CitE Enzymes Are Essential for Mycobacterium tuberculosis to Establish Infection in Macrophages and Guinea Pigs.

Bacterial citrate lyase activity has been demonstrated in various eukaryotes, bacteria and archaea, underscoring their importance in energy metabolism of the cell. While the bacterial citrate lyase comprises of three different subunits, M. tuberculosis genome lacks CitD and CitF subunits of citrate lyase complex but encodes for 2 homologs of CitE subunits, Rv2498c and Rv3075c. Using temperature sensitive mycobacteriophages, we were able to generate both single and double citE mutant strains of M. tuberculosis. The survival experiments revealed increased susceptibility of the double mutant strain to oxidative stress in comparison to the parental strain. Also, simultaneous deletion of both citE1 and citE2 in M. tuberculosis genome resulted in impairment of intracellular replication in macrophages. The double mutant strain displayed reduced growth in lungs and spleens of guinea pigs. This is the first study demonstrating that M. tuberculosis critically requires CitE subunits of citrate lyase for pathogenesis. Taken together, these findings position these enzymes as potential targets for development of anti-tubercular small molecules.

3-Hydroxy-3-methylglutaric and 3-methylglutaric acids impair redox status and energy production and transfer in rat heart: relevance for the pathophysiology of cardiac dysfunction in 3-hydroxy-3-methylglutaryl-coenzyme A lyase deficiency.

3-Hydroxy-3-methylglutaryl-coenzyme A lyase (HL) deficiency is characterized by tissue accumulation of 3-hydroxy-3-methylglutaric (HMG), and 3-methylglutaric (MGA) acids. Affected patients present cardiomyopathy, whose pathomechanisms are not yet established. We investigated the effects of HMG and MGA on energy and redox homeostasis in rat heart using in vivo and in vitro models. In vivo experiments showed that intraperitoneal administration of HMG and MGA decreased the activities of the respiratory chain complex II and creatine kinase (CK), whereas HMG also decreased the activity of complex II-III. Furthermore, HMG and MGA injection increased reactive species production and carbonyl formation, and decreased glutathione concentrations. Regarding the enzymatic antioxidant defenses, HMG and MGA increased glutathione peroxidase (GPx) and glutathione reductase (GR) activities, while only MGA diminished the activities of superoxide dismutase (SOD) and catalase, as well as the protein content of SOD1. Pre-treatment with melatonin (MEL) prevented MGA-induced decrease of CK activity and SOD1 levels. In vitro results demonstrated that HMG and MGA increased reactive species formation, induced lipid peroxidation and decreased glutathione. We also verified that reactive species overproduction and glutathione decrease provoked by HMG and MGA were abrogated by MEL and lipoic acid (LA), while only MEL prevented HMG- and MGA-induced lipoperoxidation. Allopurinol (ALP) also prevented reactive species overproduction caused by both metabolites. Our data provide solid evidence that bioenergetics dysfunction and oxidative stress are induced by HMG and MGA in heart, which may explain the cardiac dysfunction observed in HL deficiency, and also suggest that antioxidant supplementation could be considered as adjuvant therapy for affected patients.

Application of multiplex ligation-dependent probe amplification, and identification of a heterozygous Alu-associated deletion and a uniparental disomy of chromosome 1 in two patients with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL) deficiency is an autosomal recessive disorder affecting the leucine catabolic pathway and ketone body synthesis, and is clinically characterized by metabolic crises with hypoketotic hypoglycemia, metabolic acidosis and hyperammonemia. In the present study, we initially used PCR with genomic followed by direct sequencing to investigate the molecular genetic basis of HMGCL deficiency in two patients clinically diagnosed with the condition. Although we identified a mutation in each patient, the inheritance patterns of these mutations were not consistent with disease causation. Therefore, we investigated HMGCL using multiplex ligation-dependent probe amplification (MLPA) to determine the copy numbers of all exons. A heterozygous deletion that included exons 2-4 was identified in one of the patients. MLPA revealed that the other patient had two copies for all HMGCL exons. Paternal uniparental isodisomy of chromosome 1 was confirmed in this patient by microarray analysis. These findings indicate that MLPA is useful for the identification of genomic aberrations and mutations other than small-scale nucleotide alterations. To the best of our knowledge, this is the first study describing HMGCL deficiency caused by uniparental disomy.

Fat mass- and obesity-associated gene Fto affects the dietary response in mouse white adipose tissue.

Common variants of human fat mass- and obesity-associated gene Fto have been linked with higher body mass index, but the biological explanation for the link has remained obscure. Recent findings suggest that these variants affect the homeobox protein IRX3. Here we report that FTO has a role in white adipose tissue which modifies its response to high-fat feeding. Wild type and Fto-deficient mice were exposed to standard or high-fat diet for 16 weeks after which metabolism, behavior and white adipose tissue morphology were analyzed together with adipokine levels and relative expression of genes regulating white adipose tissue adipogenesis and Irx3. Our results indicate that Fto deficiency increases the expression of genes related to adipogenesis preventing adipocytes from becoming hypertrophic after high-fat diet. In addition, we report a novel finding of increased Irx3 expression in Fto-deficient mice after high-fat feeding indicating a complex link between FTO, IRX3 and fat metabolism.

The fat mass and obesity associated gene (Fto) regulates activity of the dopaminergic midbrain circuitry.

Dopaminergic (DA) signaling governs the control of complex behaviors, and its deregulation has been implicated in a wide range of diseases. Here we demonstrate that inactivation of the Fto gene, encoding a nucleic acid demethylase, impairs dopamine receptor type 2 (D2R) and type 3 (D3R) (collectively, 'D2-like receptor')-dependent control of neuronal activity and behavioral responses. Conventional and DA neuron-specific Fto knockout mice show attenuated activation of G protein-coupled inwardly-rectifying potassium (GIRK) channel conductance by cocaine and quinpirole. Impaired D2-like receptor-mediated autoinhibition results in attenuated quinpirole-mediated reduction of locomotion and an enhanced sensitivity to the locomotor- and reward-stimulatory actions of cocaine. Analysis of global N(6)-methyladenosine (m(6)A) modification of mRNAs using methylated RNA immunoprecipitation coupled with next-generation sequencing in the midbrain and striatum of Fto-deficient mice revealed increased adenosine methylation in a subset of mRNAs important for neuronal signaling, including many in the DA signaling pathway. Several proteins encoded by these mRNAs had altered expression levels. Collectively, FTO regulates the demethylation of specific mRNAs in vivo, and this activity relates to the control of DA transmission.

The 3-hydroxy-methylglutaryl coenzyme A lyase HCL1 is required for macrophage colonization by human fungal pathogen Histoplasma capsulatum.

Histoplasma capsulatum is a fungal respiratory pathogen that survives and replicates within the phagolysosome of macrophages. The molecular factors it utilizes to subvert macrophage antimicrobial defenses are largely unknown. Although the ability of H. capsulatum to prevent acidification of the macrophage phagolysosome is thought to be critical for intracellular survival, this hypothesis has not been tested since H. capsulatum mutants that experience decreased phagosomal pH have not been identified. In a screen to identify H. capsulatum genes required for lysis of bone marrow-derived macrophages (BMDMs), we identified an insertion mutation disrupting the H. capsulatum homolog of 3-hydroxy-methylglutaryl coenzyme A (HMG CoA) lyase (HCL1). In addition to its inability to lyse macrophages, the hcl1 mutant had a severe growth defect in BMDMs, indicating that HMG CoA lyase gene function is critical for macrophage colonization. In other organisms, HMG CoA lyase catalyzes the last step in the leucine catabolism pathway. In addition, both fungi and humans deficient in HMG CoA lyase accumulate acidic intermediates as a consequence of their inability to catabolize leucine. Consistent with observations in other organisms, the H. capsulatum hcl1 mutant was unable to grow on leucine as the major carbon source, caused acidification of its growth medium in vitro, and resided in an acidified vacuole within macrophages. Mice infected with the hcl1 mutant took significantly longer to succumb to infection than mice infected with the wild-type strain. Taken together, these data indicate the importance of Hcl1 function in H. capsulatum replication in the harsh growth environment of the macrophage phagosome.

Attenuation of Streptococcus suis virulence by the alteration of bacterial surface architecture.

NeuB, a sialic acid synthase catalyzes the last committed step of the de novo biosynthetic pathway of sialic acid, a major element of bacterial surface structure. Here we report a functional NeuB homologue of Streptococcus suis, a zoonotic agent, and systematically address its molecular and immunological role in bacterial virulence. Disruption of neuB led to thinner capsules and more susceptibility to pH, and cps2B inactivation resulted in complete absence of capsular polysaccharides. These two mutants both exhibited increased adhesion and invasion to Hep-2 cells and improved sensibility to phagocytosis. Not only do they retain the capability of inducing the release of host pro-inflammatory cytokines, but also result in the faster secretion of IL-8. Easier cleaning up of the mutant strains in whole blood is consistent with virulence attenuation seen with experimental infections of both mice and SPF-piglets. Therefore we concluded that altered architecture of S. suis surface attenuates its virulence.

Knockdown of the fat mass and obesity gene disrupts cellular energy balance in a cell-type specific manner.

Recent studies suggest that FTO variants strongly correlate with obesity and mainly influence energy intake with little effect on the basal metabolic rate. We suggest that FTO influences eating behavior by modulating intracellular energy levels and downstream signaling mechanisms which control energy intake and metabolism. Since FTO plays a particularly important role in adipocytes and in hypothalamic neurons, SH-SY5Y neuronal cells and 3T3-L1 adipocytes were used to understand how siRNA mediated knockdown of FTO expression alters cellular energy homeostasis. Cellular energy status was evaluated by measuring ATP levels using a luminescence assay and uptake of fluorescent glucose. FTO siRNA in SH-SY5Y cells mediated mRNA knockdown (-82%), increased ATP concentrations by up to 46% (P = 0.013) compared to controls, and decreased phosphorylation of AMPk and Akt in SH-SY5Y by -52% and -46% respectively as seen by immunoblotting. In contrast, FTO siRNA in 3T3-L1 cells decreased ATP concentration by -93% (p<0.0005), and increased AMPk and Akt phosphorylation by 204% and 70%, respectively suggesting that FTO mediates control of energy levels in a cell-type specific manner. Furthermore, glucose uptake was decreased in both SH-SY5Y (-51% p = 0.015) and 3T3-L1 cells (-30%, p = 0.0002). We also show that FTO knockdown decreases NPY mRNA expression in SH-SY5Y cells (-21%) through upregulation of pSTAT3 (118%). These results provide important evidence that FTO-variant linked obesity may be associated with altered metabolic functions through activation of downstream metabolic mediators including AMPk.

The enzyme 4-hydroxy-2-oxoglutarate aldolase is deficient in primary hyperoxaluria type 3.

BACKGROUND: Mutations in the 4-hydroxy-2-oxoglutarate aldolase (HOGA1) gene have been recently identified in patients with atypical primary hyperoxaluria (PH). However, it was not clearly established whether these mutations caused disease via loss of function or activation of the gene product. METHODS: Whole-gene sequencing of HOGA1 was conducted in 28 unrelated patients with a high clinical suspicion of PH and in whom Types 1 and 2 had been excluded. RESULTS: Fifteen patients were homozygous or compound heterozygous for mutations in HOGA1. In total, seven different mutations were identified including three novel changes: a missense mutation, c.107C > T (p.Ala36Val), and two nonsense mutations c.117C > A (p.Tyr39X) and c.208C > T (p.Arg70X) as well as the previously documented c.860G > T (p.Gly297Val), c.907C > T (p.Arg303Cys) and in-frame c.944_946delAGG (p.Glu315del) mutations. The recurrent c.700 + 5G > T splice site mutation in intron 5 was most common with a frequency of 67%. Expression studies on hepatic messenger RNA demonstrated the pathogenicity of this mutation. CONCLUSIONS: The detection of a patient with two novel nonsense mutations within exon 1 of the gene, c.117C > A (p.Tyr39X) and c.208C > T (p.Arg70X), provides definitive proof that PH Type 3 is due to deficiency of the 4-hydroxy-2-oxoglutarate aldolase enzyme.

A series of pregnancies in women with inherited metabolic disease.

In this case series we report 12 pregnancies, in women treated at four centres, illustrating some of the issues that may be encountered during pregnancy by women with inherited metabolic disease. We discuss how specific pregnancy, labour and delivery issues for mothers with methylmalonic acidemia, homocystinuria, propionic acidemia, glutaric acidemia type 1, ornithine transcarbamylase (OTC) deficiency and 3-hydroxy-3-methylglutaric(HMG)-CoA lyase deficiency were managed and the outcome for the mother and child in each case. Eight of the 12 pregnancies resulted in the successful delivery of a liveborn infant. Several women experienced decompensation of their condition during pregnancy or the post-partum period. There was one maternal death in a women with 3-hydroxy-3-methylglutaric(HMG)-CoA lyase deficiency. Pre-pregnancy counselling and co-management of high risk medical patients by obstetricians and specialist physicians with an understanding of the relationship between pregnancy and inherited metabolic disease is essential.

Requirement of purine and pyrimidine synthesis for colonization of the mouse intestine by Escherichia coli.

To study the adaptation of an intestinal bacterium to its natural environment, germfree mice were associated with commensal Escherichia coli MG1655. Two-dimensional gel electrophoresis was used to identify proteins differentially expressed in E. coli MG1655 collected from either cecal contents or anaerobic in vitro cultures. Fourteen differentially expressed proteins (>3-fold; P < 0.05) were identified, nine of which were upregulated in cecal versus in vitro-grown E. coli. Four of these proteins were investigated further for their role in gut colonization. After deletion of the corresponding genes, the resulting E. coli mutants were tested for their ability to colonize the intestines of gnotobiotic mice in competition with the wild-type strain. A mutant devoid of ydjG, which encodes a putative NADH-dependent methylglyoxal reductase, reached a 1.2-log-lower cecal concentration than the wild type. Deletion of the nanA gene encoding N-acetylneuraminate lyase affected the colonization and persistence of E. coli in the intestines of the gnotobiotic mice only slightly. A mutant devoid of 5'-phosphoribosyl 4-(N-succinocarboxamide)-5-aminoimidazole synthase, a key enzyme of purine synthesis, displayed intestinal cell counts >4 logs lower than those of the wild type. Deletion of the gene encoding aspartate carbamoyltransferase, a key enzyme of pyrimidine synthesis, even resulted in the washout of the corresponding mutant from the mouse intestinal tract. These findings indicate that E. coli needs to synthesize purines and pyrimidines to successfully colonize the mouse intestine.

FTO effect on energy demand versus food intake.

An intronic single nucleotide polymorphism (SNP) (rs9939609) close to the fat mass and obesity associated gene (FTO) was the first SNP to be discovered with common variants linked to body mass index; at least seven studies in humans have implicated this SNP with variations in food intake and satiety, and four studies have rejected an effect on energy expenditure normalized for body weight. Fischer et al. recently constructed a mouse in which the homologous Fto gene was inactivated (Fto(-/-)) and showed that these mice were protected from obesity. This observation strongly implicates the effects of the intronic SNP rs9939609 as arising due to an effect on the closest gene (FTO). However, the suggested mechanism underlying this effect in mice was opposite to that in humans. The Fto(-/-) mice showed no significant differences in food intake relative to wild-types litter-mates but had an elevated metabolic rate. The apparent contrasting effects of the gene in humans and mice is worthy of closer investigation.

[Late onset 3-HMG-CoA lyase deficiency: a rare but treatable disorder]. / Déficit en 3-HMG-CoA lyase à révélation tardive : savoir reconnaître une maladie rare mais traitable.

3-Hydroxy-3-methylglutaric aciduria is a rare autosomal recessive genetic disorder due to a deficiency of the 3-hydroxy-3-methylglutarylCoA lyase (HMG-CoA lyase), a mitochondrial enzyme involved in ketogenesis and in the final step of l-leucine catabolism. HMG-CoA lyase deficiency can lead, in particular circumstances, such as fever, prolonged fasting or digestive disorders, to brutal and severe hypoglycemia with metabolic acidosis and sometimes fatal coma. We report on a new case of 3-hydroxy-3-methylglutaric aciduria particular by its late onset in a 3-year-old patient. Molecular investigation identified two new sequence modifications in the HMGCL gene: c.494G>A (p.Arg165Gln) and c.820G>A (p.Gly274Arg). We remind about this case report that the therapeutical is mainly preventive and allows a very good prognosis for this disease. Long-term treatment consists in limited fasting time, continuous low protein diet and l-carnitine supplementation. Preventive measures are essential: prevention of fasting and emergency treatment during intercurrent infections.

Host plant genome overcomes the lack of a bacterial gene for symbiotic nitrogen fixation.

Homocitrate is a component of the iron-molybdenum cofactor in nitrogenase, where nitrogen fixation occurs. NifV, which encodes homocitrate synthase (HCS), has been identified from various diazotrophs but is not present in most rhizobial species that perform efficient nitrogen fixation only in symbiotic association with legumes. Here we show that the FEN1 gene of a model legume, Lotus japonicus, overcomes the lack of NifV in rhizobia for symbiotic nitrogen fixation. A Fix(-) (non-fixing) plant mutant, fen1, forms morphologically normal but ineffective nodules. The causal gene, FEN1, was shown to encode HCS by its ability to complement a HCS-defective mutant of Saccharomyces cerevisiae. Homocitrate was present abundantly in wild-type nodules but was absent from ineffective fen1 nodules. Inoculation with Mesorhizobium loti carrying FEN1 or Azotobacter vinelandii NifV rescued the defect in nitrogen-fixing activity of the fen1 nodules. Exogenous supply of homocitrate also recovered the nitrogen-fixing activity of the fen1 nodules through de novo nitrogenase synthesis in the rhizobial bacteroids. These results indicate that homocitrate derived from the host plant cells is essential for the efficient and continuing synthesis of the nitrogenase system in endosymbionts, and thus provide a molecular basis for the complementary and indispensable partnership between legumes and rhizobia in symbiotic nitrogen fixation.

Striatum is more vulnerable to oxidative damage induced by the metabolites accumulating in 3-hydroxy-3-methylglutaryl-CoA lyase deficiency as compared to liver.

The present work investigated the in vitro effects of 3-hydroxy-3-methylglutarate, 3-methylglutarate, 3-methylglutaconate and 3-hydroxyisovalerate, which accumulate in 3-hydroxy-3-methylglutaric aciduria, on important parameters of oxidative stress in striatum and liver of young rats, tissues that are injured in this disorder. Our results show that all metabolites induced lipid peroxidation (thiobarbituric acid-reactive substances increase) and decreased glutathione levels in striatum, whereas 3-hydroxy-3-methylglutarate, besides inducing the strongest effect, also altered thiobarbituric acid-reactive substances and glutathione levels in the liver. Furthermore, 3-hydroxy-3-methylglutarate, 3-methylglutarate and 3-methylglutaconate oxidized sulfhydryl groups in the striatum, but not in the liver. Our data indicate that 3-hydroxy-3-methylglutarate behaves as a stronger pro-oxidant agent compared to the other metabolites accumulating in 3-hydroxy-3-methylglutaric aciduria and that the striatum present higher vulnerability to oxidative damage relatively to the liver.

Inactivation of the Fto gene protects from obesity.

Several independent, genome-wide association studies have identified a strong correlation between body mass index and polymorphisms in the human FTO gene. Common variants in the first intron define a risk allele predisposing to obesity, with homozygotes for the risk allele weighing approximately 3 kilograms more than homozygotes for the low risk allele. Nevertheless, the functional role of FTO in energy homeostasis remains elusive. Here we show that the loss of Fto in mice leads to postnatal growth retardation and a significant reduction in adipose tissue and lean body mass. The leanness of Fto-deficient mice develops as a consequence of increased energy expenditure and systemic sympathetic activation, despite decreased spontaneous locomotor activity and relative hyperphagia. Taken together, these experiments provide, to our knowledge, the first direct demonstration that Fto is functionally involved in energy homeostasis by the control of energy expenditure.

3-Hydroxy-3-methylglutaryl-coenzyme A lyase deficiency: initial presentation in a young adult.

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase deficiency is a rare inborn error affecting leucine catabolism and ketogenesis, usually presenting in the neonatal period. Late forms of the disease have been detected in infancy and childhood, but not in adults. We report a case of HMG-CoA lyase deficiency with initial presentation in a 29-year-old adult with no prior history of the disease, which to our knowledge is the first case described with presentation at this age.

Molecular analysis of Taiwanese patients with 3-hydroxy-3-methylglutaryl CoA lyase deficiency.

BACKGROUND: 3-Hydroxy-3-methylglutaryl CoA lyase deficiency (HL deficiency) is a rare autosomal recessive mitochondrial disease characterized by a deficiency in the enzyme 3-Hydroxy-3-methylglutaryl CoA lyase (HMGCL). Here, we report on novel mutations identified in the HMGCL gene in 2 Taiwanese patients with HL deficiency. METHODS: Analysis of organic acids in urine was performed using gas chromatography-mass spectrometry to confirm HL deficiency in the two subjects. The mutations in their HMGCL genes then were determined by direct sequencing. In addition, the effect of a splice site mutation was determined using reverse transcription-polymerase chain reactions (RT-PCR). RESULTS: A total of 3 novel mutations in the HMGCL gene were revealed by molecular analysis: one missense mutation (c.494G>T, p.Arg165Gln) and 2 splice site mutations (IVS3+1G>A, IVS6-1G>A). The results of RT-PCR revealed that an IVS3+1G>A mutation leads to skipping of exon3. We also calculated that the incidence of HL deficiency in Taiwan is <1 per 1,000,000 live births. CONCLUSIONS: The results of this study suggest that unique HMGCL gene mutations exist in Taiwanese HL deficiency patients. Therefore, HMGCL gene profiling may be useful in genetic counseling for families affected by HL deficiency.