Transposon mobilization in the human fungal pathogen Cryptococcus is mutagenic during infection and promotes drug resistance in vitro.

When transitioning from the environment, pathogenic microorganisms must adapt rapidly to survive in hostile host conditions. This is especially true for environmental fungi that cause opportunistic infections in immunocompromised patients since these microbes are not well adapted human pathogens. Cryptococcus species are yeastlike fungi that cause lethal infections, especially in HIV-infected patients. Using Cryptococcus deneoformans in a murine model of infection, we examined contributors to drug resistance and demonstrated that transposon mutagenesis drives the development of 5-fluoroorotic acid (5FOA) resistance. Inactivation of target genes URA3 or URA5 primarily reflected the insertion of two transposable elements (TEs): the T1 DNA transposon and the TCN12 retrotransposon. Consistent with in vivo results, increased rates of mutagenesis and resistance to 5FOA and the antifungal drugs rapamycin/FK506 (rap/FK506) and 5-fluorocytosine (5FC) were found when Cryptococcus was incubated at 37° compared to 30° in vitro, a condition that mimics the temperature shift that occurs during the environment-to-host transition. Inactivation of the RNA interference (RNAi) pathway, which suppresses TE movement in many organisms, was not sufficient to elevate TE movement at 30° to the level observed at 37°. We propose that temperature-dependent TE mobilization in Cryptococcus is an important mechanism that enhances microbial adaptation and promotes pathogenesis and drug resistance in the human host.

Combination of Secondary Plant Metabolites and Micronutrients Improves Mitochondrial Function in a Cell Model of Early Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by excessive formation of beta-amyloid peptides (Aß), mitochondrial dysfunction, enhanced production of reactive oxygen species (ROS), and altered glycolysis. Since the disease is currently not curable, preventive and supportive approaches are in the focus of science. Based on studies of promising single substances, the present study used a mixture (cocktail, SC) of compounds consisting of hesperetin (HstP), magnesium-orotate (MgOr), and folic acid (Fol), as well as the combination (KCC) of caffeine (Cof), kahweol (KW) and cafestol (CF). For all compounds, we showed positive results in SH-SY5Y-APP695 cells-a model of early AD. Thus, SH-SY5Y-APP695 cells were incubated with SC and the activity of the mitochondrial respiration chain complexes were measured, as well as levels of ATP, Aß, ROS, lactate and pyruvate. Incubation of SH-SY5Y-APP695 cells with SC significantly increased the endogenous respiration of mitochondria and ATP levels, while Aß1-40 levels were significantly decreased. Incubation with SC showed no significant effects on oxidative stress and glycolysis. In summary, this combination of compounds with proven effects on mitochondrial parameters has the potential to improve mitochondrial dysfunction in a cellular model of AD.

Sea Cucumber Saponins Derivatives Alleviate Hepatic Lipid Accumulation Effectively in Fatty Acids-Induced HepG2 Cells and Orotic Acid-Induced Rats.

The sulfated echinoside A (EA) and holothurin A (HA) are two prominent saponins in sea cucumber with high hemolytic activity but also superior lipid-lowering activity. Deglycosylated derivatives EA2 and HA2 exhibit low hemolysis compared to EA and HA, but their efficacies on lipid metabolism regulation remains unknown. In this study, fatty acids-treated HepG2 cells and orotic acid-treated rats were used to investigate the lipid-lowering effects of sea cucumber saponin derivatives. Both the saponin and derivatives could effectively alleviate lipid accumulation in HepG2 model, especially EA and EA2. Moreover, though the lipid-lowering effect of EA2 was not equal with EA at the same dosage of 0.05% in diet, 0.15% dosage of EA2 significantly reduced hepatic steatosis rate, liver TC and TG contents by 76%, 41.5%, and 63.7%, respectively, compared to control and reversed liver histopathological features to normal degree according to H&E stained sections. Possible mechanisms mainly included enhancement of fatty acids ß-oxidation and cholesterol catabolism through bile acids synthesis and excretion, suppression of lipogenesis and cholesterol uptake. It revealed that the efficacy of EA2 on lipid metabolism regulation was dose-dependent, and 0.15% dosage of EA2 possessed better efficacy with lower toxicity compared to 0.05% dosage of EA.

Broad antifungal resistance mediated by RNAi-dependent epimutation in the basal human fungal pathogen Mucor circinelloides.

Mucormycosis-an emergent, deadly fungal infection-is difficult to treat, in part because the causative species demonstrate broad clinical antifungal resistance. However, the mechanisms underlying drug resistance in these infections remain poorly understood. Our previous work demonstrated that one major agent of mucormycosis, Mucor circinelloides, can develop resistance to the antifungal agents FK506 and rapamycin through a novel, transient RNA interference-dependent mechanism known as epimutation. Epimutations silence the drug target gene and are selected by drug exposure; the target gene is re-expressed and sensitivity is restored following passage without drug. This silencing process involves generation of small RNA (sRNA) against the target gene via core RNAi pathway proteins. To further elucidate the role of epimutation in the broad antifungal resistance of Mucor, epimutants were isolated that confer resistance to another antifungal agent, 5-fluoroorotic acid (5-FOA). We identified epimutant strains that exhibit resistance to 5-FOA without mutations in PyrF or PyrG, enzymes which convert 5-FOA into the active toxic form. Using sRNA hybridization as well as sRNA library analysis, we demonstrate that these epimutants harbor sRNA against either pyrF or pyrG, and further show that this sRNA is lost after reversion to drug sensitivity. We conclude that epimutation is a mechanism capable of targeting multiple genes, enabling Mucor to develop resistance to a variety of antifungal agents. Elucidation of the role of RNAi in epimutation affords a fuller understanding of mucormycosis. Furthermore, it improves our understanding of fungal pathogenesis and adaptation to stresses, including the evolution of drug resistance.

Orotic acid protects pancreatic ß cell by p53 inactivation in diabetic mouse model.

Impairment of pancreatic ß cells is a principal driver of the development of diabetes. Restoring normal insulin release from the ß cells depends on the ATP produced by the intracellular mitochondria. In maintaining mitochondrial function, the tumor suppressor p53 has emerged as a novel regulator of metabolic homeostasis and participates in adaptations to nutritional changes. In this study, we used orotic acid, an intermediate in the pathway for de novo synthesis of the pyrimidine nucleotide, to reduce genotoxicity. Administration of orotic acid reduced p53 activation of MIN6 ß cells and subsequently reduced ß cell death in the db/db mouse. Orotic acid intake helped to maintain the islet size, number of ß cells, and protected insulin secretion in the db/db mouse. In conclusion, orotic acid treatment maintained ß cell function and reduced cell death, and may therefore, be a future therapeutic strategy for the prevention and treatment of diabetes.

Hypoxia Active Platinum(IV) Prodrugs of Orotic Acid Selective to Liver Cancer Cells.

Platinum(IV) complexes of orotic acid selectively target liver cancer cells displaying enhanced activity and higher uptake in Hep G2. The comparatively higher expression of Organic Anion Transporter 2 (OAT2) in Hep G2 and decrease in toxicity in the presence of OAT2 inhibitor suggest its involvement in the uptake of the complexes. They are resistant to sequestration by the copper transporter ATP7B, unlike cisplatin and oxaliplatin.

Orotic acid-treated hepatocellular carcinoma cells resist steatosis by modification of fatty acid metabolism.

BACKGROUND: Orotic acid (OA) has been intensively utilized to induce fatty liver in rats. Although the capacity of OA to cause steatosis is species-specific, previous in vitro studies indicate that humans could also be susceptible to OA-induced fatty liver. The aim of the present study was to re-elucidate the potential of OA exposure to modulate the cellular mechanisms involved in both non-alcoholic fatty liver disease pathogenesis and cellular protection from lipid accumulation. In addition, alterations in detailed fatty acid (FA) profiles of cells and culture media were analyzed to assess the significance of lipid metabolism in these phenomena. METHODS: In our experiments, human hepatocellular carcinoma HepG2 cells were exposed to OA. Bacterial endotoxin, lipopolysaccharide (LPS), was used to mimic hepatic inflammation. The lipogenic and inflammatory effects of OA and/or LPS on cells were assessed by labeling cellular lipids with Nile red stain and by performing image quantifications. The expression levels of key enzymes involved in de novo lipogenesis (DNL) and of inflammatory markers related to the disease development were studied by qRT-PCR. FA profiles of cells and culture media were determined from total lipids with gas chromatography-mass spectrometry. RESULTS: Our data indicate that although OA possibly promotes the first stage of DNL, it does not cause a definite lipogenic transformation in HepG2 cells. Reduced proportions of 16:0, increased stearoyl-Coenzyme A desaturase 1 mRNA expression and relatively high proportions of 16:1n-7 suggest that active delta9-desaturation may limit lipogenesis and the accumulation of toxic 16:0. Inflammatory signaling could be reduced by the increased production of long-chain n-3 polyunsaturated FA (PUFA) and the active incorporation of certain FA, including 18:1n-9, into cells. In addition, increased proportions of 20:4n-6 and 22:6n-3, total PUFA and dimethyl acetal 18:0 suggest that OA exposure may cause increased secretion of lipoproteins and extracellular vesicles. CONCLUSIONS: The present data suggest that, apart from the transcription-level events reported by previous studies, modifications of FA metabolism may also be involved in the prevention of OA-mediated steatosis. Increased delta9-desaturation and secretion of lipoproteins and extracellular vesicles could offer potential mechanisms for further studies to unravel how OA-treated cells alleviate lipidosis.

In Vitro Interaction of 5-Aminoorotic Acid and Its Gallium(III) Complex with Superoxide Radical, Generated by Two Model Systems.

Increased levels of the superoxide radical are associated with oxidative damage to healthy tissues and with elimination of malignant cells in a living body. It is desirable that a chemotherapeutic combines pro-oxidant behavior around and inside tumors with antioxidant action near healthy cells. A complex consisting of a pro-oxidant cation and antioxidant ligands could be a potential anticancer agent. Ga(III) salts are known anticancer substances, and 5-aminoorotic acid (HAOA) is a ligand with antioxidant properties. The in vitro effects of HAOA and its complex with Ga(III) (gallium(III) 5-aminoorotate (GaAOA)) on the in vitro accumulation of superoxide and other free radicals were estimated. Model systems such as potassium superoxide (KO2), xanthine/xanthine oxidase (X/XO), and rat blood serum were utilized. Data suggested better antioxidant effect of GaAOA compared to HAOA. Evidently, all three ligands of GaAOA participated in the scavenging of superoxide. The effects in rat blood serum were more nuanced, considering the chemical and biochemical complexity of this model system. It was observed that the free-radical-scavenging action of both compounds investigated may be manifested via both hydrogen donation and electron transfer pathways. It was proposed that the radical-scavenging activities (RSAs) of HAOA and its complex with Ga(III) may be due to a complex process, depending on the concentration, and on the environment, nature, and size of the free radical. The electron transfer pathway was considered as more probable in comparison to hydrogen donation in the scavenging of superoxide by 5-aminoorotic acid and its gallium(III) complex.

CRISPR-Cas9 induces point mutation in the mucormycosis fungus Rhizopus delemar.

Rhizopus delemar causes devastating mucormycosis in immunodeficient individuals. Despite its medical importance, R. delemar remains understudied largely due to the lack of available genetic markers, the presence of multiple gene copies due to genome duplication, and mitotically unstable transformants resulting from conventional and limited genetic approaches. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) system induces efficient homologous and non-homologous break points and generates individual and multiple mutant alleles without requiring selective marker genes in a wide variety of organisms including fungi. Here, we have successfully adapted this technology for inducing gene-specific single nucleotide (nt) deletions in two clinical strains of R. delemar: FGSC-9543 and CDC-8219. For comparative reasons, we first screened for spontaneous uracil auxotrophic mutants resistant to 5-fluoroorotic acid (5-FOA) and obtained one substitution (f1) mutationin the FGSC-9543 strain and one deletion (f2) mutation in the CDC-8219 strain. The f2 mutant was then successfully complemented with a pyrF-dpl200 marker gene. We then introduced a vector pmCas9:tRNA-gRNA that expresses both Cas9 endonuclease and pyrF-specific gRNA into FGSC-9543 and CDC-8219 strains and obtained 34 and 42 5-FOA resistant isolates, respectively. Candidate transformants were successively transferred eight times by propagating hyphal tips prior to genotype characterization. Sequencing of the amplified pyrF allele in all transformants tested revealed a single nucleotide (nt) deletion at the 4th nucleotide before the protospacer adjacent motif (PAM) sequence, which is consistent with CRISPR-Cas9 induced gene mutation through non-homologous end joining (NHEJ). Our study provides a new research tool for investigating molecular pathogenesis mechanisms of R. delemar while also highlighting the utilization of CRISPR-Cas9 technology for generating specific mutants of Mucorales fungi.

Effects of Orotic Acid-Induced Non-Alcoholic Fatty Liver on the Pharmacokinetics of Metoprolol and its Metabolites in Rats.

PURPOSE: Preliminary study results have shown that rats with non-alcoholic fatty liver disease (NAFLD) induced by 1% orotic acid-containing diet have decreased hepatic CYP2D activity. This study aims to evaluate the possible pharmacokinetic changes in NAFLD as a result of reduced metabolic activity of CYP2D. METHODS: The pharmacokinetics of metoprolol and its metabolites, O-desmethyl metoprolol (DMM) and α-hydroxy metoprolol (HM), was investigated in NAFLD and control rats following intravenous (1 mg/kg) and oral (2 mg/kg) administration of metoprolol. The hepatic CYP2D expression was also investigated. RESULTS: NAFLD rats had lower CYP2D expression (by 36.6%) and slower intrinsic clearance (CLint) of metoprolol and formation of HM (by 40.1% and 37.2%, respectively). There were no significant changes in the pharmacokinetics of metoprolol and its metabolites following intravenous administration. In contrast, oral administration of metoprolol resulted in significantly increased total area under plasma concentration-time curve (AUC) of metoprolol (by 127%) and decreased metabolite formation ratios (AUCDMM/AUCMetoprolol [by 42.8%], AUCHM/AUCMetoprolol [by 35.0%]) in NAFLD rats. Moreover, these changes were well correlated with severity of steatosis as quantified by hepatic triglyceride contents. CONCLUSIONS: NALFD can lead to a reduction in the hepatic CLint of a drug if it is a substrate of the CYP2D subfamily. The decreased clearance may result in elevated drug concentrations and increased exposure.

A Genetic System for the Thermophilic Acetogenic Bacterium Thermoanaerobacter kivui.

Thermoanaerobacter kivui is one of the very few thermophilic acetogenic microorganisms. It grows optimally at 66°C on sugars but also lithotrophically with H2 + CO2 or with CO, producing acetate as the major product. While a genome-derived model of acetogenesis has been developed, only a few physiological or biochemical experiments regarding the function of important enzymes in carbon and energy metabolism have been carried out. To address this issue, we developed a method for targeted markerless gene deletions and for integration of genes into the genome of T. kivui The strain naturally took up plasmid DNA in the exponential growth phase, with a transformation frequency of up to 3.9 × 10-6 A nonreplicating plasmid and selection with 5-fluoroorotate was used to delete the gene encoding the orotate phosphoribosyltransferase (pyrE), resulting in a ΔpyrE uracil-auxotrophic strain, TKV002. Reintroduction of pyrE on a plasmid or insertion of pyrE into different loci within the genome restored growth without uracil. We subsequently studied fructose metabolism in T. kivui The gene fruK (TKV_c23150) encoding 1-phosphofructosekinase (1-PFK) was deleted, using pyrE as a selective marker via two single homologous recombination events. The resulting ΔfruK strain, TKV003, did not grow on fructose; however, growth on glucose (or on mannose) was unaffected. The combination of pyrE as a selective marker and the natural competence of the strain for DNA uptake will be the basis for future studies on CO2 reduction and energy conservation and their regulation in this thermophilic acetogenic bacterium.IMPORTANCE Acetogenic bacteria are currently the focus of research toward biotechnological applications due to their potential for de novo synthesis of carbon compounds such as acetate, butyrate, or ethanol from H2 + CO2 or from synthesis gas. Based on available genome sequences and on biochemical experiments, acetogens differ in their energy metabolism. Thus, there is an urgent need to understand the carbon and electron flows through the Wood-Ljungdahl pathway and their links to energy conservation, which requires genetic manipulations such as deletion or overexpression of genes encoding putative key enzymes. Unfortunately, genetic systems have been reported for only a few acetogenic bacteria. Here, we demonstrate proof of concept for the genetic modification of the thermophilic acetogenic species Thermoanaerobacter kivui The genetic system will be used to study genes involved in biosynthesis and energy metabolism, and may further be applied to metabolically engineer T. kivui to produce fuels and chemicals.

Effects of extracellular orotic acid on acute contraction-induced adaptation patterns in C2C12 cells.

Dietary administration of orotic acid (OA), an intermediate in the pyrimidine biosynthetic pathway, is considered to provide a wide range of beneficial effects, including cardioprotection and exercise adaptation. Its mechanisms of action, when applied extracellularly, however, are barely understood. In this study, we evaluated potential effects of OA on skeletal muscle using an in vitro contraction model of electrically pulse-stimulated (EPS) C2C12 myotubes. By analyzing a subset of genes representing inflammatory, metabolic, and structural adaptation pathways, we could show that OA supplementation diminishes the EPS-provoked expression of inflammatory transcripts (interleukin 6, Il6; chemokine (C-X-C Motif) ligand 5, Cxcl5), and attenuated transcript levels of nuclear receptor subfamily 4 group A member 3 (Nr4A3), early growth response 1 (Egr1), activating transcription factor 3 (Atf3), and fast-oxidative MyHC-IIA isoform (Myh2). By contrast, OA had no suppressive effect on the pathogen-provoked inflammatory gene response in skeletal muscle cells, as demonstrated by stimulation of C2C12 myotubes with bacterial LPS. In addition, we observed a suppressive effect of OA on EPS-induced phosphorylation of AMP-activated protein kinase (AMPK), whereas EPS-triggered phosphorylation/activation of the mammalian target of rapamycin (mTOR) was not affected. Finally, we demonstrate that OA positively influences glycogen levels in EP-stimulated myotubes. Taken together, our results suggest that in skeletal muscle cells, OA modulates both the inflammatory and the metabolic reaction provoked by acute contraction. These results might have important clinical implications, specifically in cardiovascular and exercise medicine.

Putative orotate transporter of Cryptococcus neoformans, Oat1, is a member of the NCS1/PRT transporter super family and its loss causes attenuation of virulence.

It is well known that 5-fluoroorotic acid (5-FOA)-resistant mutants isolated from wild-type Cryptococcus neoformans are exclusively either ura3 or ura5 mutants. Unexpectedly, many of the 5-FOA-resistant mutants isolated in our selective regime were Ura+. We identified CNM00460 as the gene responsible for these mutations. Cnm00460 belongs to the nucleobase cation symporter 1/purine-related transporter (NCS1/PRT) super family of fungal transporters, representative members of which are uracil transporter, uridine transporter and allantoin transporter of Saccharomyces cerevisiae. Since the CNM00460 gene turned out to be involved in utilization of orotic acid, most probably as transporter, we designated this gene Orotic Acid Transporter 1 (OAT1). This is the first report of orotic acid transporter in this family. C. neoformans has four members of the NCS1/PRT family, including Cnm00460, Cnm02550, Cnj00690, and Cnn02280. Since the cnm02550∆ strain showed resistance to 5-fluorouridine, we concluded that CNM02550 encodes uridine permease and designated it URidine Permease 1 (URP1). We found that oat1 mutants were sensitive to 5-FOA in the medium containing proline as nitrogen source. A mutation in the GAT1 gene, a positive transcriptional regulator of genes under the control of nitrogen metabolite repression, in the genetic background of oat1 conferred the phenotype of weak resistance to 5-FOA even in the medium using proline as nitrogen source. Thus, we proposed the existence of another orotic acid utilization system (tentatively designated OAT2) whose expression is under the control of nitrogen metabolite repression at least in part. We found that the OAT1 gene is necessary for full pathogenic activity of C. neoformans var. neoformans.

Targeted gene replacement at the URA3 locus of the basidiomycetous yeast Pseudozyma antarctica and its transformation using lithium acetate treatment.

The basidiomycetous yeast Pseudozyma antarctica is a remarkable producer of industrially valuable enzymes and extracellular glycolipids. In this study, we developed a method for targeted gene replacement in P. antarctica. In addition, transformation conditions were optimized using lithium acetate, single-stranded carrier DNA and polyethylene glycol (lithium acetate treatment), generally used for ascomycetous yeast transformation. In the rice-derived P. antarctica strain GB-4(0), PaURA3, a homologue of the Saccharomyces cerevisiae orotidine-5'-phosphate decarboxylase gene (URA3), was selected as the target locus. A disruption cassette was constructed by linking the nouseothricine resistance gene (natMX4) to homologous DNA fragments of PaURA3, then electroporated into the strain GB-4(0). We obtained strain PGB015 as one of the PaURA3 disruptants (Paura3Δ::natMX4). Then the PCR-amplified PaURA3 fragment was introduced into PGB015, and growth of transformant colonies but not background colonies was observed on selective media lacking uracil. The complementation of uracil-auxotrophy in PGB015 by introduction of PaURA3 was also performed using lithium acetate treatment, which resulted in a transformation efficiency of 985 CFU/6.8 µg DNA and a gene-targeting ratio of two among 30 transformants. Copyright © 2017 John Wiley & Sons, Ltd.

Design and synthesis of potent substrate-based inhibitors of the Trypanosoma cruzi dihydroorotate dehydrogenase.

Chagas disease, caused by the parasitic protozoan Trypanosoma cruzi, is the leading cause of heart disease in Latin America. T. cruzi dihydroorotate dehydrogenase (DHODH), which catalyzes the production of orotate, was demonstrated to be essential for T. cruzi survival, and thus has been considered as a potential drug target to combat Chagas disease. Here we report the design and synthesis of 75 compounds based on the orotate structure. A comprehensive structure-activity relationship (SAR) study revealed two 5-substituted orotate analogues (5u and 5v) that exhibit Kiapp values of several ten nanomolar level and a selectivity of more than 30,000-fold over human DHODH. The information presented here will be invaluable in the search for next-generation drug leads for Chagas disease.

Dipeptidyl peptidase IV is involved in the cellulose-responsive induction of cellulose biomass-degrading enzyme genes in Aspergillus aculeatus.

We screened for factors involved in the cellulose-responsive induction of cellulose biomass-degrading enzyme genes from approximately 12,000 Aspergillus aculeatus T-DNA insertion mutants harboring a transcriptional fusion between the FIII-avicelase gene (cbhI) promoter and the orotidine 5'-monophosphate decarboxylase gene. Analysis of 5-fluoroorodic acid (5-FOA) sensitivity, cellulose utilization, and cbhI expression of the mutants revealed that a mutant harboring T-DNA at the dipeptidyl peptidase IV (dppIV) locus had acquired 5-FOA resistance and was deficient in cellulose utilization and cbhI expression. The deletion of dppIV resulted in a significant reduction in the cellulose-responsive expression of both cbhI as well as genes controlled by XlnR-independent and XlnR-dependent signaling pathways at an early phase in A. aculeatus. In contrast, the dppIV deletion did not affect the xylose-responsive expression of genes under the control of XlnR. These results demonstrate that DppIV participates in cellulose-responsive induction in A. aculeatus.

[The influence of 'potassium orotate' on neocollagenogenesis in implantation of polypropylene endoprosthesis and polypropylene combined with polylactic acid endoprosthesis]. / Vliyanie orotata kaliya na neokollagenogenez pri implantatsii polipropilenovogo endoproteza i endoproteza iz polipropilena s polimolochnoi kislotoi v eksperimente.

AIM: To study neocollagenogenesis after implantation of polypropylene endoprosthesis and polypropylene combined with polylactic acid endoprosthesis on background of «potassium orotate¼ administration. MATERIAL AND METHODS: We used two different types of endoprosthesis in the experiment. The first type was made of just polypropylene, the second type was made of polypropylene combined with polylactic acid. Histological examination was performed using polarizing microscopy. Collagen types I and III ratio in connective tissue around the prosthesis was analyzed according to the color that was individual for each type. RESULTS: The results were significantly better in case of collagenogenesis stimulation with Potassium orotate within 30 days and later for one type of endoprosthesis. Also we revealed that collagenogenesis and paraprosthesis capsule formation were more active in case of combined endoprosthesis. We revealed stimulating action of «Potassium Orotate¼ for collegenogenesis process, this fact was proved by increased collagen I/III ratio. CONCLUSION: Optimization of collagenogenesis was based on persistent 1,37-fold increase of collagen I/III ratio in case of combined endoprosthesis after 90 days. It was manifested by accelerated formation of connective tissue capsule and facilitated early isolation of the implant from surrounding tissues.

Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy.

African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) - a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite.

Plastid uridine salvage activity is required for photoassimilate allocation and partitioning in Arabidopsis.

Nucleotides are synthesized from de novo and salvage pathways. To characterize the uridine salvage pathway, two genes, UKL1 and UKL2, that tentatively encode uridine kinase (UK) and uracil phosphoribosyltransferase (UPRT) bifunctional enzymes were studied in Arabidopsis thaliana. T-DNA insertions in UKL1 and UKL2 reduced transcript expression and increased plant tolerance to toxic analogs 5-fluorouridine and 5-fluorouracil. Enzyme activity assays using purified recombinant proteins indicated that UKL1 and UKL2 have UK but not UPRT activity. Subcellular localization using a C-terminal enhanced yellow fluorescent protein fusion indicated that UKL1 and UKL2 localize to plastids. The ukl2 mutant shows reduced transient leaf starch during the day. External application of orotate rescued this phenotype in ukl2, indicating pyrimidine pools are limiting for starch synthesis in ukl2. Intermediates for lignin synthesis were upregulated, and there was increased lignin and reduced cellulose content in the ukl2 mutant. Levels of ATP, ADP, ADP-glucose, UTP, UDP, and UDP-glucose were altered in a light-dependent manner. Seed composition of the ukl1 and ukl2 mutants included lower oil and higher protein compared with the wild type. Unlike single gene mutants, the ukl1 ukl2 double mutant has severe developmental defects and reduced biomass accumulation, indicating these enzymes catalyze redundant reactions. These findings point to crucial roles played by uridine salvage for photoassimilate allocation and partitioning.

Marker recycling in Lentinula edodes via 5-fluoroorotic acid counter-selection.

Shiitake (Lentinula edodes) contains various beneficial compounds and possesses several notable properties. However, there are few reports on its molecular breeding due to delay in development of its gene-modifying technology. Therefore, here, strain UV30 (pyrG -) was bred from the UV-irradiated protoplasts of strain M2. Strain UV30 was uracil-auxotrophic, and the phenylalanine residue in the active centre of orotidine-5-phosphate decarboxylase encoded by pyrG in the strain was substituted with a serine residue. Next, a recycling marker consisting of the upstream sequence of ku80, a repeat sequence (a portion of the downstream sequence of ku80), pyrG, and the downstream sequence of ku80 was introduced into the strain UV30. Consequently, the prototrophic strain ckp2-1, in which ku80 was replaced with the recycling marker, was obtained. After cultivation in complete medium, mycelia from the edges of ckp2-1 colonies were inoculated into a complete medium containing 5-Fluoroorotic acid (5-FOA). A 5-FOA-resistant strain KaM2, in which pyrG sequence was spliced from the recycling marker sequence via homologous recombination, was obtained. In this study, we developed the first marker recycling system for multigene targeting in L. edodes. Moreover, the resulting ∆ku80 strain may serve as a non-homologous end-joining deficient strain for further genetic manipulations.