A Dual-Mechanism Antibiotic Kills Gram-Negative Bacteria and Avoids Drug Resistance.

The rise of antibiotic resistance and declining discovery of new antibiotics has created a global health crisis. Of particular concern, no new antibiotic classes have been approved for treating Gram-negative pathogens in decades. Here, we characterize a compound, SCH-79797, that kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism of action (MoA) with undetectably low resistance frequencies. To characterize its MoA, we combined quantitative imaging, proteomic, genetic, metabolomic, and cell-based assays. This pipeline demonstrates that SCH-79797 has two independent cellular targets, folate metabolism and bacterial membrane integrity, and outperforms combination treatments in killing methicillin-resistant Staphylococcus aureus (MRSA) persisters. Building on the molecular core of SCH-79797, we developed a derivative, Irresistin-16, with increased potency and showed its efficacy against Neisseria gonorrhoeae in a mouse vaginal infection model. This promising antibiotic lead suggests that combining multiple MoAs onto a single chemical scaffold may be an underappreciated approach to targeting challenging bacterial pathogens.

The folate way to T cell fate.

The role of folate-dependent one carbon (1C) metabolism in CD4+ T cell polarization is incompletely understood. In this issue of Immunity, Sugiura et al. (2021) provide evidence that blocking the 1C metabolic enzyme MTHFD2 may curb pro-inflammatory CD4+ T cells, while redirecting them toward a regulatory T cell phenotype.

Mutation in folate metabolism causes epigenetic instability and transgenerational effects on development.

The importance of maternal folate consumption for normal development is well established, yet the molecular mechanism linking folate metabolism to development remains poorly understood. The enzyme methionine synthase reductase (Mtrr) is necessary for utilization of methyl groups from the folate cycle. We found that a hypomorphic mutation of the mouse Mtrr gene results in intrauterine growth restriction, developmental delay, and congenital malformations, including neural tube, heart, and placental defects. Importantly, these defects were dependent upon the Mtrr genotypes of the maternal grandparents. Furthermore, we observed widespread epigenetic instability associated with altered gene expression in the placentas of wild-type grandprogeny of Mtrr-deficient maternal grandparents. Embryo transfer experiments revealed that Mtrr deficiency in mice lead to two distinct, separable phenotypes: adverse effects on their wild-type daughters' uterine environment, leading to growth defects in wild-type grandprogeny, and the appearance of congenital malformations independent of maternal environment that persist for five generations, likely through transgenerational epigenetic inheritance.

Metformin retards aging in C. elegans by altering microbial folate and methionine metabolism.

The biguanide drug metformin is widely prescribed to treat type 2 diabetes and metabolic syndrome, but its mode of action remains uncertain. Metformin also increases lifespan in Caenorhabditis elegans cocultured with Escherichia coli. This bacterium exerts complex nutritional and pathogenic effects on its nematode predator/host that impact health and aging. We report that metformin increases lifespan by altering microbial folate and methionine metabolism. Alterations in metformin-induced longevity by mutation of worm methionine synthase (metr-1) and S-adenosylmethionine synthase (sams-1) imply metformin-induced methionine restriction in the host, consistent with action of this drug as a dietary restriction mimetic. Metformin increases or decreases worm lifespan, depending on E. coli strain metformin sensitivity and glucose concentration. In mammals, the intestinal microbiome influences host metabolism, including development of metabolic disease. Thus, metformin-induced alteration of microbial metabolism could contribute to therapeutic efficacy-and also to its side effects, which include folate deficiency and gastrointestinal upset.

Recognition of cyclic dinucleotides and folates by human SLC19A1.

Cyclic dinucleotides (CDNs) are ubiquitous signalling molecules in all domains of life1,2. Mammalian cells produce one CDN, 2'3'-cGAMP, through cyclic GMP-AMP synthase after detecting cytosolic DNA signals3-7. 2'3'-cGAMP, as well as bacterial and synthetic CDN analogues, can act as second messengers to activate stimulator of interferon genes (STING) and elicit broad downstream responses8-21. Extracellular CDNs must traverse the cell membrane to activate STING, a process that is dependent on the solute carrier SLC19A122,23. Moreover, SLC19A1 represents the major transporter for folate nutrients and antifolate therapeutics24,25, thereby placing SLC19A1 as a key factor in multiple physiological and pathological processes. How SLC19A1 recognizes and transports CDNs, folate and antifolate is unclear. Here we report cryo-electron microscopy structures of human SLC19A1 (hSLC19A1) in a substrate-free state and in complexes with multiple CDNs from different sources, a predominant natural folate and a new-generation antifolate drug. The structural and mutagenesis results demonstrate that hSLC19A1 uses unique yet divergent mechanisms to recognize CDN- and folate-type substrates. Two CDN molecules bind within the hSLC19A1 cavity as a compact dual-molecule unit, whereas folate and antifolate bind as a monomer and occupy a distinct pocket of the cavity. Moreover, the structures enable accurate mapping and potential mechanistic interpretation of hSLC19A1 with loss-of-activity and disease-related mutations. Our research provides a framework for understanding the mechanism of SLC19-family transporters and is a foundation for the development of potential therapeutics.

Methotrexate recognition by the human reduced folate carrier SLC19A1.

Folates are essential nutrients with important roles as cofactors in one-carbon transfer reactions, being heavily utilized in the synthesis of nucleic acids and the metabolism of amino acids during cell division1,2. Mammals lack de novo folate synthesis pathways and thus rely on folate uptake from the extracellular milieu3. The human reduced folate carrier (hRFC, also known as SLC19A1) is the major importer of folates into the cell1,3, as well as chemotherapeutic agents such as methotrexate4-6. As an anion exchanger, RFC couples the import of folates and antifolates to anion export across the cell membrane and it is a major determinant in methotrexate (antifolate) sensitivity, as genetic variants and its depletion result in drug resistance4-8. Despite its importance, the molecular basis of substrate specificity by hRFC remains unclear. Here we present cryo-electron microscopy structures of hRFC in the apo state and captured in complex with methotrexate. Combined with molecular dynamics simulations and functional experiments, our study uncovers key determinants of hRFC transport selectivity among folates and antifolate drugs while shedding light on important features of anion recognition by hRFC.

EBF1 and Pax5 safeguard leukemic transformation by limiting IL-7 signaling, Myc expression, and folate metabolism.

EBF1 and PAX5 mutations are associated with the development of B progenitor acute lymphoblastic leukemia (B-ALL) in humans. To understand the molecular networks driving leukemia in the Ebf1+/-Pax5+/- (dHet) mouse model for B-ALL, we interrogated the transcriptional profiles and chromatin status of leukemic cells, preleukemic dHet pro-B, and wild-type pro-B cells with the corresponding EBF1 and Pax5 cistromes. In dHet B-ALL cells, many EBF1 and Pax5 target genes encoding pre-BCR signaling components and transcription factors were down-regulated, whereas Myc and genes downstream from IL-7 signaling or associated with the folate pathway were up-regulated. We show that blockade of IL-7 signaling in vivo and methotrexate treatment of leukemic cells in vitro attenuate the expansion of leukemic cells. Single-cell RNA-sequencing revealed heterogeneity of leukemic cells and identified a subset of wild-type pro-B cells with reduced Ebf1 and enhanced Myc expression that show hallmarks of dHet B-ALL cells. Thus, EBF1 and Pax5 may safeguard early stage B cells from transformation to B-ALL by limiting IL-7 signaling, folate metabolism and Myc expression.

Homocysteine Modification in Protein Structure/Function and Human Disease.

Epidemiological studies established that elevated homocysteine, an important intermediate in folate, vitamin B12, and one carbon metabolism, is associated with poor health, including heart and brain diseases. Earlier studies show that patients with severe hyperhomocysteinemia, first identified in the 1960s, exhibit neurological and cardiovascular abnormalities and premature death due to vascular complications. Although homocysteine is considered to be a nonprotein amino acid, studies over the past 2 decades have led to discoveries of protein-related homocysteine metabolism and mechanisms by which homocysteine can become a component of proteins. Homocysteine-containing proteins lose their biological function and acquire cytotoxic, proinflammatory, proatherothrombotic, and proneuropathic properties, which can account for the various disease phenotypes associated with hyperhomocysteinemia. This review describes mechanisms by which hyperhomocysteinemia affects cellular proteostasis, provides a comprehensive account of the biological chemistry of homocysteine-containing proteins, and discusses pathophysiological consequences and clinical implications of their formation.

A combination of chlorzoxazone and folic acid improves recognition memory, anxiety and depression in SCA3-84Q mice.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is reported to be the most common type of autosomal dominant cerebellar ataxia (ADCA). SCA3 patients suffer from a progressive decline in motor coordination and other disease-associated symptoms. Moreover, recent studies have reported that SCA3 patients also exhibit symptoms of cerebellar cognitive affective syndrome (CCAS). We previously observed signs of CCAS in mouse model of SCA3. Particularly, SCA3-84Q mice suffer from anxiety, recognition memory decline, and also exhibit signs of low mood and aversion to activity. Here we studied the effect of long-term injections of SK channels activator chlorzoxazone (CHZ) together and separately with the folic acid (FA) on the cerebellar Purkinje cell (PC) firing and histology, and also on the motor and cognitive functions as well as mood alterations in SCA3-84Q hemizygous transgenic mice. We realized that both CHZ and CHZ-FA combination had similar positive effect on pure cerebellum impairments including PC firing precision, PC histology, and motor performance in SCA3-84Q mice. However, only the CHZ-FA combination, but not CHZ, had significantly ameliorated the signs of anxiety and depression, and also noticeably improved recognition memory in SCA3-84Q mice. Our results suggest that the combination therapy for both ataxia and non-motor symptoms is required for the complex treatment of ADCA.

Dihydrofolate reductase activity controls neurogenic transitions in the developing neocortex.

One-carbon/folate (1C) metabolism supplies methyl groups required for DNA and histone methylation, and is involved in the maintenance of self-renewal in stem cells. Dihydrofolate reductase (DHFR), a key enzyme in 1C metabolism, is highly expressed in human and mouse neural progenitors at the early stages of neocortical development. Here, we have investigated the role of DHFR in the developing neocortex and report that reducing its activity in human neural organoids and mouse embryonic neocortex accelerates indirect neurogenesis, thereby affecting neuronal composition of the neocortex. Furthermore, we show that decreasing DHFR activity in neural progenitors leads to a reduction in one-carbon/folate metabolites and correlates with modifications of H3K4me3 levels. Our findings reveal an unanticipated role for DHFR in controlling specific steps of neocortex development and indicate that variations in 1C metabolic cues impact cell fate transitions.

Role of riboflavin deficiency in malaria pathophysiology.

The emergence of resistance against antimalarials and insecticides poses a significant threat to malaria elimination strategies. It is crucial to explore potential risk factors for malaria to identify new targets and alternative therapies. Malnutrition is a well-established risk factor for malaria. Deficiencies of micronutrients such as vitamin A, zinc, iron, folic acid, and phenotypic measures of malnutrition, such as stunting and wasting, have been studied extensively in the context of malaria. Vitamin B2, also known as riboflavin, is a micronutrient involved in maintaining cellular homeostasis. Riboflavin deficiency has been shown to have an inverse correlation with malarial parasitaemia. This article reviews the role of riboflavin in maintaining redox homeostasis and probes how riboflavin deficiency could alter malaria pathogenesis by disrupting the balance between oxidants and antioxidants. Though riboflavin analogues have been explored as antimalarials, new in vivo and patient-based research is required to target riboflavin-associated pathways for antimalarial therapy.

Dietary modifications for enhanced cancer therapy.

Tumours depend on nutrients supplied by the host for their growth and survival. Modifications to the host's diet can change nutrient availability in the tumour microenvironment, which might represent a promising strategy for inhibiting tumour growth. Dietary modifications can limit tumour-specific nutritional requirements, alter certain nutrients that target the metabolic vulnerabilities of the tumour, or enhance the cytotoxicity of anti-cancer drugs. Recent reports have suggested that modification of several nutrients in the diet can alter the efficacy of cancer therapies, and some of the newest developments in this quickly expanding field are reviewed here. The results discussed indicate that the dietary habits and nutritional state of a patient must be taken into account during cancer research and therapy.

A sub-10-nm, folic acid-conjugated gold nanoparticle as self-therapeutic treatment of tubulointerstitial fibrosis.

Nanomedicines for treating chronic kidney disease (CKD) are on the horizon, yet their delivery to renal tubules where tubulointerstitial fibrosis occurs remains inefficient. We report a folic acid-conjugated gold nanoparticle that can transport into renal tubules and treat tubulointerstitial fibrosis in mice with unilateral ureteral obstruction. The 3-nm gold core allows for the dissection of bio-nano interactions in the fibrotic kidney, ensures the overall nanoparticle (~7 nm) to be small enough for glomerular filtration, and naturally inhibits the p38α mitogen-activated protein kinase in the absence of chemical or biological drugs. The folic acids support binding to selected tubule cells with overexpression of folate receptors and promote retention in the fibrotic kidney. Upon intravenous injection, this nanoparticle can selectively accumulate in the fibrotic kidney over the nonfibrotic contralateral kidney at ~3.6% of the injected dose. Delivery to the fibrotic kidney depends on nanoparticle size and disease stage. Notably, a single injection of this self-therapeutic nanoparticle reduces tissue degeneration, inhibits genes related to the extracellular matrix, and treats fibrosis more effectively than standard Captopril therapy. Our data underscore the importance of constructing CKD nanomedicines based on renal pathophysiology.

Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryoelectron microscopy.

Cobalamin-dependent methionine synthase (MetH) catalyzes the synthesis of methionine from homocysteine and 5-methyltetrahydrofolate (CH3-H4folate) using the unique chemistry of its cofactor. In doing so, MetH links the cycling of S-adenosylmethionine with the folate cycle in one-carbon metabolism. Extensive biochemical and structural studies on Escherichia coli MetH have shown that this flexible, multidomain enzyme adopts two major conformations to prevent a futile cycle of methionine production and consumption. However, as MetH is highly dynamic as well as both a photosensitive and oxygen-sensitive metalloenzyme, it poses special challenges for structural studies, and existing structures have necessarily come from a "divide and conquer" approach. In this study, we investigate E. coli MetH and a thermophilic homolog from Thermus filiformis using small-angle X-ray scattering (SAXS), single-particle cryoelectron microscopy (cryo-EM), and extensive analysis of the AlphaFold2 database to present a structural description of the full-length MetH in its entirety. Using SAXS, we describe a common resting-state conformation shared by both active and inactive oxidation states of MetH and the roles of CH3-H4folate and flavodoxin in initiating turnover and reactivation. By combining SAXS with a 3.6-Å cryo-EM structure of the T. filiformis MetH, we show that the resting-state conformation consists of a stable arrangement of the catalytic domains that is linked to a highly mobile reactivation domain. Finally, by combining AlphaFold2-guided sequence analysis and our experimental findings, we propose a general model for functional switching in MetH.

Methylenetetrahydrofolate reductase deficiency and high-dose FA supplementation disrupt embryonic development of energy balance and metabolic homeostasis in zebrafish.

Folic acid (synthetic folate, FA) is consumed in excess in North America and may interact with common pathogenic variants in methylenetetrahydrofolate reductase (MTHFR); the most prevalent inborn error of folate metabolism with wide-ranging obesity-related comorbidities. While preclinical murine models have been valuable to inform on diet-gene interactions, a recent Folate Expert panel has encouraged validation of new animal models. In this study, we characterized a novel zebrafish model of mthfr deficiency and evaluated the effects of genetic loss of mthfr function and FA supplementation during embryonic development on energy homeostasis and metabolism. mthfr-deficient zebrafish were generated using CRISPR mutagenesis and supplemented with no FA (control, 0FA) or 100 µm FA (100FA) throughout embryonic development (0-5 days postfertilization). We show that the genetic loss of mthfr function in zebrafish recapitulates key biochemical hallmarks reported in MTHFR deficiency in humans and leads to greater lipid accumulation and aberrant cholesterol metabolism as reported in the Mthfr murine model. In mthfr-deficient zebrafish, energy homeostasis was also impaired as indicated by altered food intake, reduced metabolic rate and lower expression of central energy-regulatory genes. Microglia abundance, involved in healthy neuronal development, was also reduced. FA supplementation to control zebrafish mimicked many of the adverse effects of mthfr deficiency, some of which were also exacerbated in mthfr-deficient zebrafish. Together, these findings support the translatability of the mthfr-deficient zebrafish as a preclinical model in folate research.

Folate supports IL-25-induced tuft cell expansion following enteroviral infections.

Intestinal tuft cells, a kind of epithelial immune cells, rapidly expand in response to pathogenic infections, which is associated with infection-induced interleukin 25 (IL-25) upregulation. However, the metabolic mechanism of IL-25-induced tuft cell expansion is largely unknown. Folate metabolism provides essential purine and methyl substrates for cell proliferation and differentiation. Thus, we aim to investigate the roles of folate metabolism playing in IL-25-induced tuft cell expansion by enteroviral infection and recombinant murine IL-25 (rmIL-25) protein-stimulated mouse models. At present, enteroviruses, such as EV71, CVA16, CVB3, and CVB4, upregulated IL-25 expression and induced tuft cell expansion in the intestinal tissues of mice. However, EV71 did not induce intestinal tuft cell expansion in IL-25-/- mice. Interestingly, compared to the mock group, folate was enriched in the intestinal tissues of both the EV71-infected group and the rmIL-25 protein-stimulated group. Moreover, folate metabolism supported IL-25-induced tuft cell expansion since both folate-depletion and anti-folate MTX-treated mice had a disrupted tuft cell expansion in response to rmIL-25 protein stimulation. In summary, our data suggested that folate metabolism supported intestinal tuft cell expansion in response to enterovirus-induced IL-25 expression, which provided a new insight into the mechanisms of tuft cell expansion from the perspective of folate metabolism.

Exposure to prenatal excess or imbalanced micronutrients leads to long-term perturbations in one-carbon metabolism, trimethylamine-N-oxide and DNA methylation in Wistar rat offspring.

Prenatal multivitamins, including folic acid, are commonly consumed in excess, whereas choline, an essential nutrient and an important source of labile methyl groups, is underconsumed. Here, we characterized profiles of one-carbon metabolism and related pathways and patterns of DNA methylation in offspring exposed to excess or imbalanced micronutrients prenatally. Pregnant Wistar rats were fed either recommended 1× vitamins (RV), high 10× vitamins (HV), high 10× folic acid with recommended choline (HFolRC), or high 10× folic acid with no choline (HFolNC). Offspring were weaned to a high-fat diet for 12 weeks. Circulating metabolites were analyzed with a focus on the hypothalamus, an area known to be under epigenetic regulation. HV, HFolRC, and HFolNC males had higher body weight (BW) and lower plasma choline and methionine consistent with lower hypothalamic S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) and global DNA methylation compared with RV. HV and HFolNC females had higher BW and lower plasma 5-methyltetrahydrofolate and methionine consistent with lower hypothalamic global DNA methylation compared with RV. Plasma dimethylglycine (DMG) and methionine were higher as with hypothalamic SAM:SAH and global DNA methylation in HFolRC females without changes in BW compared with RV. Plasma trimethylamine and trimethylamine-N-oxide were higher in males but lower in females from HFolRC compared with RV. Network modeling revealed a link between the folate-dependent pathway and SAH, with most connections through DMG. Final BW was negatively correlated with choline, DMG, and global DNA methylation. In conclusion, prenatal intake of excess or imbalanced micronutrients induces distinct metabolic and epigenetic perturbations in offspring that reflect long-term nutritional programming of health.

Folic acid antioxidant supplementation to binge drinking adolescent rats improves hydric-saline balance and blood pressure, but fails to increase renal NO availability and glomerular filtration rate.

Binge drinking (BD) is an especially pro-oxidant pattern of alcohol consumption, particularly widespread in the adolescent population. In the kidneys, it affects the glomerular filtration rate (GFR), leading to high blood pressure. BD exposure also disrupts folic acid (FA) homeostasis and its antioxidant properties. The aim of this study is to test a FA supplementation as an effective therapy against the oxidative, nitrosative, and apoptotic damage as well as the renal function alteration occurred after BD in adolescence. Four groups of adolescent rats were used: control, BD (exposed to intraperitoneal alcohol), control FA-supplemented group and BD FA-supplemented group. Dietary FA content in control groups was 2 ppm, and 8 ppm in supplemented groups. BD provoked an oxidative imbalance in the kidneys by dysregulating antioxidant enzymes and increasing the enzyme NADPH oxidase 4 (NOX4), which led to an increase in caspase-9. BD also altered the renal nitrosative status affecting the expression of the three nitric oxide (NO) synthase (NOS) isoforms, leading to a decrease in NO levels. Functionally, BD produced a hydric-electrolytic imbalance, a low GFR and an increase in blood pressure. FA supplementation to BD adolescent rats improved the oxidative, nitrosative, and apoptotic balance, recovering the hydric-electrolytic equilibrium and blood pressure. However, neither NO levels nor GFR were recovered, showing in this study for the first time that NO availability in the kidneys plays a crucial role in GFR regulation that the antioxidant effects of FA cannot repair.

Association of embryonic inositol status with susceptibility to neural tube defects, metabolite profile, and maternal inositol intake.

Maternal nutrition contributes to gene-environment interactions that influence susceptibility to common congenital anomalies such as neural tube defects (NTDs). Supplemental myo-inositol (MI) can prevent NTDs in some mouse models and shows potential for prevention of human NTDs. We investigated effects of maternal MI intake on embryonic MI status and metabolism in curly tail mice, which are genetically predisposed to NTDs that are inositol-responsive but folic acid resistant. Dietary MI deficiency caused diminished MI in maternal plasma and embryos, showing that de novo synthesis is insufficient to maintain MI levels in either adult or embryonic mice. Under normal maternal dietary conditions, curly tail embryos that developed cranial NTDs had significantly lower MI content than unaffected embryos, revealing an association between diminished MI status and failure of cranial neurulation. Expression of inositol-3-phosphate synthase 1, required for inositol biosynthesis, was less abundant in the cranial neural tube than at other axial levels. Supplemental MI or d-chiro-inositol (DCI) have previously been found to prevent NTDs in curly tail embryos. Here, we investigated the metabolic effects of MI and DCI treatments by mass spectrometry-based metabolome analysis. Among inositol-responsive metabolites, we noted a disproportionate effect on nucleotides, especially purines. We also found altered proportions of 5-methyltetrahydrolate and tetrahydrofolate in MI-treated embryos suggesting altered folate metabolism. Treatment with nucleotides or the one-carbon donor formate has also been found to prevent NTDs in curly tail embryos. Together, these findings suggest that the protective effect of inositol may be mediated through the enhanced supply of nucleotides during neural tube closure.

Folate regulation of planar cell polarity pathway and F-actin through folate receptor alpha.

Folate deficiency contribute to neural tube defects (NTDs) which could be rescued by folate supplementation. However, the underlying mechanisms are still not fully understood. Besides, there is considerable controversy concerning the forms of folate used for supplementation. To address this controversy, we prepared culture medium with different forms of folate, folic acid (FA), and 5-methyltetrahydrofolate (5mTHF), at concentrations of 5 µM, 500 nM, 50 nM, and folate free, respectively. Mouse embryonic fibroblasts (MEFs) were treated with different folates continuously for three passages, and cell proliferation and F-actin were monitored. We determined that compared to 5mTHF, FA showed stronger effects on promoting cell proliferation and F-actin formation. We also found that FOLR1 protein level was positively regulated by folate concentration and the non-canonical Wnt/planar cell polarity (PCP) pathway signaling was significantly enriched among different folate conditions in RNA-sequencing analyses. We demonstrated for the first time that FOLR1 could promote the transcription of Vangl2, one of PCP core genes. The transcription of Vangl2 was down-regulated under folate-deficient condition, which resulted in a decrease in PCP activity and F-actin formation. In summary, we identified a distinct advantage of FA in cell proliferation and F-actin formation over 5mTHF, as well as demonstrating that FOLR1 could promote transcription of Vangl2 and provide a new mechanism by which folate deficiency can contribute to the etiology of NTDs.