Glutamate controls tPA recycling by astrocytes, which in turn influences glutamatergic signals.

Tissue-type plasminogen activator (tPA) regulates physiological processes in the brain, such as learning and memory, and plays a critical role in neuronal survival and neuroinflammation in pathological conditions. Here we demonstrate, by combining mouse in vitro and in vivo data, that tPA is an important element of the cross talk between neurons and astrocytes. The data show that tPA released by neurons is constitutively endocytosed by astrocytes via the low-density lipoprotein-related protein receptor, and is then exocytosed in a regulated manner. The exocytotic recycling of tPA by astrocytes is inhibited in the presence of extracellular glutamate. Kainate receptors of astrocytes act as sensors of extracellular glutamate and, via a signaling pathway involving protein kinase C, modulate the exocytosis of tPA. Further, by thus capturing extracellular tPA, astrocytes serve to reduce NMDA-mediated responses potentiated by tPA. Overall, this work provides the first demonstration that the neuromodulator, tPA, may also be considered as a gliotransmitter.

Temperature-dependent regulation of mycolic acid cyclopropanation in saprophytic mycobacteria: role of the Mycobacterium smegmatis 1351 gene (MSMEG_1351) in CIS-cyclopropanation of alpha-mycolates.

The cell envelope is a crucial determinant of virulence and drug resistance in Mycobacterium tuberculosis. Several features of pathogenesis and immunomodulation of host responses are attributable to the structural diversity in cell wall lipids, particularly in the mycolic acids. Structural modification of the alpha-mycolic acid by introduction of cyclopropane rings as catalyzed by the methyltransferase, PcaA, is essential for a lethal, persistent infection and the cording phenotype in M. tuberculosis. Here, we demonstrate the presence of cyclopropanated cell wall mycolates in the nonpathogenic strain Mycobacterium smegmatis and identify MSMEG_1351 as a gene encoding a PcaA homologue. Interestingly, alpha-mycolic acid cyclopropanation was inducible in cultures grown at 25 degrees C. The growth temperature modulation of the cyclopropanating activity was determined by high resolution magic angle spinning NMR analyses on whole cells. In parallel, quantitative reverse transcription-PCR analysis showed that MSMEG_1351 gene expression is up-regulated at 25 degrees C compared with 37 degrees C. An MSMEG_1351 knock-out strain of M. smegmatis, generated by recombineering, exhibited a deficiency in cyclopropanation of alpha-mycolates. The functional equivalence of PcaA and MSMEG_1351 was established by cross-complementation in the MSMEG_1351 knock-out mutant and also in a DeltapcaA strain of Mycobacterium bovis BCG. Overexpression of MSMEG_1351 restored the wild-type mycolic acid profile and the cording phenotype in BCG. Although the biological significance of mycolic acid cyclopropanation in nonpathogenic mycobacteria remains unclear, it likely represents a mechanism of adaptation of cell wall structure and composition to cope with environmental factors.

Mycolic acid methyltransferase, MmaA4, is necessary for thiacetazone susceptibility in Mycobacterium tuberculosis.

Susceptibility of Mycobacterium tuberculosis to the second-line antitubercular drug thiacetazone (TAC) requires activation by the monoxygenase, EthA. Here, we report isolation of spontaneous mutants in Mycobacterium bovis BCG that are highly resistant to TAC, but carry a functional EthA. Unexpectedly, a majority of the TAC-resistant mutants lacked keto-mycolic acids, which are long-chain fatty acids associated with the cell wall and which contribute significantly to the physiopathology of tuberculosis. Predictably, causative mutations in the above mutants were in the gene encoding methyltransferase MmaA4, which is required for synthesis of keto- and methoxy-mycolic acids. Drug-resistant phenotype of the BCG mutants was reproduced in a mmaA4, but not in a mmaA3 null mutant of M. tuberculosis CDC1551. Susceptibility to TAC could be restored by complementation with a functional mmaA4 gene. Interestingly, overexpression of MmaA4 in M. bovis BCG made it more susceptible to TAC. We provide novel mechanistic insights into antitubercular drug activation by co-ordinated actions of EthA and MmaA4. This study is the first demonstration of the participation of an enzyme linked to the synthesis of oxygenated mycolates in a drug activation process in M. tuberculosis, and highlights the interplay between mycolic acid synthesis, drug activation and mycobacterial virulence.

Cloning and overexpression of alkaline phosphatase PhoK from Sphingomonas sp. strain BSAR-1 for bioprecipitation of uranium from alkaline solutions.

Cells of Sphingomonas sp. strain BSAR-1 constitutively expressed an alkaline phosphatase, which was also secreted in the extracellular medium. A null mutant lacking this alkaline phosphatase activity was isolated by Tn5 random mutagenesis. The corresponding gene, designated phoK, was cloned and overexpressed in Escherichia coli strain BL21(DE3). The resultant E. coli strain EK4 overexpressed cellular activity 55 times higher and secreted extracellular PhoK activity 13 times higher than did BSAR-1. The recombinant strain very rapidly precipitated >90% of input uranium in less than 2 h from alkaline solutions (pH, 9 +/- 0.2) containing 0.5 to 5 mM of uranyl carbonate, compared to BSAR-1, which precipitated uranium in >7 h. In both strains BSAR-1 and EK4, precipitated uranium remained cell bound. The EK4 cells exhibited a much higher loading capacity of 3.8 g U/g dry weight in <2 h compared to only 1.5 g U/g dry weight in >7 h in BSAR-1. The data demonstrate the potential utility of genetically engineering PhoK for the bioprecipitation of uranium from alkaline solutions.

Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria.

Successful treatment of human tuberculosis requires 6-9 months' therapy with multiple antibiotics. Incomplete clearance of tubercle bacilli frequently results in disease relapse, presumably as a result of reactivation of persistent drug-tolerant Mycobacterium tuberculosis cells, although the nature and location of these persisters are not known. In other pathogens, antibiotic tolerance is often associated with the formation of biofilms--organized communities of surface-attached cells--but physiologically and genetically defined M. tuberculosis biofilms have not been described. Here, we show that M. tuberculosis forms biofilms with specific environmental and genetic requirements distinct from those for planktonic growth, which contain an extracellular matrix rich in free mycolic acids, and harbour an important drug-tolerant population that persist despite exposure to high levels of antibiotics.

Thiacetazone, an antitubercular drug that inhibits cyclopropanation of cell wall mycolic acids in mycobacteria.

BACKGROUND: Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets. METHODOLOGY/PRINCIPLE FINDINGS: We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strain Mycobacterium bovis BCG, as well as in the related pathogenic species Mycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified from drug-treated mycobacteria showed a significant loss of cyclopropanation in both the alpha- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs. CONCLUSIONS/SIGNIFICANCE: This is a first report on the mechanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment.

The N-terminal domain of OmpATb is required for membrane translocation and pore-forming activity in mycobacteria.

OmpATb is the prototype of a new family of porins in Mycobacterium tuberculosis and Mycobacterium bovis BCG. Although the pore-forming activity of this protein has been clearly established by using recombinant protein produced in Escherichia coli, characterization of the native porin has been hampered by the scarce amount of protein present in the M. tuberculosis detergent extracts. To this aim, we have developed a protocol to overproduce and obtain high yields of OmpATb in both Mycobacterium smegmatis and M. bovis BCG. The protein could be extracted and purified from the cell wall fraction and subsequently used for analysis of the pore-forming activity in multichannel and single-channel conductance experiments. Our results indicate that OmpATb produced in mycobacteria presents an average conductance value of 1,600+/-100 pS, slightly higher than that of OmpATb produced in E. coli, suggesting the occurrence of OmpATb in a highly ordered organization within the mycobacterial cell wall. In contrast to OmpATb, a truncated form lacking the first 72 amino acids (OmpATb73-326) was essentially found in the cytosol and was not active in planar lipid bilayers. This suggested that the N-terminal domain of OmpATb could participate in targeting of OmpATb to the cell wall. This was further confirmed by analyzing M. smegmatis clones expressing a chimeric protein consisting of a fusion between the N-terminal domain of OmpATb and the E. coli PhoA reporter. The present study shows for the first time that the N terminus of OmpATb is required for targeting the porin to the cell wall and also appears to be essential for its pore-forming activity.

EthA, a common activator of thiocarbamide-containing drugs acting on different mycobacterial targets.

Many of the current antimycobacterial agents require some form of cellular activation unmasking reactive groups, which in turn will bind to their specific targets. Therefore, understanding the mechanisms of activation of current antimycobacterials not only helps to decipher mechanisms of drug resistance but may also facilitate the development of alternative activation strategies or of analogues that do not require such processes. Herein, through the use of genetically defined strains of Mycobacterium bovis BCG we provide evidence that EthA, previously shown to activate ethionamide, also converts isoxyl (ISO) and thiacetazone (TAC) into reactive species. These results were further supported by the development of an in vitro assay using purified recombinant EthA, which allowed direct assessment of the metabolism of ISO. Interestingly, biochemical analysis of [(14)C]acetate-labeled cultures suggested that all of these EthA-activated drugs inhibit mycolic acid biosynthesis via different mechanisms through binding to specific targets. This report is also the first description of the molecular mechanism of action of TAC, a thiosemicarbazone antimicrobial agent that is still used in the treatment of tuberculosis as a second-line drug in many developing countries. Altogether, the results suggest that EthA is a common activator of thiocarbamide-containing drugs. The broad specificity of EthA can now be used to improve the activation process of these drugs, which may help overcome the toxicity problems associated with clinical thiocarbamide use.

Cloning and characterization of a fur homologue from Azospirillum brasilense Sp7.

A homologue of the ferric uptake regulator gene, fur, was identified from a Azospirillum brasilense Sp7 genomic DNA clone. Experiments performed with transcriptional lacZ fusions demonstrated that the A. brasilense fur homologue regulated the expression of two fur regulated Escherichia coli genes: fiu (ferric iron uptake) and fhuF (ferric hydroxamate uptake). A differential regulation by the cognate Fur and the heterologous Fur homologue in response to the iron status of the growth medium was also observed.

A novel potassium deficiency-induced stimulon in Anabaena torulosa.

Potassium deficiency enhanced the synthesis of fifteen proteins in the nitrogen-fixing cyanobacterium Anabaena torulosa and of nine proteins in Escherichia coli. These were termed potassium deficiency-induced proteins or PDPs and constitute hitherto unknown potassium deficiency-induced stimulons. Potassium deficiency also enhanced the synthesis of certain osmotic stress-induced proteins. Addition of K+ repressed the synthesis of a majority of the osmotic stress-induced proteins and of PDPs in these bacteria. These proteins contrast with the dinitrogenase reductase of A. torulosa and the glycine betaine-binding protein of E. coli, both of which were osmo-induced to a higher level in potassium-supplemented conditions. The data demonstrate the occurrence of novel potassium deficiency-induced stimulons and a wider role of K+ in regulation of gene expression and stress responses in bacteria

Pleiotropic effects of potassium deficiency in a heterocystous, nitrogen-fixing cyanobacterium, Anabaena torulosa.

Omission of potassium from the growth medium caused multiple metabolic impairments and resulted in cessation of growth of the filamentous, heterocystous, nitrogen-fixing cyanobacterium Anabaena torulosa, during both diazotrophic and nitrogen-supplemented growth. Prominent defects observed during potassium deprivation were: (i) the loss of photosynthetic pigments, (ii) impairment of photosynthetic functions, (iii) reduced synthesis of dinitrogenase reductase (Fe-protein), (iv) inhibition of nitrogenase activity, and (v) specific qualitative modifications of protein synthesis leading to the repression of twelve polypeptides and synthesis and accumulation of nine novel polypeptides. The observed metabolic defects were reversible, and growth arrested under prolonged potassium deficiency was fully restored upon re-addition of potassium. Such pleiotropic effects of potassium deficiency demonstrate that apart from its well-known requirement for pH and turgor homeostasis, K+ plays other vital specific roles in cyanobacterial growth and metabolism.