Mycobacterium tuberculosis Induces Irg1 in Murine Macrophages by a Pathway Involving Both TLR-2 and STING/IFNAR Signaling and Requiring Bacterial Phagocytosis.

Irg1 is an enzyme that generates itaconate, a metabolite that plays a key role in the regulation of inflammatory responses. Previous studies have implicated Irg1 as an important mediator in preventing excessive inflammation and tissue damage in Mycobacterium tuberculosis (Mtb) infection. Here, we investigated the pattern recognition receptors and signaling pathways by which Mtb triggers Irg1 gene expression by comparing the responses of control and genetically deficient BMDMs. Using this approach, we demonstrated partial roles for TLR-2 (but not TLR-4 or -9), MyD88 and NFκB signaling in Irg1 induction by Mtb bacilli. In addition, drug inhibition studies revealed major requirements for phagocytosis and endosomal acidification in Irg1 expression triggered by Mtb but not LPS or PAM3CSK4. Importantly, the Mtb-induced Irg1 response was highly dependent on the presence of the bacterial ESX-1 secretion system, as well as host STING and Type I IFN receptor (IFNAR) signaling with Type II IFN (IFN-γ) signaling playing only a minimal role. Based on these findings we hypothesize that Mtb induces Irg1 expression in macrophages via the combination of two independent triggers both dependent on bacterial phagocytosis: 1) a major signal stimulated by phagocytized Mtb products released by an ESX-1-dependent mechanism into the cytosol where they activate the STING pathway leading to Type I-IFN production, and 2) a secondary TLR-2, MyD88 and NFκB dependent signal that enhances Irg1 production independently of Type I IFN induction.

Oleate Hydratase in Lactobacillus delbrueckii subsp. bulgaricus LBP UFSC 2230 Catalyzes the Reversible Conversion between Linoleic Acid and Ricinoleic Acid.

Conjugated linoleic acid (CLA) has been the subject of numerous studies in recent decades because of its associated health benefits. CLA is an intermediate product of the biohydrogenation pathway of linoleic acid (LA) in bacteria. Several bacterial species capable of efficiently converting LA into CLA have been widely reported in the literature, among them Lactobacillus delbrueckii subsp. bulgaricus LBP UFSC 2230. Over the last few years, a multicomponent enzymatic system consisting of three enzymes involved in the biohydrogenation process of LA has been proposed. Sequencing the genome of L. delbrueckii subsp. bulgaricus LBP UFSC 2230 revealed only one gene capable of encoding an oleate hydratase (OleH), unlike the presence of multiple genes typically found in similar strains. This study investigated the biological effect of the OleH enzyme of L. delbrueckii subsp. bulgaricus LBP UFSC 2230 on the hydration of LA and dehydration of ricinoleic acid (RA) and its possible role in the production of CLA. The OleH was cloned, expressed, purified, and characterized. Fatty acid measurements were made by an internal standard method using a gas chromatography-coupled flame ionization detector (GC-FID) system. It was found that the enzyme is a hydratase/dehydratase, leading to a reversible transformation between LA and RA. In addition, the results showed that L. delbrueckii subsp. bulgaricus LBP UFSC 2230 OleH protein plays a role in stress tolerance in Escherichia coli. In conclusion, the OleH of L. delbrueckii subsp. bulgaricus LBP UFSC 2230 catalyzes the initial stage of saturation metabolism of LA, although it has not converted the substrates directly into CLA. IMPORTANCE This study provides insight into the enzymatic mechanism of CLA synthesis in L. delbrueckii subsp. bulgaricus and broadens our understanding of the bioconversion of LA and RA by OleH. The impact of OleH on the production of the c9, t11 CLA isomer and stress tolerance by E. coli has been assisted. The results provide an understanding of the factors which influence OleH activity. L. delbrueckii subsp. bulgaricus LBP UFSC 2230 OleH presented two putative fatty acid-binding sites. Recombinant OleH catalyzed both LA hydration and RA dehydration. OleH was shown to play a role in bacterial growth performance in the presence of LA.

Molecular modeling of a series of dehydroquinate dehydratase type II inhibitors of Mycobacterium tuberculosis and design of new binders.

Tuberculosis, caused by Mycobacterium tuberculosis (M. tuberculosis), is still responsible for a large number of fatal cases, especially in developing countries with alarming rates of incidence and prevalence worldwide. Mycobacterium tuberculosis has a remarkable ability to develop new resistance mechanisms to the conventional antimicrobials treatment. Because of this, there is an urgent need for novel bioactive compounds for its treatment. The dehydroquinate dehydratase II (DHQase II) is considered a key enzyme of shikimate pathway, and it can be used as a promising target for the design of new bioactive compounds with antibacterial action. The aim of this work was the construction of QSAR models to aid the design of new potential DHQase II inhibitors. For that purpose, various molecular modeling approaches, such as activity cliff, QSAR models and computer-aided ligand design were utilized. A predictive in silico 4D-QSAR model was built using a database comprising 86 inhibitors of DHQase II, and the model was used to predict the activity of the designed ligands. The obtained model proved to predict well the DHQase II inhibition for an external validation dataset ([Formula: see text] = 0.72). Also, the Activity Cliff analysis shed light on important structural features applied to the ligand design.

Early life fluoxetine treatment causes long-term lean phenotype in skeletal muscle of rats exposed to maternal lard-based high-fat diet.

There is a concern about early life exposure to Selective Serotonin Reuptake Inhibitors (SSRI) in child development and motor system maturation. Little is known, however, about the interaction of environmental factors, such as maternal nutrition, associated with early exposure to SSRI. The increased maternal consumption of high-fat diets is worrisome and affects serotonin system development with repercussions in body phenotype. This study aimed to assess the short- and long-term effects of neonatal fluoxetine treatment on the body and skeletal muscle phenotype of rats exposed to a maternal lard-based high-fat (H) diet during the perinatal period. A maternal lard-based high-fat diet causes reduced birth weight, a short-term reduction in type IIA fibers in the soleus muscle, and in type IIB fibers in the Extensor Digitorum Longus (EDL) muscle, reducing Lactate Dehydrogenase (LDH) activity in both muscles. In the long-term, the soleus showed reduced muscle weight, smaller area and perimeter of muscle fibers, while the EDL muscle showed reduced Citrate Synthase (CS) activity in offspring from the rats on the maternal lard-based high-fat diet. Early-life exposure to fluoxetine reduced body weight and growth and reduced soleus weight and enzymatic activity in young rats. Exposure to neonatal fluoxetine in adult rats caused a decreased body mass index, less food intake, and reduced muscle weight with reduced CS and LDH activity. Neonatal fluoxetine in young rats exposed to a maternal lard-based high-fat diet caused reduced body weight and growth, reduced soleus weight as well as area and perimeter of type I muscle fibers. In adulthood, there was a reduction in food intake, increased proportion of IIA type fibers, reduced area and perimeter of type IIB, and reduction in levels of CS activity in EDL muscle. Neonatal fluoxetine treatment in rats exposed to a maternal lard-based, high-fat diet induces a reduction in muscle weight, an increase in the proportion of oxidative fibers and greater oxidative enzymatic activity in adulthood.

The structure of a prokaryotic feruloyl-CoA hydratase-lyase from a lignin-degrading consortium with high oligomerization stability under extreme pHs.

In the context of increasing demand for renewable alternatives of fuels and chemicals, the valorization of lignin emerges as a value-adding strategy in biorefineries and an alternative to petroleum-derived molecules. One of the compounds derived from lignin is ferulic acid (FA), which can be converted into valuable molecules such as vanillin. In microorganisms, FA biotransformation into vanillin can occur via a two-step reaction catalyzed by the sequential activity of a feruloyl-CoA synthetase (FCS) and an feruloyl-CoA hydratase-lyase (FCHL), which could be exploited industrially. In this study, a prokaryotic FCHL derived from a lignin-degrading microbial consortium (named LM-FCHL) was cloned, successfully expressed in soluble form and purified. The crystal structure was solved and refined at 2.1 Å resolution. The LM-FCHL is a hexamer composed of a dimer of trimers, which showed to be quite stable under extreme pH conditions. Finally, small angle X-ray scattering corroborates the hexameric state in solution and indicates flexibility in the protein structure. The present study contributes to the field of lignin valorization to valuable molecules by establishing the biophysical and structural characterization for a novel FCHL member of unique characteristics.

Relative expression of stress-related genes during acclimation at low temperature of psychrotrophic Oenococcus oeni strains from Patagonian wine.

In the present study, we evaluated the transcriptional response of four stress-related genes in three Oenococcus oeni strains after acclimation at two different temperatures. Gene expression was analyzed at time zero and after 48 h acclimation at 18 and 21 °C. After the acclimation period cells were inoculated into sterile Pinot noir wine and MLF was followed for 25 days to investigate if different acclimation temperatures could influence cell survival and MLF performance. L-malic acid consumption, population survival, and transcriptional behavior were different upon the acclimation temperature. rmlB and hsp20 genes presented a considerable increase in their expression level when strains were acclimated at 18 °C particularly in the psychrotrophic strains UNQOe19 and UNQOe4 isolated from Patagonian Pinot noir wine in comparison with the control strain (ATCC 27310). The increase in rmlB and hsp20 expression could account for the better survival of these strains in Pinot noir in comparison with the control strain. In addition, Patagonian populations acclimated at 18 °C were able to consume a higher percentage of L-malic acid in comparison with cells acclimated at 21 °C. Our results suggest that gene expression analysis of cells acclimated at sub-optimal temperatures could benefit the selection of psychrotrophic strains aimed as starter cultures.

Development of machine learning models to predict inhibition of 3-dehydroquinate dehydratase.

In this study, we describe the development of new machine learning models to predict inhibition of the enzyme 3-dehydroquinate dehydratase (DHQD). This enzyme is the third step of the shikimate pathway and is responsible for the synthesis of chorismate, which is a natural precursor of aromatic amino acids. The enzymes of shikimate pathway are absent in humans, which make them protein targets for the design of antimicrobial drugs. We focus our study on the crystallographic structures of DHQD in complex with competitive inhibitors, for which experimental inhibition constant data is available. Application of supervised machine learning techniques was able to elaborate a robust DHQD-targeted model to predict binding affinity. Combination of high-resolution crystallographic structures and binding information indicates that the prevalence of intermolecular electrostatic interactions between DHQD and competitive inhibitors is of pivotal importance for the binding affinity against this enzyme. The present findings can be used to speed up virtual screening studies focused on the DHQD structure.

Long non-coding RNA-ROR aggravates myocardial ischemia/reperfusion injury.

Long non-coding RNAs (lncRNAs) play an important role in the pathogenesis of cardiovascular diseases, especially in myocardial infarction and ischemia/reperfusion (I/R). However, the underlying molecular mechanism remains unclear. In this study, we determined the role and the possible underlying molecular mechanism of lncRNA-ROR in myocardial I/R injury. H9c2 cells and human cardiomyocytes (HCM) were subjected to either hypoxia/reoxygenation (H/R), I/R or normal conditions (normoxia). The expression levels of lncRNA-ROR were detected in serum of myocardial I/R injury patients, H9c2 cells, and HCM by qRT-PCR. Then, levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) were measured by kits. Cell viability, apoptosis, apoptosis-associated factors, and p38/MAPK pathway were examined by MTT, flow cytometry, and western blot assays. Furthermore, reactive oxygen species (ROS) production was determined by H2DCF-DA and MitoSOX Red probes with flow cytometry. NADPH oxidase activity and NOX2 protein levels were measured by lucigenin chemiluminescence and western blot. Results showed that lncRNA-ROR expression was increased in I/R patients and in H/R treatment of H9c2 cells and HCM. Moreover, lncRNA-ROR significantly promoted H/R-induced myocardial injury via stimulating release of LDH, MDA, SOD, and GSH-PX. Furthermore, lncRNA-ROR decreased cell viability, increased apoptosis, and regulated expression of apoptosis-associated factors. Additionally, lncRNA-ROR increased phosphorylation of p38 and ERK1/2 expression and inhibition of p38/MAPK, and rescued lncRNA-ROR-induced cell injury in H9c2 cells and HCM. ROS production, NADPH oxidase activity, and NOX2 protein levels were promoted by lncRNA-ROR. These data suggested that lncRNA-ROR acted as a therapeutic agent for the treatment of myocardial I/R injury.

Occurrence of virulence-associated genes among Staphylococcus saprophyticus isolated from different sources.

Staphylococcus saprophyticus is an important pathogen responsible for community urinary tract infections (UTI). Besides composing the human microbiota, this species is widely distributed in the environment and the origins of this organism for human infection is not fully characterized. Although some virulence determinants are known, such as d-serine deaminase (DsdA), urease and cell-wall associated proteins, few studies investigated the distribution of virulence-associated genes and analyzed the pathogenic potential of S. saprophyticus strains from different sources. The aim of the present study was to detect the presence of S. saprophyticus genes encoding surface proteins UafA, Aas, Ssp, SdrI, SssF as well as the DsdA and urease enzymes. A total of 142 S. saprophyticus strains were obtained from four sources: UTI, colonization, water and food. It was found, in every tested strain, the presence of genes encoding the surface proteins UafA, Aas, Ssp and SssF and the DsdA and urease enzymes. In contrast, the gene encoding SdrI surface protein was not detected in any of the strains of S. saprophyticus. These results provide a better understanding of the characteristics of S. saprophyticus strains and suggest that isolates from non-human sources have a potential to colonize the urinary tract.

Fused dimerization increases expression, solubility, and activity of bacterial dehydratase enzymes.

FabA and FabZ are the two dehydratase enzymes in Escherichia coli that catalyze the dehydration of acyl intermediates in the biosynthesis of fatty acids. Both enzymes form obligate dimers in which the active site contains key amino acids from both subunits. While FabA is a soluble protein that has been relatively straightforward to express and to purify from cultured E. coli, FabZ has shown to be mostly insoluble and only partially active. In an effort to increase the solubility and activity of both dehydratases, we made constructs consisting of two identical subunits of FabA or FabZ fused with a naturally occurring peptide linker, so as to force their dimerization. The fused dimer of FabZ (FabZ-FabZ) was expressed as a soluble enzyme with an ninefold higher activity in vitro than the unfused FabZ. This construct exemplifies a strategy for the improvement of enzymes from the fatty acid biosynthesis pathways, many of which function as dimers, catalyzing critical steps for the production of fatty acids.

Long non-coding RNA-ROR aggravates myocardial ischemia/reperfusion injury

Long non-coding RNAs (lncRNAs) play an important role in the pathogenesis of cardiovascular diseases, especially in myocardial infarction and ischemia/reperfusion (I/R). However, the underlying molecular mechanism remains unclear. In this study, we determined the role and the possible underlying molecular mechanism of lncRNA-ROR in myocardial I/R injury. H9c2 cells and human cardiomyocytes (HCM) were subjected to either hypoxia/reoxygenation (H/R), I/R or normal conditions (normoxia). The expression levels of lncRNA-ROR were detected in serum of myocardial I/R injury patients, H9c2 cells, and HCM by qRT-PCR. Then, levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) were measured by kits. Cell viability, apoptosis, apoptosis-associated factors, and p38/MAPK pathway were examined by MTT, flow cytometry, and western blot assays. Furthermore, reactive oxygen species (ROS) production was determined by H2DCF-DA and MitoSOX Red probes with flow cytometry. NADPH oxidase activity and NOX2 protein levels were measured by lucigenin chemiluminescence and western blot. Results showed that lncRNA-ROR expression was increased in I/R patients and in H/R treatment of H9c2 cells and HCM. Moreover, lncRNA-ROR significantly promoted H/R-induced myocardial injury via stimulating release of LDH, MDA, SOD, and GSH-PX. Furthermore, lncRNA-ROR decreased cell viability, increased apoptosis, and regulated expression of apoptosis-associated factors. Additionally, lncRNA-ROR increased phosphorylation of p38 and ERK1/2 expression and inhibition of p38/MAPK, and rescued lncRNA-ROR-induced cell injury in H9c2 cells and HCM. ROS production, NADPH oxidase activity, and NOX2 protein levels were promoted by lncRNA-ROR. These data suggested that lncRNA-ROR acted as a therapeutic agent for the treatment of myocardial I/R injury.

Characterization and role of the 3-methylglutaconyl coenzyme A hidratase in Trypanosoma brucei.

Trypanosoma brucei, the agent of African Trypanosomiasis, is a flagellated protozoan parasite that develops in tsetse flies and in the blood of various mammals. The parasite acquires nutrients such as sugars, lipids and amino acids from their hosts. Amino acids are used to generate energy and for protein and lipid synthesis. However, it is still unknown how T. brucei catabolizes most of the acquired amino acids. Here we explored the role of an enzyme of the leucine catabolism, the 3-methylglutaconyl-Coenzyme A hydratase (3-MGCoA-H). It catalyzes the hydration of 3-methylglutaconyl-Coenzyme A (3-MGCoA) into 3-hydroxymethylglutaryl-Coenzyme A (3-HMGCoA). We found that 3-MGCoA-H localizes in the mitochondrial matrix and is expressed in both insect and mammalian bloodstream forms of the parasite. The depletion of 3-MGCoA-H by RNA interference affected minimally the proliferation of both forms. However, an excess of leucine in the culture medium caused growth defects in cells depleted of 3-MGCoA-H, which could be reestablished by mevalonate, a precursor of isoprenoids and steroids. Indeed, procyclics depleted of the 3-MGCoA-H presented reduced levels of synthesized steroids relative to cholesterol that is scavenged by the parasite, and these levels were also reestablished by mevalonate. These results suggest that accumulation of leucine catabolites could affect the level of mevalonate and consequently inhibit the sterol biosynthesis, required for T. brucei growth.

In Azospirillum brasilense, mutations in flmA or flmB genes affect polar flagellum assembly, surface polysaccharides, and attachment to maize roots.

Azospirillum brasilense is a soil bacterium capable of promoting plant growth. Several surface components were previously reported to be involved in the attachment of A. brasilense to root plants. Among these components are the exopolysaccharide (EPS), lipopolysaccharide (LPS) and the polar flagellum. Flagellin from polar flagellum is glycosylated and it was suggested that genes involved in such a posttranslational modification are the same ones involved in the biosynthesis of sugars present in the O-antigen of the LPS. In this work, we report on the characterization of two homologs present in A. brasilense Cd, to the well characterized flagellin modification genes, flmA and flmB, from Aeromonas caviae. We show that mutations in either flmA or flmB genes of A. brasilense resulted in non-motile cells due to alterations in the polar flagellum assembly. Moreover, these mutations also affected the capability of A. brasilense cells to adsorb to maize roots and to produce LPS and EPS. By generating a mutant containing the polar flagellum affected in their rotation, we show the importance of the bacterial motility for the early colonization of maize roots.

A high-throughput screening assay for distinguishing nitrile hydratases from nitrilases.

A modified colorimetric high-throughput screen based on pH changes combined with an amidase inhibitor capable of distinguishing between nitrilases and nitrile hydratases. This enzymatic screening is based on a binary response and is suitable for the first step of hierarchical screening projects.

A high-throughput screening assay for distinguishing nitrile hydratases from nitrilases

A modified colorimetric high-throughput screen based on pH changes combined with an amidase inhibitor capable of distinguishing between nitrilases and nitrile hydratases. This enzymatic screening is based on a binary response and is suitable for the first step of hierarchical screening projects.

A high-throughput screening assay for distinguishing nitrile hydratases from nitrilases

A modified colorimetric high-throughput screen based on pH changes combined with an amidase inhibitor capable of distinguishing between nitrilases and nitrile hydratases. This enzymatic screening is based on a binary response and is suitable for the first step of hierarchical screening projects.(AU)

Adenoviral transfer of human aquaporin-1 gene to rat liver improves bile flow in estrogen-induced cholestasis.

Estrogens can cause liver cholestatic disease. As downregulation of hepatocyte canalicular aquaporin-8 (AQP8) water channels has been involved in estrogen-induced bile secretory failure, we tested whether the archetypal water channel AQP1 improves 17α-ethinylestradiol (EE)-induced cholestasis. EE administration to rats reduced bile flow by 50%. A recombinant adenoviral (Ad) vector encoding human AQP1 (hAQP1), AdhAQP1, or a control vector was administered by retrograde bile ductal infusion. Hepatocyte canalicular hAQP1 expression was confirmed by liver immunostaining and immunoblotting in purified membrane fractions. Accordingly, canalicular osmotic water permeability was markedly increased. Bile flow, either basal or bile salt-stimulated was significantly augmented by over 50%. The choleretic efficiency of endogenous bile salts (that is, volume of bile per µmol of excreted bile salt) was significantly increased by 45% without changes in the biliary bile salt composition. Our data suggest that the adenoviral transfer of hAQP1 gene to the livers of EE-induced cholestatic rats improves bile flow by enhancing the AQP-mediated bile salt-induced canalicular water secretion. This novel finding might have potential therapeutic implications for cholestatic diseases.

Yeast two-hybrid liquid screening.

Yeast two-hybrid (YTH) method consists of a genetic trap that selects for "prey" cDNA products within a library that interact with a "bait" protein of interest. Here, we provide a protocol for YTH screening using a liquid medium screening method, which improves the sensitivity of this technique and streamlines the laborious classic screening in solid medium plates. The method uses a simple series of dilutions with established yeast strains transformed with diverse baits and complex cDNA libraries. This allows for prompt detection of positive clones revealed by liquid growth, due to activation of HIS3 reporter gene. Activation of a second reporter gene and reconstruction of the YTH interaction is highly reproducible using this system. This approach can either be performed using culture flasks or deep-well 96-well plates and the number of interactions obtained is similar, when compared to the classic method. In addition, the liquid screening method is faster and more economical for YTH screening and has the added benefit of automation if 96-well plates are used.

Identification of novel protein domains required for the expression of an active dehydratase fragment from a polyunsaturated fatty acid synthase.

Polyunsaturated fatty acids (PUFAs) are made in some strains of deep-sea bacteria by multidomain proteins that catalyze condensation, ketoreduction, dehydration, and enoyl-reduction. In this work, we have used the Udwary-Merski Algorithm sequence analysis tool to define the boundaries that enclose the dehydratase (DH) domains in a PUFA multienzyme. Sequence analysis revealed the presence of four areas of high structure in a region that was previously thought to contain only two DH domains as defined by FabA-homology. The expression of the protein fragment containing all four protein domains resulted in an active enzyme, while shorter protein fragments were not soluble. The tetradomain fragment was capable of catalyzing the conversion of crotonyl-CoA to ß-hydroxybutyryl-CoA efficiently, as shown by UV absorbance change as well as by chromatographic retention of reaction products. Sequence alignments showed that the two novel domains contain as much sequence conservation as the FabA-homology domains, suggesting that they too may play a functional role in the overall reaction. Structure predictions revealed that all domains belong to the hotdog protein family: two of them contain the active site His70 residue present in FabA-like DHs, while the remaining two do not. Replacing the active site His residues in both FabA domains for Ala abolished the activity of the tetradomain fragment, indicating that the DH activity is contained within the FabA-homology regions. Taken together, these results provide a first glimpse into a rare arrangement of DH domains which constitute a defining feature of the PUFA synthases.

Reduction of the monounsaturated fatty acid content of Escherichia coli results in increased resistance to oxidative damage.

Reactive oxygen species (ROSs) affect several macromolecules and cellular components in eukaryotic and prokaryotic cells. In this work, the effect of various ROS-generating compounds on the Escherichia coli membrane was studied. Membrane fatty acid profiles, oxidative damage levels and bacterial resistance to these toxicants were determined. Studies included wild-type cells as well as a strain exhibiting a modified monounsaturated fatty acid (MUFA) profile (accomplished by overexpressing the ß-hydroxyacyl acyl carrier protein dehydratase-encoding gene, fabA). Levels of membrane MUFAs and oxidative damage markers decreased slightly upon toxicant exposure with a concomitant increase in cell resistance to these ROS-generating compounds. A direct relationship between MUFAs and lipid peroxidation was observed. The lower the MUFA the lower the peroxide levels, suggesting that MUFAs are targets for membrane lipid oxidation.