Lipid droplets and peroxisomes are co-regulated to drive lifespan extension in response to mono-unsaturated fatty acids.

Dietary mono-unsaturated fatty acids (MUFAs) are linked to longevity in several species. But the mechanisms by which MUFAs extend lifespan remain unclear. Here we show that an organelle network involving lipid droplets and peroxisomes is critical for MUFA-induced longevity in Caenorhabditis elegans. MUFAs upregulate the number of lipid droplets in fat storage tissues. Increased lipid droplet number is necessary for MUFA-induced longevity and predicts remaining lifespan. Lipidomics datasets reveal that MUFAs also modify the ratio of membrane lipids and ether lipids-a signature associated with decreased lipid oxidation. In agreement with this, MUFAs decrease lipid oxidation in middle-aged individuals. Intriguingly, MUFAs upregulate not only lipid droplet number but also peroxisome number. A targeted screen identifies genes involved in the co-regulation of lipid droplets and peroxisomes, and reveals that induction of both organelles is optimal for longevity. Our study uncovers an organelle network involved in lipid homeostasis and lifespan regulation, opening new avenues for interventions to delay aging.

Cross-Laboratory Standardization of Preclinical Lipidomics Using Differential Mobility Spectrometry and Multiple Reaction Monitoring.

Modern biomarker and translational research as well as personalized health care studies rely heavily on powerful omics' technologies, including metabolomics and lipidomics. However, to translate metabolomics and lipidomics discoveries into a high-throughput clinical setting, standardization is of utmost importance. Here, we compared and benchmarked a quantitative lipidomics platform. The employed Lipidyzer platform is based on lipid class separation by means of differential mobility spectrometry with subsequent multiple reaction monitoring. Quantitation is achieved by the use of 54 deuterated internal standards and an automated informatics approach. We investigated the platform performance across nine laboratories using NIST SRM 1950-Metabolites in Frozen Human Plasma, and three NIST Candidate Reference Materials 8231-Frozen Human Plasma Suite for Metabolomics (high triglyceride, diabetic, and African-American plasma). In addition, we comparatively analyzed 59 plasma samples from individuals with familial hypercholesterolemia from a clinical cohort study. We provide evidence that the more practical methyl-tert-butyl ether extraction outperforms the classic Bligh and Dyer approach and compare our results with two previously published ring trials. In summary, we present standardized lipidomics protocols, allowing for the highly reproducible analysis of several hundred human plasma lipids, and present detailed molecular information for potentially disease relevant and ethnicity-related materials.

In-depth triacylglycerol profiling using MS3 Q-Trap mass spectrometry.

Total triacylglycerol (TAG) level is a key clinical marker of metabolic and cardiovascular diseases. However, the roles of individual TAGs have not been thoroughly explored in part due to their extreme structural complexity. We present a targeted mass spectrometry-based method combining multiple reaction monitoring (MRM) and multiple stage mass spectrometry (MS3) for the comprehensive qualitative and semiquantitative profiling of TAGs. This method referred as TriP-MS3 - triacylglycerol profiling using MS3 - screens for more than 6,700 TAG species in a fully automated fashion. TriP-MS3 demonstrated excellent reproducibility (median interday CV âˆ¼ 0.15) and linearity (median R2 = 0.978) and detected 285 individual TAG species in human plasma. The semiquantitative accuracy of the method was validated by comparison with a state-of-the-art reverse phase liquid chromatography (RPLC)-MS (R2 = 0.83), which is the most commonly used approach for TAGs profiling. Finally, we demonstrate the utility and the versatility of the method by characterizing the effects of a fatty acid desaturase inhibitor on TAG profiles in vitro and by profiling TAGs in Caenorhabditis elegans.

Characterization of key enzymes involved in triacylglycerol biosynthesis in mycobacteria.

Phosphatidic acid phosphatase (PAP) catalyzes the dephosphorylation of phosphatidic acid (PA) yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. PAP activity has a key role in the regulation of PA flux towards TAG or glycerophospholipid synthesis. In this work we have characterized two Mycobacterium smegmatis genes encoding for functional PAP proteins. Disruption of both genes provoked a sharp reduction in de novo TAG biosynthesis in early growth phase cultures under stress conditions. In vivo labeling experiments demonstrated that TAG biosynthesis was restored in the ∆PAP mutant when bacteria reached exponential growth phase, with a concomitant reduction of phospholipid synthesis. In addition, comparative lipidomic analysis showed that the ∆PAP strain had increased levels of odd chain fatty acids esterified into TAGs, suggesting that the absence of PAP activity triggered other rearrangements of lipid metabolism, like phospholipid recycling, in order to maintain the wild type levels of TAG. Finally, the lipid changes observed in the ∆PAP mutant led to defective biofilm formation. Understanding the interaction between TAG synthesis and the lipid composition of mycobacterial cell envelope is a key step to better understand how lipid homeostasis is regulated during Mycobacterium tuberculosis infection.

Correction: Pleiotropic Effect of AccD5 and AccE5 Depletion in Acyl-Coenzyme A Carboxylase Activity and in Lipid Biosynthesis in Mycobacteria.

[This corrects the article DOI: 10.1371/journal.pone.0099853.].

FasR Regulates Fatty Acid Biosynthesis and Is Essential for Virulence of Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is the world's leading cause of death from an infectious disease. One of the main features of this pathogen is the complex and dynamic lipid composition of the cell envelope, which adapts to the variable host environment and defines the fate of infection by actively interacting with and modulating immune responses. However, while much has been learned about the enzymes of the numerous lipid pathways, little knowledge is available regarding the proteins and metabolic signals regulating lipid metabolism during M. tuberculosis infection. In this work, we constructed and characterized a FasR-deficient mutant in M. tuberculosis and demonstrated that FasR positively regulates fas and acpS expression. Lipidomic analysis of the wild type and mutant strains revealed complete rearrangement of most lipid components of the cell envelope, with phospholipids, mycolic acids, sulfolipids, and phthiocerol dimycocerosates relative abundance severely altered. As a consequence, replication of the mutant strain was impaired in macrophages leading to reduced virulence in a mouse model of infection. Moreover, we show that the fasR mutant resides in acidified cellular compartments, suggesting that the lipid perturbation caused by the mutation prevented M. tuberculosis inhibition of phagolysosome maturation. This study identified FasR as a novel factor involved in regulation of mycobacterial virulence and provides evidence for the essential role that modulation of lipid homeostasis plays in the outcome of M. tuberculosis infection.

Analysing nonsynonymous mutations between two Mycobacterium bovis strains with contrasting pathogenic profiles.

Mycobacterium bovis (M. bovis) is the causative agent of bovine tuberculosis, a chronic infectious disease that can affect cattle, other domesticated species, wild animals and humans. This disease produces important economic losses worldwide. Two M. bovis strains (04-303 and 534) have been isolated in Argentina. Whereas the 04-303 strain was isolated from a wild boar, the 534 strain was obtained from cattle. In a previous study, six weeks after infection, the 04-303 strain induced 100% mortality in mice. By contrast, mice infected with the 534 strain survived, with limited tissue damage, after four months. In this study we compared all predictive proteins encoded in both M. bovis genomes. The comparative analysis revealed 141 polymorphic proteins between both strains. From these proteins, nine virulence proteins showed polymorphisms in 04-303, whereas five did it in the 534 strain. Remarkably, both strains contained a high level of polymorphism in proteins related to phthiocerol dimycocerosate (PDIM) synthesis or transport. Further experimental evidence indicated that only mutations in the 534 strain have an impact on PDIM synthesis. The observed reduction in PDIM content in the 534 strain, together with its low capacity to induce phagosome arrest, may be associated with the reported deficiency of this strain to replicate and survive inside bovine macrophages. The findings of this study could contribute to a better understanding of pathogenicity and virulence aspects of M. bovis, which is essential for further studies aiming at developing new vaccines and diagnostic techniques for bovines.

Role of long-chain acyl-CoAs in the regulation of mycolic acid biosynthesis in mycobacteria.

One of the dominant features of the biology of Mycobacterium tuberculosis, and other mycobacteria, is the mycobacterial cell envelope with its exceptional complex composition. Mycolic acids are major and very specific components of the cell envelope and play a key role in its architecture and impermeability. Biosynthesis of mycolic acid (MA) precursors requires two types of fatty acid synthases, FAS I and FAS II, which should work in concert in order to keep lipid homeostasis tightly regulated. Both FAS systems are regulated at their transcriptional level by specific regulatory proteins. FasR regulates components of the FAS I system, whereas MabR and FadR regulate components of the FAS II system. In this article, by constructing a tight mabR conditional mutant in Mycobacterium smegmatis mc2155, we demonstrated that sub-physiological levels of MabR lead to a downregulation of the fasII genes, inferring that this protein is a transcriptional activator of the FAS II system. In vivo labelling experiments and lipidomic studies carried out in the wild-type and the mabR conditional mutant demonstrated that under conditions of reduced levels of MabR, there is a clear inhibition of biosynthesis of MAs, with a concomitant change in their relative composition, and of other MA-containing molecules. These studies also demonstrated a change in the phospholipid composition of the membrane of the mutant strain, with a significant increase of phosphatidylinositol. Gel shift assays carried out with MabR and PfasII as a probe in the presence of different chain-length acyl-CoAs strongly suggest that molecules longer than C18 can be sensed by MabR to modulate its affinity for the operator sequences that it recognizes, and in that way switch on or off the MabR-dependent promoter. Finally, we demonstrated the direct role of MabR in the upregulation of the fasII operon genes after isoniazid treatment.

Integration of Temperature and Blue-Light Sensing in Acinetobacter baumannii Through the BlsA Sensor.

BlsA is a BLUF photoreceptor present in Acinetobacter baumannii, responsible for modulation of motility, biofilm formation and virulence by light. In this work, we have combined physiological and biophysical evidences to begin to understand the basis of the differential photoregulation observed as a function of temperature. Indeed, we show that blsA expression is reduced at 37°C, which correlates with negligible photoreceptor levels in the cells, likely accounting for absence of photoregulation at this temperature. Another point of control occurs on the functionality of the BlsA photocycle itself at different temperatures, which occurs with an average quantum yield of photoactivation of the signaling state of 0.20 ± 0.03 at 15°C < T < 25°C, but is practically inoperative at T > 30°C, as a result of conformational changes produced in the nanocavity of FAD. This effect would be important when the photoreceptor is already present in the cell to avoid almost instantaneously further signaling process when it is no longer necessary, for example under circumstances of temperature changes possibly faced by the bacteria. This complex interplay between light and temperature would provide the bacteria clues of environmental location and dictate/modulate light photosensing in A. baumannii.

A conditional mutant of the fatty acid synthase unveils unexpected cross talks in mycobacterial lipid metabolism.

Unlike most bacteria, mycobacteria rely on the multi-domain enzyme eukaryote-like fatty acid synthase I (FAS I) to make fatty acids de novo. These metabolites are precursors of the biosynthesis of most of the lipids present both in the complex mycobacteria cell wall and in the storage lipids inside the cell. In order to study the role of the type I FAS system in Mycobacterium lipid metabolism in vivo, we constructed a conditional mutant in the fas-acpS operon of Mycobacterium smegmatis and analysed in detail the impact of reduced de novo fatty acid biosynthesis on the global architecture of the cell envelope. As expected, the mutant exhibited growth defect in the non-permissive condition that correlated well with the lower expression of fas-acpS and the concomitant reduction of FAS I, confirming that FAS I is essential for survival. The reduction observed in FAS I provoked an accumulation of its substrates, acetyl-CoA and malonyl-CoA, and a strong reduction of C12 to C18 acyl-CoAs, but not of long-chain acyl-CoAs (C19 to C24). The most intriguing result was the ability of the mutant to keep synthesizing mycolic acids when fatty acid biosynthesis was impaired. A detailed comparative lipidomic analysis showed that although reduced FAS I levels had a strong impact on fatty acid and phospholipid biosynthesis, mycolic acids were still being synthesized in the mutant, although with a different relative species distribution. However, when triacylglycerol degradation was inhibited, mycolic acid biosynthesis was significantly reduced, suggesting that storage lipids could be an intracellular reservoir of fatty acids for the biosynthesis of complex lipids in mycobacteria. Understanding the interaction between FAS I and the metabolic pathways that rely on FAS I products is a key step to better understand how lipid homeostasis is regulated in this microorganism and how this regulation could play a role during infection in pathogenic mycobacteria.

Analysis of coenzyme A activated compounds in actinomycetes.

Acyl-CoAs are crucial compounds involved in essential metabolic pathways such as the Krebs cycle and lipid, carbohydrate, and amino acid metabolisms, and they are also key signal molecules involved in the transcriptional regulation of lipid biosynthesis in many organisms. In this study, we took advantage of the high selectivity of mass spectrometry and developed an ion-pairing reverse-phase high-pressure liquid chromatography electrospray ionization high-resolution mass spectrometry (IP-RP-HPLC/ESI-HRMS) method to carry on a comprehensive analytical determination of the wide range of fatty acyl-CoAs present in actinomycetes. The advantage of using a QTOF spectrometer resides in the excellent mass accuracy over a wide dynamic range and measurements of the true isotope pattern that can be used for molecular formula elucidation of unknown analytes. As a proof of concept, we used this assay to determine the composition of the fatty acyl-CoA pools in Mycobacterium, Streptomyces, and Corynebacterium species, revealing an extraordinary difference in fatty acyl-CoA amounts and species distribution between the three genera and between the two species of mycobacteria analyzed, including the presence of different chain-length carboxy-acyl-CoAs, key substrates of mycolic acid biosynthesis. The method was also used to analyze the impact of two fatty acid synthase inhibitors on the acyl-CoA profile of Mycobacterium smegmatis, which showed some unexpected low levels of C24 acyl-CoAs in the isoniazid-treated cells. This robust, sensitive, and reliable method should be broadly applicable in the studies of the wide range of bacteria metabolisms in which acyl-CoA molecules participate.

Pleiotropic effect of AccD5 and AccE5 depletion in acyl-coenzyme A carboxylase activity and in lipid biosynthesis in mycobacteria.

Mycobacteria contain a large variety of fatty acids which are used for the biosynthesis of several complex cell wall lipids that have been implicated in the ability of the organism to resist host defenses. The building blocks for the biosynthesis of all these lipids are provided by a fairly complex set of acyl-CoA carboxylases (ACCases) whose subunit composition and roles within these organisms have not yet been clearly established. Previous biochemical and structural studies provided strong evidences that ACCase 5 from Mycobacterium tuberculosis is formed by the AccA3, AccD5 and AccE5 subunits and that this enzyme complex carboxylates acetyl-CoA and propionyl-CoA with a clear substrate preference for the latest. In this work we used a genetic approach to unambiguously demonstrate that the products of both accD5 and accE5 genes are essential for the viability of Mycobacterium smegmatis. By obtaining a conditional mutant on the accD5-accE5 operon, we also demonstrated that the main physiological role of this enzyme complex was to provide the substrates for fatty acid and mycolic acid biosynthesis. Furthermore, enzymatic and biochemical analysis of the conditional mutant provided strong evidences supporting the notion that AccD5 and/or AccE5 have an additional role in the carboxylation of long chain acyl-CoA prior to mycolic acid condensation. These studies represent a significant step towards a better understanding of the roles of ACCases in mycobacteria and confirm ACCase 5 as an interesting target for the development of new antimycobacterial drugs.

The opportunistic human pathogen Acinetobacter baumannii senses and responds to light.

Light is a ubiquitous environmental signal that many organisms sense and respond to by modulating their physiological responses accordingly. While this is an expected response among phototrophic microorganisms, the ability of chemotrophic prokaryotes to sense and react to light has become a puzzling and novel issue in bacterial physiology, particularly among bacterial pathogens. In this work, we show that the opportunistic pathogen Acinetobacter baumannii senses and responds to blue light. Motility and formation of biofilms and pellicles were observed only when bacterial cells were incubated in darkness. In contrast, the killing of Candida albicans filaments was enhanced when they were cocultured with bacteria under light. These bacterial responses depend on the expression of the A. baumannii ATCC 17978 A1S_2225 gene, which codes for an 18.6-kDa protein that contains an N-terminal blue-light-sensing-using flavin (BLUF) domain and lacks a detectable output domain(s). Spectral analyses of the purified recombinant protein showed its ability to sense light by a red shift upon illumination. Therefore, the A1S_2225 gene, which is present in several members of the Acinetobacter genus, was named blue-light-sensing A (blsA). Interestingly, temperature plays a role in the ability of A. baumannii to sense and respond to light via the BlsA photoreceptor protein.