Uncoupled glycerol-3-phosphate shuttle in kidney cancer reveals that cytosolic GPD is essential to support lipid synthesis.

The glycerol-3-phosphate shuttle (G3PS) is a major NADH shuttle that regenerates reducing equivalents in the cytosol and produces energy in the mitochondria. Here, we demonstrate that G3PS is uncoupled in kidney cancer cells where the cytosolic reaction is ∼4.5 times faster than the mitochondrial reaction. The high flux through cytosolic glycerol-3-phosphate dehydrogenase (GPD) is required to maintain redox balance and support lipid synthesis. Interestingly, inhibition of G3PS by knocking down mitochondrial GPD (GPD2) has no effect on mitochondrial respiration. Instead, loss of GPD2 upregulates cytosolic GPD on a transcriptional level and promotes cancer cell proliferation by increasing glycerol-3-phosphate supply. The proliferative advantage of GPD2 knockdown tumor can be abolished by pharmacologic inhibition of lipid synthesis. Taken together, our results suggest that G3PS is not required to run as an intact NADH shuttle but is instead truncated to support complex lipid synthesis in kidney cancer.

IL-7-Induced Glycerol Transport and TAG Synthesis Promotes Memory CD8+ T Cell Longevity.

Memory T cells are critical for long-term immunity against reinfection and require interleukin-7 (IL-7), but the mechanisms by which IL-7 controls memory T cell survival, particularly metabolic fitness, remain elusive. We discover that IL-7 induces expression of the glycerol channel aquaporin 9 (AQP9) in virus-specific memory CD8+ T cells, but not naive cells, and that AQP9 is vitally required for their long-term survival. AQP9 deficiency impairs glycerol import into memory CD8+ T cells for fatty acid esterification and triglyceride (TAG) synthesis and storage. These defects can be rescued by ectopic expression of TAG synthases, which restores lipid stores and memory T cell survival. Finally, we find that TAG synthesis is a central component of IL-7-mediated survival of human and mouse memory CD8+T cells. This study uncovers the metabolic mechanisms by which IL-7 tailors the metabolism of memory T cells to promote their longevity and fast response to rechallenge.

Discovery, structure and mechanism of a tetraether lipid synthase.

Archaea synthesize isoprenoid-based ether-linked membrane lipids, which enable them to withstand extreme environmental conditions, such as high temperatures, high salinity, and low or high pH values1-5. In some archaea, such as Methanocaldococcus jannaschii, these lipids are further modified by forming carbon-carbon bonds between the termini of two lipid tails within one glycerophospholipid to generate the macrocyclic archaeol or forming two carbon-carbon bonds between the termini of two lipid tails from two glycerophospholipids to generate the macrocycle glycerol dibiphytanyl glycerol tetraether (GDGT)1,2. GDGT contains two 40-carbon lipid chains (biphytanyl chains) that span both leaflets of the membrane, providing enhanced stability to extreme conditions. How these specialized lipids are formed has puzzled scientists for decades. The reaction necessitates the coupling of two completely inert sp3-hybridized carbon centres, which, to our knowledge, has not been observed in nature. Here we show that the gene product of mj0619 from M. jannaschii, which encodes a radical S-adenosylmethionine enzyme, is responsible for biphytanyl chain formation during synthesis of both the macrocyclic archaeol and GDGT membrane lipids6. Structures of the enzyme show the presence of four metallocofactors: three [Fe4S4] clusters and one mononuclear rubredoxin-like iron ion. In vitro mechanistic studies show that Csp3-Csp3 bond formation takes place on fully saturated archaeal lipid substrates and involves an intermediate bond between the substrate carbon and a sulfur of one of the [Fe4S4] clusters. Our results not only establish the biosynthetic route for tetraether formation but also improve the use of GDGT in GDGT-based paleoclimatology indices7-10.

Large enrichments in fatty acid 2H/1H ratios distinguish respiration from aerobic fermentation in yeast Saccharomyces cerevisiae.

Shifts in the hydrogen stable isotopic composition (2H/1H ratio) of lipids relative to water (lipid/water 2H-fractionation) at natural abundances reflect different sources of the central cellular reductant, NADPH, in bacteria. Here, we demonstrate that lipid/water 2H-fractionation (2εfattyacid/water) can also constrain the relative importance of key NADPH pathways in eukaryotes. We used the metabolically flexible yeast Saccharomyces cerevisiae, a microbial model for respiratory and fermentative metabolism in industry and medicine, to investigate 2εfattyacid/water. In chemostats, fatty acids from glycerol-respiring cells were >550‰ 2H-enriched compared to those from cells aerobically fermenting sugars via overflow metabolism, a hallmark feature in cancer. Faster growth decreased 2H/1H ratios, particularly in glycerol-respiring cells by 200‰. Variations in the activities and kinetic isotope effects among NADP+-reducing enzymes indicate cytosolic NADPH supply as the primary control on 2εfattyacid/water. Contributions of cytosolic isocitrate dehydrogenase (cIDH) to NAPDH production drive large 2H-enrichments with substrate metabolism (cIDH is absent during fermentation but contributes up to 20 percent NAPDH during respiration) and slower growth on glycerol (11 percent more NADPH from cIDH). Shifts in NADPH demand associated with cellular lipid abundance explain smaller 2εfattyacid/water variations (<30‰) with growth rate during fermentation. Consistent with these results, tests of murine liver cells had 2H-enriched lipids from slower-growing, healthy respiring cells relative to fast-growing, fermenting hepatocellular carcinoma. Our findings point to the broad potential of lipid 2H/1H ratios as a passive natural tracker of eukaryotic metabolism with applications to distinguish health and disease, complementing studies that rely on complex isotope-tracer addition methods.

A single gene mutation underpins metabolic adaptation and acquisition of filamentous competence in the emerging fungal pathogen Candida auris.

Filamentous cell growth is a vital property of fungal pathogens. The mechanisms of filamentation in the emerging multidrug-resistant fungal pathogen Candida auris are poorly understood. Here, we show that exposure of C. auris to glycerol triggers a rod-like filamentation-competent (RL-FC) phenotype, which forms elongated filamentous cells after a prolonged culture period. Whole-genome sequencing analysis reveals that all RL-FC isolates harbor a mutation in the C2H2 zinc finger transcription factor-encoding gene GFC1 (Gfc1 variants). Deletion of GFC1 leads to an RL-FC phenotype similar to that observed in Gfc1 variants. We further demonstrate that GFC1 mutation causes enhanced fatty acid ß-oxidation metabolism and thereby promotes RL-FC/filamentous growth. This regulation is achieved through a Multiple Carbon source Utilizer (Mcu1)-dependent mechanism. Interestingly, both the evolved RL-FC isolates and the gfc1Δ mutant exhibit an enhanced ability to colonize the skin. Our results reveal that glycerol-mediated GFC1 mutations are beneficial during C. auris skin colonization and infection.

In situ analysis of osmolyte mechanisms of proteome thermal stabilization.

Organisms use organic molecules called osmolytes to adapt to environmental conditions. In vitro studies indicate that osmolytes thermally stabilize proteins, but mechanisms are controversial, and systematic studies within the cellular milieu are lacking. We analyzed Escherichia coli and human protein thermal stabilization by osmolytes in situ and across the proteome. Using structural proteomics, we probed osmolyte effects on protein thermal stability, structure and aggregation, revealing common mechanisms but also osmolyte- and protein-specific effects. All tested osmolytes (trimethylamine N-oxide, betaine, glycerol, proline, trehalose and glucose) stabilized many proteins, predominantly via a preferential exclusion mechanism, and caused an upward shift in temperatures at which most proteins aggregated. Thermal profiling of the human proteome provided evidence for intrinsic disorder in situ but also identified potential structure in predicted disordered regions. Our analysis provides mechanistic insight into osmolyte function within a complex biological matrix and sheds light on the in situ prevalence of intrinsically disordered regions.

Systematic comparison of unilamellar vesicles reveals that archaeal core lipid membranes are more permeable than bacterial membranes.

One of the deepest branches in the tree of life separates the Archaea from the Bacteria. These prokaryotic groups have distinct cellular systems including fundamentally different phospholipid membrane bilayers. This dichotomy has been termed the lipid divide and possibly bestows different biophysical and biochemical characteristics on each cell type. Classic experiments suggest that bacterial membranes (formed from lipids extracted from Escherichia coli, for example) show permeability to key metabolites comparable to archaeal membranes (formed from lipids extracted from Halobacterium salinarum), yet systematic analyses based on direct measurements of membrane permeability are absent. Here, we develop a new approach for assessing the membrane permeability of approximately 10 µm unilamellar vesicles, consisting of an aqueous medium enclosed by a single lipid bilayer. Comparing the permeability of 18 metabolites demonstrates that diether glycerol-1-phosphate lipids with methyl branches, often the most abundant membrane lipids of sampled archaea, are permeable to a wide range of compounds useful for core metabolic networks, including amino acids, sugars, and nucleobases. Permeability is significantly lower in diester glycerol-3-phosphate lipids without methyl branches, the common building block of bacterial membranes. To identify the membrane characteristics that determine permeability, we use this experimental platform to test a variety of lipid forms bearing a diversity of intermediate characteristics. We found that increased membrane permeability is dependent on both the methyl branches on the lipid tails and the ether bond between the tails and the head group, both of which are present on the archaeal phospholipids. These permeability differences must have had profound effects on the cell physiology and proteome evolution of early prokaryotic forms. To explore this further, we compare the abundance and distribution of transmembrane transporter-encoding protein families present on genomes sampled from across the prokaryotic tree of life. These data demonstrate that archaea tend to have a reduced repertoire of transporter gene families, consistent with increased membrane permeation. These results demonstrate that the lipid divide demarcates a clear difference in permeability function with implications for understanding some of the earliest transitions in cell origins and evolution.

Breast cancer cell-derived exosome-delivered microRNA-155 targets UBQLN1 in adipocytes and facilitates cancer cachexia-related fat loss.

Cachexia occurrence and development are associated with loss of white adipose tissues, which may be involved with cancer-derived exosomes. This study attempted to characterize the functional mechanisms of breast cancer (BC) cell-derived exosome-loaded microRNA (miR)-155 in cancer cachexia-related fat loss. Exosomes were incubated with preadipocytes and cellular lipid droplet accumulation was observed using Oil Red O staining. Western blotting evaluated the cellular levels of lipogenesis marker peroxisome proliferator activated receptor gamma (PPARγ) and adiponectin, C1Q and collagen domain containing (AdipoQ). Differentiated adipocytes were incubated with exosomes, and phosphate hormone sensitive lipase (P-HSL), adipose triglyceride lipase (ATGL) and glycerol were detected in adipocytes, in addition to uncoupling protein 1 (UCP1) and leptin levels. A mouse model of cancer cachexia was established where cancer exosomes were injected intravenously. The changes in body weight and tumor-free body weights were recorded and serum glycerol levels and lipid accumulation in adipose tissues were determined. Also, the relationship between miR-155 and UBQLN1 was predicted and verified. BC exosome treatment reduced PPARγ and AdipoQ protein levels, promoted the levels of P-HSL and ATGL proteins, facilitated glycerol release, increased UCP1 expression and lowered leptin expression in adipocytes. Exosomal miR-155 inhibited lipogenesis in preadipocytes and boosted the browning of white adipose tissues. miR-155 downregulation alleviated cancer exosome-induced browning of white adipose tissues and fat loss. Mechanistically, miR-155 targeted UBQLN1, and UBQLN1 upregulation reversed the impacts of cancer exosomes. miR-155 loaded by BC cell-derived exosomes significantly affects white adipose browning and inhibition of cancer-derived exosomes.

Systematic sequence engineering enhances the induction strength of the glucose-regulated GTH1 promoter of Komagataella phaffii.

The promoter of the high-affinity glucose transporter Gth1 (PGTH1) is tightly repressed on glucose and glycerol surplus, and strongly induced in glucose-limitation, thus enabling regulated methanol-free production processes in the yeast production host Komagataella phaffii. To further improve this promoter, an intertwined approach of nucleotide diversification through random and rational engineering was pursued. Random mutagenesis and fluorescence activated cell sorting of PGTH1 yielded five variants with enhanced induction strength. Reverse engineering of individual point mutations found in the improved variants identified two single point mutations with synergistic action. Sequential deletions revealed the key promoter segments for induction and repression properties, respectively. Combination of the single point mutations and the amplification of key promoter segments led to a library of novel promoter variants with up to 3-fold higher activity. Unexpectedly, the effect of gaining or losing a certain transcription factor binding site (TFBS) was highly dependent on its context within the promoter. Finally, the applicability of the novel promoter variants for biotechnological production was proven for the secretion of different recombinant model proteins in fed batch cultivation, where they clearly outperformed their ancestors. In addition to advancing the toolbox for recombinant protein production and metabolic engineering of K. phaffii, we discovered single nucleotide positions and correspondingly affected TFBS that distinguish between glycerol- and glucose-mediated repression of the native promoter.

Krüppel-like factor 15 integrated autophagy and gluconeogenesis to maintain glucose homeostasis under 20-hydroxyecdysone regulation.

The regulation of glycometabolism homeostasis is vital to maintain health and development of animal and humans; however, the molecular mechanisms by which organisms regulate the glucose metabolism homeostasis from a feeding state switching to a non-feeding state are not fully understood. Using the holometabolous lepidopteran insect Helicoverpa armigera, cotton bollworm, as a model, we revealed that the steroid hormone 20-hydroxyecdysone (20E) upregulated the expression of transcription factor Krüppel-like factor (identified as Klf15) to promote macroautophagy/autophagy, apoptosis and gluconeogenesis during metamorphosis. 20E via its nuclear receptor EcR upregulated Klf15 transcription in the fat body during metamorphosis. Knockdown of Klf15 using RNA interference delayed pupation and repressed autophagy and apoptosis of larval fat body during metamorphosis. KLF15 promoted autophagic flux and transiting to apoptosis. KLF15 bound to the KLF binding site (KLF bs) in the promoter of Atg8 (autophagy-related gene 8/LC3) to upregulate Atg8 expression. Knockdown Atg8 reduced free fatty acids (FFAs), glycerol, free amino acids (FAAs) and glucose levels. However, knockdown of Klf15 accumulated FFAs, glycerol, and FAAs. Glycolysis was switched to gluconeogenesis, trehalose and glycogen synthesis were changed to degradation during metamorphosis, which were accompanied by the variation of the related genes expression. KLF15 upregulated phosphoenolpyruvate carboxykinase (Pepck) expression by binding to KLF bs in the Pepck promoter for gluconeogenesis, which utilised FFAs, glycerol, and FAAs directly or indirectly to increase glucose in the hemolymph. Taken together, 20E via KLF15 integrated autophagy and gluconeogenesis by promoting autophagy-related and gluconeogenesis-related genes expression.

Metformin, phenformin, and galegine inhibit complex IV activity and reduce glycerol-derived gluconeogenesis.

SignificanceMetformin is the most commonly prescribed drug for the treatment of type 2 diabetes mellitus, yet the mechanism by which it lowers plasma glucose concentrations has remained elusive. Most studies to date have attributed metformin's glucose-lowering effects to inhibition of complex I activity. Contrary to this hypothesis, we show that inhibition of complex I activity in vitro and in vivo does not reduce plasma glucose concentrations or inhibit hepatic gluconeogenesis. We go on to show that metformin, and the related guanides/biguanides, phenformin and galegine, inhibit complex IV activity at clinically relevant concentrations, which, in turn, results in inhibition of glycerol-3-phosphate dehydrogenase activity, increased cytosolic redox, and selective inhibition of glycerol-derived hepatic gluconeogenesis both in vitro and in vivo.

Cryptic specialized metabolites drive Streptomyces exploration and provide a competitive advantage during growth with other microbes.

Streptomyces bacteria have a complex life cycle that is intricately linked with their remarkable metabolic capabilities. Exploration is a recently discovered developmental innovation of these bacteria, that involves the rapid expansion of a structured colony on solid surfaces. Nutrient availability impacts exploration dynamics, and we have found that glycerol can dramatically increase exploration rates and alter the metabolic output of exploring colonies. We show here that glycerol-mediated growth acceleration is accompanied by distinct transcriptional signatures and by the activation of otherwise cryptic metabolites including the orange-pigmented coproporphyrin, the antibiotic chloramphenicol, and the uncommon, alternative siderophore foroxymithine. Exploring cultures are also known to produce the well-characterized desferrioxamine siderophore. Mutational studies of single and double siderophore mutants revealed functional redundancy when strains were cultured on their own; however, loss of the alternative foroxymithine siderophore imposed a more profound fitness penalty than loss of desferrioxamine during coculture with the yeast Saccharomyces cerevisiae. Notably, the two siderophores displayed distinct localization patterns, with desferrioxamine being confined within the colony area, and foroxymithine diffusing well beyond the colony boundary. The relative fitness advantage conferred by the alternative foroxymithine siderophore was abolished when the siderophore piracy capabilities of S. cerevisiae were eliminated (S. cerevisiae encodes a ferrioxamine-specific transporter). Our work suggests that exploring Streptomyces colonies can engage in nutrient-targeted metabolic arms races, deploying alternative siderophores that allow them to successfully outcompete other microbes for the limited bioavailable iron during coculture.

Dual-function AzuCR RNA modulates carbon metabolism.

SignificanceWhile most small, regulatory RNAs are thought to be "noncoding," a few have been found to also encode a small protein. Here we describe a 164-nucleotide RNA that encodes a 28-amino acid, amphipathic protein, which interacts with aerobic glycerol-3-phosphate dehydrogenase and increases dehydrogenase activity but also base pairs with two mRNAs to reduce expression. The coding and base-pairing sequences overlap, and the two regulatory functions compete.

Glycerol metabolism contributes to competition by oral streptococci through production of hydrogen peroxide.

As a biological byproduct from both humans and microbes, glycerol's contribution to microbial homeostasis in the oral cavity remains understudied. In this study, we examined glycerol metabolism by Streptococcus sanguinis, a commensal associated with oral health. Genetic mutants of glucose-PTS enzyme II (manL), glycerol metabolism (glp and dha pathways), and transcriptional regulators were characterized with regard to glycerol catabolism, growth, production of hydrogen peroxide (H2O2), transcription, and competition with Streptococcus mutans. Biochemical assays identified the glp pathway as a novel source for H2O2 production by S. sanguinis that is independent of pyruvate oxidase (SpxB). Genetic analysis indicated that the glp pathway requires glycerol and a transcriptional regulator, GlpR, for expression and is negatively regulated by PTS, but not the catabolite control protein, CcpA. Conversely, deletion of either manL or ccpA increased the expression of spxB and a second, H2O2-non-producing glycerol metabolic pathway (dha), indicative of a mode of regulation consistent with conventional carbon catabolite repression (CCR). In a plate-based antagonism assay and competition assays performed with planktonic and biofilm-grown cells, glycerol greatly benefited the competitive fitness of S. sanguinis against S. mutans. The glp pathway appears to be conserved in several commensal streptococci and actively expressed in caries-free plaque samples. Our study suggests that glycerol metabolism plays a more significant role in the ecology of the oral cavity than previously understood. Commensal streptococci, though not able to use glycerol as a sole carbohydrate source for growth, benefit from the catabolism of glycerol through production of both ATP and H2O2. IMPORTANCE: Glycerol is an abundant carbohydrate in the oral cavity. However, little is understood regarding the metabolism of glycerol by commensal streptococci, some of the most abundant oral bacteria. This was in part because most streptococci cannot grow on glycerol as the sole carbon source. In this study, we show that Streptococcus sanguinis, a commensal associated with dental health, can degrade glycerol for persistence and competition through two pathways, one of which generates hydrogen peroxide at levels capable of inhibiting Streptococcus mutans. Preliminary studies suggest that several additional commensal streptococci are also able to catabolize glycerol, and glycerol-related genes are actively expressed in human dental plaque samples. Our findings reveal the potential of glycerol to significantly impact microbial homeostasis, which warrants further exploration.

BadR directly represses the expression of the glycerol utilization operon in the Lyme disease pathogen.

Glycerol utilization as a carbohydrate source by Borreliella burgdorferi, the Lyme disease spirochete, is critical for its successful colonization and persistence in the tick vector. The expression of the glpFKD (glp) operon, which encodes proteins for glycerol uptake/utilization, must be tightly regulated during the enzootic cycle of B. burgdorferi. Previous studies have established that the second messenger cyclic di-GMP (c-di-GMP) is required for the activation of glp expression, while an alternative sigma factor RpoS acts as a negative regulator for glp expression. In the present study, we report identification of a cis element within the 5´ untranslated region of glp that exerts negative regulation of glp expression. Further genetic screen of known and predicted DNA-binding proteins encoded in the genome of B. burgdorferi uncovered that overexpressing Borrelia host adaptation regulator (BadR), a known global regulator, dramatically reduced glp expression. Similarly, the badR mutant significantly increased glp expression. Subsequent electrophoretic mobility shift assay analyses demonstrated that BadR directly binds to this cis element, thereby repressing glp independent of RpoS-mediated repression. The efficiency of BadR binding was further assessed in the presence of c-di-GMP and various carbohydrates. This finding highlights multi-layered positive and negative regulatory mechanisms employed by B. burgdorferi to synchronize glp expression throughout its enzootic cycle.IMPORTANCEBorreliella burgdorferi, the Lyme disease pathogen, must modulate its gene expression differentially to adapt successfully to its two disparate hosts. Previous studies have demonstrated that the glycerol uptake and utilization operon, glpFKD, plays a crucial role in spirochetal survival within ticks. However, the glpFKD expression must be repressed when B. burgdorferi transitions to the mammalian host. In this study, we identified a specific cis element responsible for the repression of glpFKD. We further pinpointed Borrelia host adaptation regulator as the direct binding protein to this cis element, thereby repressing glpFKD expression. This discovery paves the way for a deeper exploration of how zoonotic pathogens sense distinct hosts and switch their carbon source utilization during transmission.

Two activating phosphorylation sites of Pbs2 MAP2K in the yeast HOG pathway are differentially dephosphorylated by four PP2C phosphatases Ptc1-Ptc4.

To cope with an increased external osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase (MAPK) through the high-osmolarity glycerol (HOG) pathway, which governs adaptive responses to osmostress. In the HOG pathway, two apparently redundant upstream branches, termed SLN1 and SHO1, activate cognate MAP3Ks (MAPKK kinase) Ssk2/22 and Ste11, respectively. These MAP3Ks, when activated, phosphorylate and thus activate the Pbs2 MAP2K (MAPK kinase), which in turn phosphorylates and activates Hog1. Previous studies have shown that protein tyrosine phosphatases and the serine/threonine protein phosphatases type 2C negatively regulate the HOG pathway to prevent its excessive and inappropriate activation, which is detrimental to cell growth. The tyrosine phosphatases Ptp2 and Ptp3 dephosphorylate Hog1 at Tyr-176, whereas the protein phosphatase type 2Cs Ptc1 and Ptc2 dephosphorylate Hog1 at Thr-174. In contrast, the identities of phosphatases that dephosphorylate Pbs2 remained less clear. Here, we examined the phosphorylation status of Pbs2 at the activating phosphorylation sites Ser-514 and Thr-518 (S514 and T518) in various mutants, both in the unstimulated and osmostressed conditions. Thus, we found that Ptc1-Ptc4 collectively regulate Pbs2 negatively, but each Ptc acts differently to the two phosphorylation sites in Pbs2. T518 is predominantly dephosphorylated by Ptc1, while S514 can be dephosphorylated by any of Ptc1-4 to an appreciable extent. We also show that Pbs2 dephosphorylation by Ptc1 requires the adaptor protein Nbp2 that recruits Ptc1 to Pbs2, thus highlighting the complex processes involved in regulating adaptive responses to osmostress.

Mycobacterial phosphodiesterase Rv0805 is a virulence determinant and its cyclic nucleotide hydrolytic activity is required for propionate detoxification.

Cyclic AMP (cAMP) signaling is essential to Mycobacterium tuberculosis (Mtb) pathogenesis. However, the roles of phosphodiesterases (PDEs) Rv0805, and the recently identified Rv1339, in cAMP homeostasis and Mtb biology are unclear. We found that Rv0805 modulates Mtb growth within mice, macrophages and on host-associated carbon sources. Mycobacterium bovis BCG grown on a combination of propionate and glycerol as carbon sources showed high levels of cAMP and had a strict requirement for Rv0805 cNMP hydrolytic activity. Supplementation with vitamin B12 or spontaneous genetic mutations in the pta-ackA operon restored the growth of BCGΔRv0805 and eliminated propionate-associated cAMP increases. Surprisingly, reduction of total cAMP levels by ectopic expression of Rv1339 restored only 20% of growth, while Rv0805 complementation fully restored growth despite a smaller effect on total cAMP levels. Deletion of an Rv0805 localization domain also reduced BCG growth in the presence of propionate and glycerol. We propose that localized Rv0805 cAMP hydrolysis modulates activity of a specialized pathway associated with propionate metabolism, while Rv1339 has a broader role in cAMP homeostasis. Future studies will address the biological roles of Rv0805 and Rv1339, including their impacts on metabolism, cAMP signaling and Mtb pathogenesis.

Loss of glycerol catabolism confers carbon-source-dependent artemisinin resistance in Mycobacterium tuberculosis.

In view of the urgent need for new antibiotics to treat human infections caused by multidrug-resistant pathogens, drug repurposing is gaining strength due to the relatively low research costs and shorter clinical trials. Such is the case of artemisinin, an antimalarial drug that has recently been shown to display activity against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. To gain insight into how Mtb is affected by artemisinin, we used RNAseq to assess the impact of artemisinin on gene expression profiles, revealing the induction of several efflux pumps and the KstR2 regulon. To anticipate the artemisinin resistance-conferring mutations that could arise in clinical Mtb strains, we performed an in vitro evolution experiment in the presence of lethal concentrations of artemisinin. We obtained artemisinin-resistant isolates displaying different growth kinetics and drug phenotypes, suggesting that resistance evolved through different pathways. Whole-genome sequencing of nine isolates revealed alterations in the glpK and glpQ1 genes, both involved in glycerol metabolism, in seven and one strains, respectively. We then constructed a glpK mutant and found that loss of glpK increases artemisinin resistance only when glycerol is present as a major carbon source. Our results suggest that mutations in glycerol catabolism genes could be selected during the evolution of resistance to artemisinin when glycerol is available as a carbon source. These results add to recent findings of mutations and phase variants that reduce drug efficacy in carbon-source-dependent ways.

γ-Glutamylation of Isopropylamine by Fermentation.

Glutamylation yields N-functionalized amino acids in several natural pathways. γ-Glutamylated amino acids may exhibit improved properties for their industrial application, e. g., as taste enhancers or in peptide drugs. γ-Glutamyl-isopropylamide (GIPA) can be synthesized from isopropylamine (IPA) and l-glutamate. In Pseudomonas sp. strain KIE171, GIPA is an intermediate in the biosynthesis of l-alaninol (2-amino-1-propanol), a precursor of the fluorochinolone antibiotic levofloxacin and of the chloroacetanilide herbicide metolachlor. In this study, fermentative production of GIPA with metabolically engineered Pseudomonas putida KT2440 using γ-glutamylmethylamide synthetase (GMAS) from Methylorubrum extorquens was established. Upon addition of IPA during growth with glycerol as carbon source in shake flasks, the recombinant strain produced up to 21.8 mM GIPA. In fed-batch bioreactor cultivations, GIPA accumulated to a titer of 11 g L-1 with a product yield of 0.11 g g-1 glycerol and a volumetric productivity of 0.24 g L-1 h-1 . To the best of our knowledge, this is the first fermentative production of GIPA.

Mitochondrial Glycerol-3-Phosphate Dehydrogenase Restricts HBV Replication via the TRIM28-Mediated Degradation of HBx.

Hepatitis B virus (HBV) infection affects hepatic metabolism. Serum metabolomics studies have suggested that HBV possibly hijacks the glycerol-3-phosphate (G3P) shuttle. In this study, the two glycerol-3-phosphate dehydrogenases (GPD1 and GPD2) in the G3P shuttle were analyzed for determining their role in HBV replication and the findings revealed that GPD2 and not GPD1 inhibited HBV replication. The knockdown of GPD2 expression upregulated HBV replication, while GPD2 overexpression reduced HBV replication. Moreover, the overexpression of GPD2 significantly reduced HBV replication in hydrodynamic injection-based mouse models. Mechanistically, this inhibitory effect is related to the GPD2-mediated degradation of HBx protein by recruiting the E3 ubiquitin ligase TRIM28 and not to the alterations in G3P metabolism. In conclusion, this study revealed GPD2, a key enzyme in the G3P shuttle, as a host restriction factor in HBV replication. IMPORTANCE The glycerol-3-phosphate (G3P) shuttle is important for the delivery of cytosolic reducing equivalents into mitochondria for oxidative phosphorylation. The study analyzed two key components of the G3P shuttle and identified GPD2 as a restriction factor in HBV replication. The findings revealed a novel mechanism of GPD2-mediated inhibition of HBV replication via the recruitment of TRIM28 for degrading HBx, and the HBx-GPD2 interaction could be another potential therapeutic target for anti-HBV drug development.