mice also have defects in cranial mesenchyme formation. In this work, mass spectrometry imaging was used to obtain ion maps of the cranial mesenchyme that identified the spatial distribution and relative abundance of metabolites in wild-type and Mthfd1lz/z embryos. The relative abundances of purine and thymidylate derivatives, as well as amino acids, were diminished in the cranial mesenchyme of Mthfd1lz/z embryos. Loss of Mthfd1l activity in this region also led to abnormal levels of methionine and dysregulated energy metabolism. These alterations in metabolism suggest possible approaches to preventing NTDs in humans.

hnRNPC regulates cancer-specific alternative cleavage and polyadenylation profiles.

Alternative cleavage and polyadenylation (APA) can occur at more than half of all human genes, greatly enhancing the cellular repertoire of mRNA isoforms. As these isoforms can have altered stability, localisation and coding potential, deregulation of APA can disrupt gene expression and this has been linked to many diseases including cancer progression. How APA generates cancer-specific isoform profiles and what their physiological consequences are, however, is largely unclear. Here we use a subcellular fractionation approach to determine the nuclear and cytoplasmic APA profiles of successive stages of colon cancer using a cell line-based model. Using this approach, we show that during cancer progression specific APA profiles are established. We identify that overexpression of hnRNPC has a critical role in the establishment of APA profiles characteristic for metastatic colon cancer cells, by regulating poly(A) site selection in a subset of genes that have been implicated in cancer progression including MTHFD1L.

Assimilation of formic acid and CO2 by engineered Escherichia coli equipped with reconstructed one-carbon assimilation pathways.

Gaseous one-carbon (C1) compounds or formic acid (FA) converted from CO2 can be an attractive raw material for bio-based chemicals. Here, we report the development of Escherichia coli strains assimilating FA and CO2 through the reconstructed tetrahydrofolate (THF) cycle and reverse glycine cleavage (gcv) pathway. The Methylobacterium extorquens formate-THF ligase, methenyl-THF cyclohydrolase, and methylene-THF dehydrogenase genes were expressed to allow FA assimilation. The gcv reaction was reversed by knocking out the repressor gene (gcvR) and overexpressing the gcvTHP genes. This engineered strain synthesized 96% and 86% of proteinogenic glycine and serine, respectively, from FA and CO2 in a glucose-containing medium. Native serine deaminase converted serine to pyruvate, showing 4.5% of pyruvate-forming flux comes from FA and CO2 The pyruvate-forming flux from FA and CO2 could be increased to 14.9% by knocking out gcvR, pflB, and serA, chromosomally expressing gcvTHP under trc, and overexpressing the reconstructed THF cycle, gcvTHP, and lpd genes in one vector. To reduce glucose usage required for energy and redox generation, the Candida boidinii formate dehydrogenase (Fdh) gene was expressed. The resulting strain showed specific glucose, FA, and CO2 consumption rates of 370.2, 145.6, and 14.9 mg⋅g dry cell weight (DCW)-1⋅h-1, respectively. The C1 assimilation pathway consumed 21.3 wt% of FA. Furthermore, cells sustained slight growth using only FA and CO2 after glucose depletion, suggesting that combined use of the C1 assimilation pathway and C. boidinii Fdh will be useful for eventually developing a strain capable of utilizing FA and CO2 without an additional carbon source such as glucose.

Biochemical properties and crystal structure of formate-tetrahydrofolate ligase from Methylobacterium extorquens CM4.

Methylobacterium extorquens is a methylotroph model organism that has the ability to assimilate formate using the tetrahydrofolate (THF) pathway. The formate-tetrahydrofolate ligase from M. extorquens (MeFtfL) is an enzyme involved in the THF pathway that catalyzes the conversion of formate, THF, and ATP into formyltetrahydrofolate and ADP. To investigate the biochemical properties of MeFtfL, we evaluated the metal usage and enzyme kinetics of the enzyme. MeFtfL uses the Mg ion for catalytic activity, but also has activity for Mn and Ca ions. The enzyme kinetics analysis revealed that Km value of farmate was much higher than THF and ATP, which shows that the ligation activity of MeFtfL is highly dependent on formation concentration. We also determined the crystal structure of MeFtfL at 2.8 Å resolution. MeFtfL functions as a tetramer, and each monomer consists of three domains. The structural superposition of MeFtfL with FtfL from Moorella thermoacetica allowed us to predict the substrate binding site of the enzyme.

MTHFD1L as a folate cycle enzyme correlates with prognostic outcome and its knockdown impairs cell invasive behaviors in osteosarcoma via mediating the AKT/mTOR pathway.

Osteosarcoma (OS) is the most frequent primary malignancy initially in bone with multiple genomic aberrations. Methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) is linked with the progression of diverse tumors. However, its function in OS is not understood completely. The expression pattern and prognostic significance of MTHFD1L in OS tissues were analyzed based on GEO database. The expression level of MTHFD1L in OS cell lines was explored by qRT-PCR. The cell proliferation, colony formation ability, invasion as well as migration in OS cells after MTHFD1L knockdown were determined using cell counting kit 8 (CCK-8) assay, colony formation and transwell methods. GSEA analysis was performed to predict the underlying mechanisms of MTHFD1L in OS development. Furthermore, the western blot was utilized to study the influence of MTHFD1L on AKT/mTOR pathway. Our results indicated that MTHFD1L expression was significantly up-regulated in OS tissues and cells compared with normal tissues and cells. High expression of MTHFD1L could lead to poor prognosis of OS patients. Cell proliferation, colony formation ability, migration and invasion were blocked because of reduced MTHFD1L in vitro. Moreover, cell cycle and AKT/mTOR pathway were all associated with MTHFD1L expression. In conclusion, the findings revealed that MTHFD1L might promote the development of OS via mediating cell cycle and AKT/mTOR pathway, indicating that MTHFD1L might act as a promising therapeutic target for OS treatment.

Arsenic trioxide targets MTHFD1 and SUMO-dependent nuclear de novo thymidylate biosynthesis.

Arsenic exposure increases risk for cancers and is teratogenic in animal models. Here we demonstrate that small ubiquitin-like modifier (SUMO)- and folate-dependent nuclear de novo thymidylate (dTMP) biosynthesis is a sensitive target of arsenic trioxide (As2O3), leading to uracil misincorporation into DNA and genome instability. Methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) and serine hydroxymethyltransferase (SHMT) generate 5,10-methylenetetrahydrofolate for de novo dTMP biosynthesis and translocate to the nucleus during S-phase, where they form a multienzyme complex with thymidylate synthase (TYMS) and dihydrofolate reductase (DHFR), as well as the components of the DNA replication machinery. As2O3 exposure increased MTHFD1 SUMOylation in cultured cells and in in vitro SUMOylation reactions, and increased MTHFD1 ubiquitination and MTHFD1 and SHMT1 degradation. As2O3 inhibited de novo dTMP biosynthesis in a dose-dependent manner, increased uracil levels in nuclear DNA, and increased genome instability. These results demonstrate that MTHFD1 and SHMT1, which are key enzymes providing one-carbon units for dTMP biosynthesis in the form of 5,10-methylenetetrahydrofolate, are direct targets of As2O3-induced proteolytic degradation, providing a mechanism for arsenic in the etiology of cancer and developmental anomalies.

Deletion of the neural tube defect-associated gene Mthfd1l disrupts one-carbon and central energy metabolism in mouse embryos.

One-carbon (1C) metabolism is a universal folate-dependent pathway essential for de novo purine and thymidylate synthesis, amino acid interconversion, universal methyl-donor production, and regeneration of redox cofactors. Homozygous deletion of the 1C pathway gene Mthfd1l encoding methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 1-like, which catalyzes mitochondrial formate production from 10-formyltetrahydrofolate, results in 100% penetrant embryonic neural tube defects (NTDs), underscoring the central role of mitochondrially derived formate in embryonic development and providing a mechanistic link between folate and NTDs. However, the specific metabolic processes that are perturbed by Mthfd1l deletion are not known. Here, we performed untargeted metabolomics on whole Mthfd1l-null and wildtype mouse embryos in combination with isotope tracer analysis in mouse embryonic fibroblast (MEF) cell lines to identify Mthfd1l deletion-induced disruptions in 1C metabolism, glycolysis, and the TCA cycle. We found that maternal formate supplementation largely corrects these disruptions in Mthfd1l-null embryos. Serine tracer experiments revealed that Mthfd1l-null MEFs have altered methionine synthesis, indicating that Mthfd1l deletion impairs the methyl cycle. Supplementation of Mthfd1l-null MEFs with formate, hypoxanthine, or combined hypoxanthine and thymidine restored their growth to wildtype levels. Thymidine addition alone was ineffective, suggesting a purine synthesis defect in Mthfd1l-null MEFs. Tracer experiments also revealed lower proportions of labeled hypoxanthine and inosine monophosphate in Mthfd1l-null than in wildtype MEFs, suggesting that Mthfd1l deletion results in increased reliance on the purine salvage pathway. These results indicate that disruptions of mitochondrial 1C metabolism have wide-ranging consequences for many metabolic processes, including those that may not directly interact with 1C metabolism.

EWS-FLI1 reprograms the metabolism of Ewing sarcoma cells via positive regulation of glutamine import and serine-glycine biosynthesis.

Ewing sarcoma (EWS) is a soft tissue and bone tumor that occurs primarily in adolescents and young adults. In most cases of EWS, the chimeric transcription factor, EWS-FLI1 is the primary oncogenic driver. The epigenome of EWS cells reflects EWS-FLI1 binding and activation or repression of transcription. Here, we demonstrate that EWS-FLI1 positively regulates the expression of proteins required for serine-glycine biosynthesis and uptake of the alternative nutrient source glutamine. Specifically, we show that EWS-FLI1 activates expression of PHGDH, PSAT1, PSPH, and SHMT2. Using cell-based studies, we also establish that EWS cells are dependent on glutamine for cell survival and that EWS-FLI1 positively regulates expression of the glutamine transporter, SLC1A5 and two enzymes involved in the one-carbon cycle, MTHFD2 and MTHFD1L. Inhibition of serine-glycine biosynthesis in EWS cells impacts their redox state leading to an accumulation of reactive oxygen species, DNA damage, and apoptosis. Importantly, analysis of EWS primary tumor transcriptome data confirmed that the aforementioned genes we identified as regulated by EWS-FLI1 exhibit increased expression compared with normal tissues. Furthermore, retrospective analysis of an independent data set generated a significant stratification of the overall survival of EWS patients into low- and high-risk groups based on the expression of PHGDH, PSAT1, PSPH, SHMT2, SLC1A5, MTHFD2, and MTHFD1L. In summary, our study demonstrates that EWS-FLI1 reprograms the metabolism of EWS cells and that serine-glycine metabolism or glutamine uptake are potential targetable vulnerabilities in this tumor type.

Low Dietary Folate Interacts with MTHFD1 Synthetase Deficiency in Mice, a Model for the R653Q Variant, to Increase Incidence of Developmental Delays and Defects.

Background: Suboptimal folate intake, a risk factor for birth defects, is common even in areas with folate fortification. A polymorphism in methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), R653Q (MTHFD1 c.1958 G > A), has also been associated with increased birth defect risk, likely through reduced purine synthesis. Objective: We aimed to determine if the interaction of MTHFD1 synthetase deficiency and low folate intake increases developmental abnormalities in a mouse model for MTHFD1 R653Q. Methods: Female Mthfd1S+/+ and Mthfd1S+/- mice were fed control or low-folate diets (2 and 0.3 mg folic acid/kg diet, respectively) before mating and during pregnancy. Embryos and placentas were examined for anomalies at embryonic day 10.5. Maternal 1-carbon metabolites were measured in plasma and liver. Results: Delays and defects doubled in litters of Mthfd1S+/- females fed low-folate diets compared to wild-type females fed either diet, or Mthfd1S+/- females fed control diets [P values (defects): diet 0.003, maternal genotype 0.012, diet × maternal genotype 0.014]. These adverse outcomes were associated with placental dysmorphology. Intrauterine growth restriction was increased by embryonic Mthfd1S+/- genotype, folate deficiency, and interaction of maternal Mthfd1S+/- genotype with folate deficiency (P values: embryonic genotype 0.045, diet 0.0081, diet × maternal genotype 0.0019). Despite a 50% increase in methylenetetrahydrofolate reductase expression in low-folate maternal liver (P diet = 0.0007), methyltetrahydrofolate concentration decreased 70% (P diet <0.0001) and homocysteine concentration doubled in plasma (P diet = 0.0001); S-adenosylmethionine decreased 40% and S-adenosylhomocysteine increased 20% in low-folate maternal liver (P diet = 0.002 and 0.0002, respectively). Conclusions: MTHFD1 synthetase-deficient mice are more sensitive to low folate intake than wild-type mice during pregnancy. Reduced purine synthesis due to synthetase deficiency and altered methylation potential due to low folate may increase pregnancy complications. Further studies and individualized intake recommendations may be required for women homozygous for the MTHFD1 R653Q variant.

The role of mitochondrial folate enzyme MTHFD1L in esophageal squamous cell carcinoma.

OBJECTIVE: The lack of novel therapeutic targets poses the major challenge to prolong survival and improve the quality of life for esophageal squamous cell carcinoma (ESCC). Methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) plays critical roles in folate cycle maintenance. However, little information is available concerning the role of MTHFD1L in cancer cells, and no studies have addressed such issues in esophageal cancer. MATERIALS AND METHOD: Surgical cancer and adjacent normal esophageal tissues were obtained from patients with esophagectomy and esophagogastrostomy for ESCC. Western blot, immunohistochemistry and Quantitative RT-PCR were performed to evaluate protein and RNA expression levels of MTHFD1L. Knockdown of MTHFD1L expression was achieved by using short hairpin RNA. The effects of MTHFD1L silencing on ESCC cell proliferation and apoptosis were assessed by the MTT assay, Celigo assays, Annexin V FACS assay and Caspase-3/7 array in vitro. RESULTS: Twenty-three paired cancer and adjacent normal esophageal tissues from patients with ESCC were included in this study. MTHFD1L protein and RNA expression levels were significantly upregulated in ESCC tissue as compared with normal tissue. High expression of MTHFD1 was also detected in two esophageal cancer cell lines (TE-1 and EC109). Knockdown of MTHFD1L expression inhibited the proliferation of TE-1 cells, and the apoptosis was distinctly increased following shMTHFD1L infection. CONCLUSIONS: Our preliminary study highlighted for the first time that MTHFD1L might be involved in the development of ESCC, which may provide a new potential tumor-specific therapeutic targeting for anti-folate agents.

Association of main folate metabolic pathway gene polymorphisms with neural tube defects in Han population of Northern China.

PURPOSE: Neural tube defects (NTDs) are one of the most prevalent and the most severe congenital malformations worldwide. Studies have confirmed that folic acid supplementation could effectively reduce NTDs risk, but the genetic mechanism remains unclear. In this study, we explored association of single nucleotide polymorphisms (SNP) within folate metabolic pathway genes with NTDs in Han population of Northern China. METHODS: We performed a case-control study to compare genotype and allele distributions of SNPs in 152 patients with NTDs and 169 controls. A total of 16 SNPs within five genes were genotyped by the Sequenom MassARRAY assay. RESULTS: Our results indicated that three SNPs associated significantly with NTDs (P<0.05). For rs2236225 within MTHFD1, children with allele A or genotype AA had a high NTDs risk (OR=1.500, 95%CI=1.061~2.120; OR=2.862, 95%CI=1.022~8.015, respectively). For rs1801133 within MTHFR, NTDs risk markedly increased in patients with allele T or genotype TT (OR=1.552, 95%CI=1.130~2.131; OR=2.344, 95%CI=1.233~4.457, respectively). For rs1801394 within MTRR, children carrying allele G and genotype GG had a higher NTDs risk (OR=1.533, 95%CI=1.102~2.188; OR=2.355, 95%CI=1.044~5.312, respectively). CONCLUSIONS: Our results suggest that rs2236225 of MTHFD1 gene, rs1801133 of MTHFR gene and rs1801394 of MTRR gene were associated with NTDs in Han population of Northern China.

Diversity of formyltetrahydrofolate synthetase genes in the rumens of roe deer (Capreolus pygargus) and sika deer (Cervus nippon) fed different diets.

Reductive acetogenesis by homoacetogens represents an alternative pathway to methanogenesis to remove metabolic hydrogen during rumen fermentation. In this study, we investigated the occurrence of homoacetogen in the rumens of pasture-fed roe deer (Capreolus pygargus) and sika deer (Cervus nippon) fed either oak-leaf-based (tannin-rich, 100 mg/kg dried matter), corn-stover-based, or corn-silage-based diets, by using formyltetrahydrofolate synthetase (FTHFS) gene sequences as a marker. The diversity and richness of FTHFS sequences was lowest in animals fed oak leaf, indicating that tannin-containing plants may affect rumen homoacetogen diversity. FTHFS amino acid sequences in the rumen of roe deer significantly differed from those of sika deer. The phylogenetic analyses showed that 44.8% of sequences in pasture-fed roe deer, and 72.1%, 81.1%, and 37.5% of sequences in sika deer fed oak-leaf-, corn-stover-, and corn-silage-based diets, respectively, may represent novel bacteria that have not yet been cultured. These results demonstrate that the rumens of roe deer and sika deer harbor potentially novel homoacetogens and that diet may influence homoacetogen community structure.

Physiological role of FolD (methylenetetrahydrofolate dehydrogenase), FchA (methenyltetrahydrofolate cyclohydrolase) and Fhs (formyltetrahydrofolate synthetase) from Clostridium perfringens in a heterologous model of Escherichia coli.

Most organisms possess bifunctional FolD [5,10-methylenetetrahydrofolate (5,10-CH2-THF) dehydrogenase-cyclohydrolase] to generate NADPH and 10-formyltetrahdrofolate (10-CHO-THF) required in various metabolic steps. In addition, some organisms including Clostridium perfringens possess another protein, Fhs (formyltetrahydrofolate synthetase), to synthesize 10-CHO-THF. Here, we show that unlike the bifunctional FolD of Escherichia coli (EcoFolD), and contrary to its annotated bifunctional nature, C. perfringens FolD (CpeFolD) is a monofunctional 5,10-CH2-THF dehydrogenase. The dehydrogenase activity of CpeFolD is about five times more efficient than that of EcoFolD. The 5,10-methenyltetrahydrofolate (5,10-CH+-THF) cyclohydrolase activity in C. perfringens is provided by another protein, FchA (5,10-CH+-THF cyclohydrolase), whose cyclohydrolase activity is ∼ 10 times more efficient than that of EcoFolD. Kinetic parameters for CpeFhs were also determined for utilization of all of its substrates. Both CpeFolD and CpeFchA are required to substitute for the single bifunctional FolD in E. coli. The simultaneous presence of CpeFolD and CpeFchA is also necessary to rescue an E. coli folD deletion strain (harbouring CpeFhs support) for its formate and glycine auxotrophies, and to alleviate its susceptibility to trimethoprim (an antifolate drug) or UV light. The presence of the three clostridial proteins (FolD, FchA and Fhs) is required to maintain folate homeostasis in the cell.

Formate-tetrahydrofolate ligase is involved in the virulence of Streptococcus suis serotype 2.

Streptococcus suis is an emerging zoonotic pathogen that causes severe infections in pigs and humans. However, the pathogenesis of S. suis remains unclear. The present study targeted a putative virulence-associated factor (fhs, encoding the formate-tetrahydrofolate ligase) of S. suis. To investigate the role of fhs in the virulence potential of S. suis serotype 2, an fhs deletion mutant (Δfhs) and the corresponding complementation strain (CΔfhs) were generated. The Δfhs mutant displayed similar growth compared to that of the wild-type and complementation strains. Using murine and pig infection models, we demonstrated for the first time that the formate-tetrahydrofolate ligase is required for the full virulence of S. suis 2. Our findings provide a new insight into the pathogenesis of S. suis 2.