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William R Jacobs - One of the best experts on this subject based on the ideXlab platform.
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biological evaluation of potent triclosan derived inhibitors of the enoyl acyl carrier protein reductase INHA in drug sensitive and drug resistant strains of mycobacterium tuberculosis
ChemMedChem, 2014Co-Authors: Catherine Vilcheze, William R Jacobs, Jozef Stec, Shichun Lun, Alexander L Perryman, Xin Wang, Joel S Freundlich, William R Bishai, Alan P KozikowskiAbstract:New triclosan (TRC) analogues were evaluated for their activity against the enoyl-acyl carrier protein reductase INHA in Mycobacterium tuberculosis (Mtb). TRC is a well-known inhibitor of INHA, and specific modifications to its positions 5 and 4' afforded 27 derivatives; of these compounds, seven derivatives showed improved potency over that of TRC. These analogues were active against both drug-susceptible and drug-resistant Mtb strains. The most active compound in this series, 4-(n-butyl)-1,2,3-triazolyl TRC derivative 3, had an MIC value of 0.6 μg mL(-1) (1.5 μM) against wild-type Mtb. At a concentration equal to its MIC, this compound inhibited purified INHA by 98 %, and showed an IC50 value of 90 nM. Compound 3 and the 5-methylisoxazole-modified TRC 14 were able to inhibit the biosynthesis of mycolic acids. Furthermore, mc(2) 4914, an Mtb strain overexpressing INHA, was found to be less susceptible to compounds 3 and 14, supporting the notion that INHA is the likely molecular target of the TRC derivatives presented herein.
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Transfer of a point mutation in Mycobacterium tuberculosis INHA resolves the target of isoniazid.
Nature Medicine, 2006Co-Authors: Catherine Vilcheze, Laurent Kremer, Manzour Hernando Hazbon, David Alland, James C Sacchettini, Torin R Weisbrod, Feng Wang, Masayoshi Arai, Roberto Colangeli, William R JacobsAbstract:Isoniazid is one of the most effective antituberculosis drugs, yet its precise mechanism of action is still controversial. Using specialized linkage transduction, a single point mutation allele (S94A) within the putative target gene INHA was transferred in Mycobacterium tuberculosis. The INHA(S94A) allele was sufficient to confer clinically relevant levels of resistance to isoniazid killing and inhibition of mycolic acid biosynthesis. This resistance correlated with the decreased binding of the INH-NAD inhibitor to INHA, as shown by enzymatic and X-ray crystallographic analyses, and establishes INHA as the primary target of isoniazid action in M. tuberculosis.
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conditional depletion of kasa a key enzyme of mycolic acid biosynthesis leads to mycobacterial cell lysis
Journal of Bacteriology, 2005Co-Authors: Apoorva Bhatt, Laurent Kremer, James C Sacchettini, Annie Z Dai, William R JacobsAbstract:Inhibition or inactivation of INHA, a fatty acid synthase II (FASII) enzyme, leads to mycobacterial cell lysis. To determine whether inactivation of other enzymes of the mycolic acid-synthesizing FASII complex also leads to lysis, we characterized the essentiality of two beta-ketoacyl-acyl carrier protein synthases, KasA and KasB, in Mycobacterium smegmatis. Using specialized transduction for allelic exchange, null kasB mutants, but not kasA mutants, could be generated in Mycobacterium smegmatis, suggesting that unlike kasB, kasA is essential. To confirm the essentiality of kasA, and to detail the molecular events that occur following depletion of KasA, we developed CESTET (conditional expression specialized transduction essentiality test), a genetic tool that combines conditional gene expression and specialized transduction. Using CESTET, we were able to generate conditional null INHA and kasA mutants. We studied the effects of depletion of KasA in M. smegmatis using the former strain as a reference. Depletion of either INHA or KasA led to cell lysis, but with different biochemical and morphological events prior to lysis. While INHA depletion led to the induction of an 80-kDa complex containing both KasA and AcpM, the mycobacterial acyl carrier protein, KasA depletion did not induce the same complex. Depletion of either INHA or KasA led to inhibition of alpha and epoxy mycolate biosynthesis and to accumulation of alpha'-mycolates. Furthermore, scanning electron micrographs revealed that KasA depletion resulted in the cell surface having a "crumpled" appearance, in contrast to the blebs observed on INHA depletion. Thus, our studies support the further exploration of KasA as a target for mycobacterial-drug development.
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inactivation of the INHA encoded fatty acid synthase ii fasii enoyl acyl carrier protein reductase induces accumulation of the fasi end products and cell lysis of mycobacterium smegmatis
Journal of Bacteriology, 2000Co-Authors: Catherine Vilcheze, James C Sacchettini, Hector R Morbidoni, Torin R Weisbrod, Hiroyuki Iwamoto, William R JacobsAbstract:The mechanism of action of isoniazid (INH), a first-line antituberculosis drug, is complex, as mutations in at least five different genes (katG, INHA, ahpC, kasA, and ndh) have been found to correlate with isoniazid resistance. Despite this complexity, a preponderance of evidence implicates INHA, which codes for an enoyl-acyl carrier protein reductase of the fatty acid synthase II (FASII), as the primary target of INH. However, INH treatment of Mycobacterium tuberculosis causes the accumulation of hexacosanoic acid (C26:0), a result unexpected for the blocking of an enoyl-reductase. To test whether inactivation of INHA is identical to INH treatment of mycobacteria, we isolated a temperature-sensitive mutation in the INHA gene of Mycobacterium smegmatis that rendered INHA inactive at 42°C. Thermal inactivation of INHA in M. smegmatis resulted in the inhibition of mycolic acid biosynthesis, a decrease in hexadecanoic acid (C16:0) and a concomitant increase of tetracosanoic acid (C24:0) in a manner equivalent to that seen in INH-treated cells. Similarly, INH treatment of Mycobacterium bovis BCG caused an inhibition of mycolic acid biosynthesis, a decrease in C16:0, and a concomitant accumulation of C26:0. Moreover, the INHA-inactivated cells, like INH-treated cells, underwent a drastic morphological change, leading to cell lysis. These data show that INHA inactivation, alone, is sufficient to induce the accumulation of saturated fatty acids, cell wall alterations, and cell lysis and are consistent with INHA being a primary target of INH.
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inactivation of the INHA encoded fatty acid synthase ii fasii enoyl acyl carrier protein reductase induces accumulation of the fasi end products and cell lysis of mycobacterium smegmatis
Journal of Bacteriology, 2000Co-Authors: Catherine Vilcheze, James C Sacchettini, Hector R Morbidoni, Torin R Weisbrod, Hiroyuki Iwamoto, Mack Kuo, William R JacobsAbstract:The mechanism of action of isoniazid (INH), a first-line antituberculosis drug, is complex, as mutations in at least five different genes (katG, INHA, ahpC, kasA, and ndh) have been found to correlate with isoniazid resistance. Despite this complexity, a preponderance of evidence implicates INHA, which codes for an enoyl-acyl carrier protein reductase of the fatty acid synthase II (FASII), as the primary target of INH. However, INH treatment of Mycobacterium tuberculosis causes the accumulation of hexacosanoic acid (C(26:0)), a result unexpected for the blocking of an enoyl-reductase. To test whether inactivation of INHA is identical to INH treatment of mycobacteria, we isolated a temperature-sensitive mutation in the INHA gene of Mycobacterium smegmatis that rendered INHA inactive at 42 degrees C. Thermal inactivation of INHA in M. smegmatis resulted in the inhibition of mycolic acid biosynthesis, a decrease in hexadecanoic acid (C(16:0)) and a concomitant increase of tetracosanoic acid (C(24:0)) in a manner equivalent to that seen in INH-treated cells. Similarly, INH treatment of Mycobacterium bovis BCG caused an inhibition of mycolic acid biosynthesis, a decrease in C(16:0), and a concomitant accumulation of C(26:0). Moreover, the INHA-inactivated cells, like INH-treated cells, underwent a drastic morphological change, leading to cell lysis. These data show that INHA inactivation, alone, is sufficient to induce the accumulation of saturated fatty acids, cell wall alterations, and cell lysis and are consistent with INHA being a primary target of INH.
Aner Gurvitz - One of the best experts on this subject based on the ideXlab platform.
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function of heterologous mycobacterium tuberculosis INHA a type 2 fatty acid synthase enzyme involved in extending c20 fatty acids to c60 to c90 mycolic acids during de novo lipoic acid synthesis in saccharomyces cerevisiae
Applied and Environmental Microbiology, 2008Co-Authors: Aner Gurvitz, Kalervo J Hiltunen, Alexander J KastaniotisAbstract:We describe the physiological function of heterologously expressed Mycobacterium tuberculosis INHA during de novo lipoic acid synthesis in yeast (Saccharomyces cerevisiae) mitochondria. INHA, representing 2-trans-enoyl-acyl carrier protein reductase and the target for the front-line antituberculous drug isoniazid, is involved in the activity of dissociative type 2 fatty acid synthase (FASII) that extends associative type 1 fatty acid synthase (FASI)-derived C20 fatty acids to form C60-to-C90 mycolic acids. Mycolic acids are major constituents of the protective layer around the pathogen that contribute to virulence and resistance to certain antimicrobials. Unlike FASI, FASII is thought to be incapable of de novo biosynthesis of fatty acids. Here, the genes for INHA (Rv1484) and four similar proteins (Rv0927c, Rv3485c, Rv3530c, and Rv3559c) were expressed in S. cerevisiae etr1Δ cells lacking mitochondrial 2-trans-enoyl-thioester reductase activity. The phenotype of the yeast mutants includes the inability to produce sufficient levels of lipoic acid, form mitochondrial cytochromes, respire, or grow on nonfermentable carbon sources. Yeast etr1Δ cells expressing mitochondrial INHA were able to respire, grow on glycerol, and produce lipoic acid. Commensurate with a role in mitochondrial de novo fatty acid biosynthesis, INHA could accept in vivo much shorter acyl-thioesters (C4 to C8) than was previously thought (>C12). Moreover, INHA functioned in the absence of AcpM or protein-protein interactions with its native FASII partners KasA, KasB, FabD, and FabH. None of the four proteins similar to INHA complemented the yeast mutant phenotype. We discuss the implications of our findings with reference to lipoic acid synthesis in M. tuberculosis and the potential use of yeast FASII mutants for investigating the physiological function of drug-targeted pathogen enzymes involved in fatty acid biosynthesis.
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function of heterologous mycobacterium tuberculosis INHA a type 2 fatty acid synthase enzyme involved in extending c20 fatty acids to c60 to c90 mycolic acids during de novo lipoic acid synthesis in saccharomyces cerevisiae
Applied and Environmental Microbiology, 2008Co-Authors: Aner Gurvitz, Kalervo J Hiltunen, Alexander J KastaniotisAbstract:Mycobacterium tuberculosis is the leading cause of mortality due to an infectious agent worldwide. The World Health Organization estimates that approximately 2 billion people have tuberculosis (53). M. tuberculosis is therefore the biggest killer among human pathogens; it is thought that about 1.7 million people die from tuberculosis every year (www.who.int/tb/publications). Moreover, increasing multiple-drug resistance has contributed significantly to the number of incurable cases, and in some countries up to 36% of patients with tuberculosis are infected with strains resistant to isoniazid (INH) or rifampin (42). These two compounds have been used clinically for several decades and are two of only a few first-line antituberculous drugs. The primary target of INH is INHA (3), and this fact alone elevates INHA to the position of one of most medically significant pathogen proteins. Hence, it is important to use new molecular approaches to study INHA in order to identify novel ways of targeting this enzyme, as well as the processes in which it is involved. INHA participates in fatty acid biosynthesis (3). Unlike the situation in Escherichia coli, the mycobacterial process is comprised of two systems. M. tuberculosis has a prokaryotic dissociative type 2 fatty acid synthase (FASII) system, in which individual reactions are catalyzed by discrete polypeptides, a process that has been characterized extensively in E. coli and plant plastids. Additionally, M. tuberculosis has an associative type 1 fatty acid synthase (FASI) system (5), which includes several enzymatic activities within a multifunctional homohexamer and resembles the cytosolic synthase in eukaryotes (47). The two systems combine to produce mycolic acids, which are very-long-chain (C54 to C63) α-branched, β-hydroxylated fatty acids that act with other factors to form the protective layer around the pathogen, thereby adding to its persistence despite lengthy treatment, and are also associated with its virulence (48). Based on its fully sequenced genome (13), it has been proposed that M. tuberculosis contains the entire complement of FASII components (48). The 2-trans-enoyl-acyl carrier protein (2-trans-enoyl-ACP) reductase of FASII is represented by INHA, which carries out the final step of the fatty acid elongation process. The M. tuberculosis genome harbors genes for four additional proteins, Rv0927c, Rv3485c, Rv3530c, and Rv3559c, that are all similar to INHA (48) and exhibit about 24 to 26% sequence identity to the latter protein (Fig. (Fig.1).1). It has been proposed that in M. tuberculosis, INHA catalyzes the reduction of 2-trans-enoyl-ACPs with a chain length greater than C12, whereas INHA in Mycobacterium smegmatis acts on C16 thioesters (35). FIG. 1. Comparison of M. tuberculosis INHA with four proteins most similar to it: Genedoc-based comparison of the deduced amino acid sequences of INHA (Rv1484) and Rv3485c, Rv0927c, Rv3530c, and Rv3559c. Dashes were added to the sequences to obtain the best fit. ... Yeast (Saccharomyces cerevisiae), mammals, and other higher eukaryotes have traditionally been considered organisms that are capable of synthesizing fatty acids only through FASI. This view has very recently been completely overhauled, since an additional mitochondrial FASII has been discovered in both yeast and mammals (2, 28, 32, 37, 50, 55, 56). In the first committed step of S. cerevisiae FASII activity, Hfa1p, representing mitochondrial acetyl coenzyme A (acetyl-CoA) carboxylase (29), converts acetyl-CoA to malonyl-CoA. A malonyl-CoA transferase, mitochondrial Mct1p (45), transfers the C3 moiety to ACP, a mitochondrial protein encoded by ACP1 (46). Chain elongation begins with the condensation of acetyl-ACP and malonyl-ACP by Cem1p (23), which acts as a mitochondrial 3-oxoacyl-ACP synthase. Mitochondrial Oar1p (45), a 3-oxoacyl-ACP reductase, produces the 3-hydroxyacyl-ACP intermediate, which is then dehydrated by mitochondrial Htd2p (32), a 3-hydroxyacyl-thioester dehydratase, to generate the 2-trans-enoyl-ACP species. The last step in each round of elongation is catalyzed by mitochondrial Etr1p representing 2-trans-enoyl-thioester reductase (50). A yeast mutant that lacks Etr1p contains abnormally small mitochondria, does not assemble respiratory complexes, and is exclusively fermentative (50, 54). This phenotype can be rescued by supplying the mutant with the gene for fungal or human mitochondrial 2-trans-enoyl-ACP reductase (37, 50). In addition, the etr1Δ mutant phenotype can also be rescued with a mitochondrially targeted E. coli FabI protein (50), representing a structurally unrelated FASII enoyl-ACP reductase (4). Whereas it has been proposed that mitochondrial FASII is involved in de novo production of the C8 precursor of lipoic acid (20), mycobacterial FASII is thought to be incapable of de novo synthesis (5). Here, we used S. cerevisiae as a surrogate for hosting M. tuberculosis protein genes (17). To examine whether INHA could physiologically metabolize short-chain enoyl-ACP substrates and to determine whether there are functional INHA homologues in M. tuberculosis, INHA and the four similar proteins were expressed in the yeast etr1Δ mutant, and transformed mutant cells were compared to cells of an otherwise isogenic strain expressing the corresponding native enzyme in terms of growth on glycerol, lipoic acid production, assembly of cytochrome complexes, respiration, and the presence of 2-trans-enoyl-thioester reductase activity. The implications of our finding that INHA can participate in de novo lipoic acid synthesis in yeast mitochondria for fatty acid biosynthesis in M. tuberculosis are briefly discussed below.
Catherine Vilcheze - One of the best experts on this subject based on the ideXlab platform.
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biological evaluation of potent triclosan derived inhibitors of the enoyl acyl carrier protein reductase INHA in drug sensitive and drug resistant strains of mycobacterium tuberculosis
ChemMedChem, 2014Co-Authors: Catherine Vilcheze, William R Jacobs, Jozef Stec, Shichun Lun, Alexander L Perryman, Xin Wang, Joel S Freundlich, William R Bishai, Alan P KozikowskiAbstract:New triclosan (TRC) analogues were evaluated for their activity against the enoyl-acyl carrier protein reductase INHA in Mycobacterium tuberculosis (Mtb). TRC is a well-known inhibitor of INHA, and specific modifications to its positions 5 and 4' afforded 27 derivatives; of these compounds, seven derivatives showed improved potency over that of TRC. These analogues were active against both drug-susceptible and drug-resistant Mtb strains. The most active compound in this series, 4-(n-butyl)-1,2,3-triazolyl TRC derivative 3, had an MIC value of 0.6 μg mL(-1) (1.5 μM) against wild-type Mtb. At a concentration equal to its MIC, this compound inhibited purified INHA by 98 %, and showed an IC50 value of 90 nM. Compound 3 and the 5-methylisoxazole-modified TRC 14 were able to inhibit the biosynthesis of mycolic acids. Furthermore, mc(2) 4914, an Mtb strain overexpressing INHA, was found to be less susceptible to compounds 3 and 14, supporting the notion that INHA is the likely molecular target of the TRC derivatives presented herein.
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Transfer of a point mutation in Mycobacterium tuberculosis INHA resolves the target of isoniazid.
Nature Medicine, 2006Co-Authors: Catherine Vilcheze, Laurent Kremer, Manzour Hernando Hazbon, David Alland, James C Sacchettini, Torin R Weisbrod, Feng Wang, Masayoshi Arai, Roberto Colangeli, William R JacobsAbstract:Isoniazid is one of the most effective antituberculosis drugs, yet its precise mechanism of action is still controversial. Using specialized linkage transduction, a single point mutation allele (S94A) within the putative target gene INHA was transferred in Mycobacterium tuberculosis. The INHA(S94A) allele was sufficient to confer clinically relevant levels of resistance to isoniazid killing and inhibition of mycolic acid biosynthesis. This resistance correlated with the decreased binding of the INH-NAD inhibitor to INHA, as shown by enzymatic and X-ray crystallographic analyses, and establishes INHA as the primary target of isoniazid action in M. tuberculosis.
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Inhibition of INHA activity, but not KasA activity, induces formation of a KasA-containing complex in mycobacteria.
Journal of Biological Chemistry, 2003Co-Authors: Laurent Kremer, Catherine Vilcheze, Hector R Morbidoni, Lynn G. Dover, William N. Maughan, Alain R. Baulard, Nadine Honoré, Vojo Deretic, James C SacchettiniAbstract:Isoniazid (INH) remains one of the key drugs used to control tuberculosis, with the enoyl-AcpM reductase INHA being the primary target. However, based on the observation that INH-treated Mycobacterium tuberculosis overproduces KasA, an enzyme involved in the biosynthesis of mycolic acids, and induces the formation of a covalent complex consisting of AcpM, KasA, and INH, it has been proposed that KasA represents the primary target of INH. However, the relevance of this complex to INH action remains obscure. This study was aimed at clarifying the role of INHA and KasA in relation to INH activity. By using anti-KasA antibodies we detected the KasA-containing complex in INH-treated Mycobacterium smegmatis. In addition, INH-treated cells also produced constant levels of KasA that were not sequestered in the complex and presumably were sufficient to ensure mycolic acid biosynthesis. Interestingly, a furA-lacking strain induced the complex at lower concentrations of INH compared with the control strain, whereas higher INH concentrations were necessary to induce the complex in a strain that lacks katG, suggesting that INH needs to be activated by KatG to induce the KasA-containing complex. The INHA inhibitors ethionamide and diazaborine also induced the complex; thus, its formation was not specifically relevant to INH action but was because of INHA inhibition. In addition, in vitro assays using purified INHA and KasA demonstrated that KatG-activated INH, triclosan, and diazaborine inhibited INHA but not KasA activity. Moreover, several thermosensitive INHA mutant strains of M. smegmatis constitutively expressed the KasA-containing complex. This study provides the biochemical and genetic evidence. 1) Only inhibition of INHA, but not KasA, induces the KasA-containing complex. 2) INH is not part of the complex. 3) INH does not target KasA, consistent with INHA being the primary target of INH.
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overexpression of INHA but not kasa confers resistance to isoniazid and ethionamide in mycobacterium smegmatis m bovis bcg and m tuberculosis
Molecular Microbiology, 2002Co-Authors: Michelle H Larsen, Catherine Vilcheze, Laurent Kremer, Gurdyal S Besra, Linda M Parsons, Max Salfinger, Leonid Heifets, Manzour Hernando Hazbon, David Alland, James C SacchettiniAbstract:Summary The INHA and kasA genes of Mycobacterium tuberculosis have each been proposed to encode the primary target of the antibiotic isoniazid (INH). Previous studies investigating whether overexpressed INHA or kasA could confer resistance to INH yielded disparate results. In this work, multicopy plasmids expressing either INHA or kasA genes were transformed into M. smegmatis, M. bovis BCG and three different M. tuberculosis strains. The resulting transformants, as well as previously published M. tuberculosis strains with multicopy INHA or kasAB plasmids, were tested for their resistance to INH, ethionamide (ETH) or thiolactomycin (TLM). Mycobacteria containing INHA plasmids uniformly exhibited 20-fold or greater increased resistance to INH and 10-fold or greater increased resistance to ETH. In contrast, the kasA plasmid conferred no increased resistance to INH or ETH in any of the five strains, but it did confer resistance to thiolactomycin, a known KasA inhibitor. INH is known to increase the expression of kasA in INH-susceptible M. tuberculosis strains. Using molecular beacons, quantified INHA and kasA mRNA levels showed that increased INHA mRNA levels corre-lated with INH resistance, whereas kasA mRNA levels did not. In summary, analysis of strains harbouring INHA or kasA plasmids yielded the same conclusion: overexpressed INHA, but not kasA, confers INH and ETH resistance to M. smegmatis, M. bovis BCG and M. tuberculosis. Therefore, INHA is the primary target of action of INH and ETH in all three species.
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inactivation of the INHA encoded fatty acid synthase ii fasii enoyl acyl carrier protein reductase induces accumulation of the fasi end products and cell lysis of mycobacterium smegmatis
Journal of Bacteriology, 2000Co-Authors: Catherine Vilcheze, James C Sacchettini, Hector R Morbidoni, Torin R Weisbrod, Hiroyuki Iwamoto, William R JacobsAbstract:The mechanism of action of isoniazid (INH), a first-line antituberculosis drug, is complex, as mutations in at least five different genes (katG, INHA, ahpC, kasA, and ndh) have been found to correlate with isoniazid resistance. Despite this complexity, a preponderance of evidence implicates INHA, which codes for an enoyl-acyl carrier protein reductase of the fatty acid synthase II (FASII), as the primary target of INH. However, INH treatment of Mycobacterium tuberculosis causes the accumulation of hexacosanoic acid (C26:0), a result unexpected for the blocking of an enoyl-reductase. To test whether inactivation of INHA is identical to INH treatment of mycobacteria, we isolated a temperature-sensitive mutation in the INHA gene of Mycobacterium smegmatis that rendered INHA inactive at 42°C. Thermal inactivation of INHA in M. smegmatis resulted in the inhibition of mycolic acid biosynthesis, a decrease in hexadecanoic acid (C16:0) and a concomitant increase of tetracosanoic acid (C24:0) in a manner equivalent to that seen in INH-treated cells. Similarly, INH treatment of Mycobacterium bovis BCG caused an inhibition of mycolic acid biosynthesis, a decrease in C16:0, and a concomitant accumulation of C26:0. Moreover, the INHA-inactivated cells, like INH-treated cells, underwent a drastic morphological change, leading to cell lysis. These data show that INHA inactivation, alone, is sufficient to induce the accumulation of saturated fatty acids, cell wall alterations, and cell lysis and are consistent with INHA being a primary target of INH.
James C Sacchettini - One of the best experts on this subject based on the ideXlab platform.
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Transfer of a point mutation in Mycobacterium tuberculosis INHA resolves the target of isoniazid.
Nature Medicine, 2006Co-Authors: Catherine Vilcheze, Laurent Kremer, Manzour Hernando Hazbon, David Alland, James C Sacchettini, Torin R Weisbrod, Feng Wang, Masayoshi Arai, Roberto Colangeli, William R JacobsAbstract:Isoniazid is one of the most effective antituberculosis drugs, yet its precise mechanism of action is still controversial. Using specialized linkage transduction, a single point mutation allele (S94A) within the putative target gene INHA was transferred in Mycobacterium tuberculosis. The INHA(S94A) allele was sufficient to confer clinically relevant levels of resistance to isoniazid killing and inhibition of mycolic acid biosynthesis. This resistance correlated with the decreased binding of the INH-NAD inhibitor to INHA, as shown by enzymatic and X-ray crystallographic analyses, and establishes INHA as the primary target of isoniazid action in M. tuberculosis.
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conditional depletion of kasa a key enzyme of mycolic acid biosynthesis leads to mycobacterial cell lysis
Journal of Bacteriology, 2005Co-Authors: Apoorva Bhatt, Laurent Kremer, James C Sacchettini, Annie Z Dai, William R JacobsAbstract:Inhibition or inactivation of INHA, a fatty acid synthase II (FASII) enzyme, leads to mycobacterial cell lysis. To determine whether inactivation of other enzymes of the mycolic acid-synthesizing FASII complex also leads to lysis, we characterized the essentiality of two beta-ketoacyl-acyl carrier protein synthases, KasA and KasB, in Mycobacterium smegmatis. Using specialized transduction for allelic exchange, null kasB mutants, but not kasA mutants, could be generated in Mycobacterium smegmatis, suggesting that unlike kasB, kasA is essential. To confirm the essentiality of kasA, and to detail the molecular events that occur following depletion of KasA, we developed CESTET (conditional expression specialized transduction essentiality test), a genetic tool that combines conditional gene expression and specialized transduction. Using CESTET, we were able to generate conditional null INHA and kasA mutants. We studied the effects of depletion of KasA in M. smegmatis using the former strain as a reference. Depletion of either INHA or KasA led to cell lysis, but with different biochemical and morphological events prior to lysis. While INHA depletion led to the induction of an 80-kDa complex containing both KasA and AcpM, the mycobacterial acyl carrier protein, KasA depletion did not induce the same complex. Depletion of either INHA or KasA led to inhibition of alpha and epoxy mycolate biosynthesis and to accumulation of alpha'-mycolates. Furthermore, scanning electron micrographs revealed that KasA depletion resulted in the cell surface having a "crumpled" appearance, in contrast to the blebs observed on INHA depletion. Thus, our studies support the further exploration of KasA as a target for mycobacterial-drug development.
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Inhibition of INHA activity, but not KasA activity, induces formation of a KasA-containing complex in mycobacteria.
Journal of Biological Chemistry, 2003Co-Authors: Laurent Kremer, Catherine Vilcheze, Hector R Morbidoni, Lynn G. Dover, William N. Maughan, Alain R. Baulard, Nadine Honoré, Vojo Deretic, James C SacchettiniAbstract:Isoniazid (INH) remains one of the key drugs used to control tuberculosis, with the enoyl-AcpM reductase INHA being the primary target. However, based on the observation that INH-treated Mycobacterium tuberculosis overproduces KasA, an enzyme involved in the biosynthesis of mycolic acids, and induces the formation of a covalent complex consisting of AcpM, KasA, and INH, it has been proposed that KasA represents the primary target of INH. However, the relevance of this complex to INH action remains obscure. This study was aimed at clarifying the role of INHA and KasA in relation to INH activity. By using anti-KasA antibodies we detected the KasA-containing complex in INH-treated Mycobacterium smegmatis. In addition, INH-treated cells also produced constant levels of KasA that were not sequestered in the complex and presumably were sufficient to ensure mycolic acid biosynthesis. Interestingly, a furA-lacking strain induced the complex at lower concentrations of INH compared with the control strain, whereas higher INH concentrations were necessary to induce the complex in a strain that lacks katG, suggesting that INH needs to be activated by KatG to induce the KasA-containing complex. The INHA inhibitors ethionamide and diazaborine also induced the complex; thus, its formation was not specifically relevant to INH action but was because of INHA inhibition. In addition, in vitro assays using purified INHA and KasA demonstrated that KatG-activated INH, triclosan, and diazaborine inhibited INHA but not KasA activity. Moreover, several thermosensitive INHA mutant strains of M. smegmatis constitutively expressed the KasA-containing complex. This study provides the biochemical and genetic evidence. 1) Only inhibition of INHA, but not KasA, induces the KasA-containing complex. 2) INH is not part of the complex. 3) INH does not target KasA, consistent with INHA being the primary target of INH.
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overexpression of INHA but not kasa confers resistance to isoniazid and ethionamide in mycobacterium smegmatis m bovis bcg and m tuberculosis
Molecular Microbiology, 2002Co-Authors: Michelle H Larsen, Catherine Vilcheze, Laurent Kremer, Gurdyal S Besra, Linda M Parsons, Max Salfinger, Leonid Heifets, Manzour Hernando Hazbon, David Alland, James C SacchettiniAbstract:Summary The INHA and kasA genes of Mycobacterium tuberculosis have each been proposed to encode the primary target of the antibiotic isoniazid (INH). Previous studies investigating whether overexpressed INHA or kasA could confer resistance to INH yielded disparate results. In this work, multicopy plasmids expressing either INHA or kasA genes were transformed into M. smegmatis, M. bovis BCG and three different M. tuberculosis strains. The resulting transformants, as well as previously published M. tuberculosis strains with multicopy INHA or kasAB plasmids, were tested for their resistance to INH, ethionamide (ETH) or thiolactomycin (TLM). Mycobacteria containing INHA plasmids uniformly exhibited 20-fold or greater increased resistance to INH and 10-fold or greater increased resistance to ETH. In contrast, the kasA plasmid conferred no increased resistance to INH or ETH in any of the five strains, but it did confer resistance to thiolactomycin, a known KasA inhibitor. INH is known to increase the expression of kasA in INH-susceptible M. tuberculosis strains. Using molecular beacons, quantified INHA and kasA mRNA levels showed that increased INHA mRNA levels corre-lated with INH resistance, whereas kasA mRNA levels did not. In summary, analysis of strains harbouring INHA or kasA plasmids yielded the same conclusion: overexpressed INHA, but not kasA, confers INH and ETH resistance to M. smegmatis, M. bovis BCG and M. tuberculosis. Therefore, INHA is the primary target of action of INH and ETH in all three species.
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inactivation of the INHA encoded fatty acid synthase ii fasii enoyl acyl carrier protein reductase induces accumulation of the fasi end products and cell lysis of mycobacterium smegmatis
Journal of Bacteriology, 2000Co-Authors: Catherine Vilcheze, James C Sacchettini, Hector R Morbidoni, Torin R Weisbrod, Hiroyuki Iwamoto, William R JacobsAbstract:The mechanism of action of isoniazid (INH), a first-line antituberculosis drug, is complex, as mutations in at least five different genes (katG, INHA, ahpC, kasA, and ndh) have been found to correlate with isoniazid resistance. Despite this complexity, a preponderance of evidence implicates INHA, which codes for an enoyl-acyl carrier protein reductase of the fatty acid synthase II (FASII), as the primary target of INH. However, INH treatment of Mycobacterium tuberculosis causes the accumulation of hexacosanoic acid (C26:0), a result unexpected for the blocking of an enoyl-reductase. To test whether inactivation of INHA is identical to INH treatment of mycobacteria, we isolated a temperature-sensitive mutation in the INHA gene of Mycobacterium smegmatis that rendered INHA inactive at 42°C. Thermal inactivation of INHA in M. smegmatis resulted in the inhibition of mycolic acid biosynthesis, a decrease in hexadecanoic acid (C16:0) and a concomitant increase of tetracosanoic acid (C24:0) in a manner equivalent to that seen in INH-treated cells. Similarly, INH treatment of Mycobacterium bovis BCG caused an inhibition of mycolic acid biosynthesis, a decrease in C16:0, and a concomitant accumulation of C26:0. Moreover, the INHA-inactivated cells, like INH-treated cells, underwent a drastic morphological change, leading to cell lysis. These data show that INHA inactivation, alone, is sufficient to induce the accumulation of saturated fatty acids, cell wall alterations, and cell lysis and are consistent with INHA being a primary target of INH.
Alexander J Kastaniotis - One of the best experts on this subject based on the ideXlab platform.
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function of heterologous mycobacterium tuberculosis INHA a type 2 fatty acid synthase enzyme involved in extending c20 fatty acids to c60 to c90 mycolic acids during de novo lipoic acid synthesis in saccharomyces cerevisiae
Applied and Environmental Microbiology, 2008Co-Authors: Aner Gurvitz, Kalervo J Hiltunen, Alexander J KastaniotisAbstract:We describe the physiological function of heterologously expressed Mycobacterium tuberculosis INHA during de novo lipoic acid synthesis in yeast (Saccharomyces cerevisiae) mitochondria. INHA, representing 2-trans-enoyl-acyl carrier protein reductase and the target for the front-line antituberculous drug isoniazid, is involved in the activity of dissociative type 2 fatty acid synthase (FASII) that extends associative type 1 fatty acid synthase (FASI)-derived C20 fatty acids to form C60-to-C90 mycolic acids. Mycolic acids are major constituents of the protective layer around the pathogen that contribute to virulence and resistance to certain antimicrobials. Unlike FASI, FASII is thought to be incapable of de novo biosynthesis of fatty acids. Here, the genes for INHA (Rv1484) and four similar proteins (Rv0927c, Rv3485c, Rv3530c, and Rv3559c) were expressed in S. cerevisiae etr1Δ cells lacking mitochondrial 2-trans-enoyl-thioester reductase activity. The phenotype of the yeast mutants includes the inability to produce sufficient levels of lipoic acid, form mitochondrial cytochromes, respire, or grow on nonfermentable carbon sources. Yeast etr1Δ cells expressing mitochondrial INHA were able to respire, grow on glycerol, and produce lipoic acid. Commensurate with a role in mitochondrial de novo fatty acid biosynthesis, INHA could accept in vivo much shorter acyl-thioesters (C4 to C8) than was previously thought (>C12). Moreover, INHA functioned in the absence of AcpM or protein-protein interactions with its native FASII partners KasA, KasB, FabD, and FabH. None of the four proteins similar to INHA complemented the yeast mutant phenotype. We discuss the implications of our findings with reference to lipoic acid synthesis in M. tuberculosis and the potential use of yeast FASII mutants for investigating the physiological function of drug-targeted pathogen enzymes involved in fatty acid biosynthesis.
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function of heterologous mycobacterium tuberculosis INHA a type 2 fatty acid synthase enzyme involved in extending c20 fatty acids to c60 to c90 mycolic acids during de novo lipoic acid synthesis in saccharomyces cerevisiae
Applied and Environmental Microbiology, 2008Co-Authors: Aner Gurvitz, Kalervo J Hiltunen, Alexander J KastaniotisAbstract:Mycobacterium tuberculosis is the leading cause of mortality due to an infectious agent worldwide. The World Health Organization estimates that approximately 2 billion people have tuberculosis (53). M. tuberculosis is therefore the biggest killer among human pathogens; it is thought that about 1.7 million people die from tuberculosis every year (www.who.int/tb/publications). Moreover, increasing multiple-drug resistance has contributed significantly to the number of incurable cases, and in some countries up to 36% of patients with tuberculosis are infected with strains resistant to isoniazid (INH) or rifampin (42). These two compounds have been used clinically for several decades and are two of only a few first-line antituberculous drugs. The primary target of INH is INHA (3), and this fact alone elevates INHA to the position of one of most medically significant pathogen proteins. Hence, it is important to use new molecular approaches to study INHA in order to identify novel ways of targeting this enzyme, as well as the processes in which it is involved. INHA participates in fatty acid biosynthesis (3). Unlike the situation in Escherichia coli, the mycobacterial process is comprised of two systems. M. tuberculosis has a prokaryotic dissociative type 2 fatty acid synthase (FASII) system, in which individual reactions are catalyzed by discrete polypeptides, a process that has been characterized extensively in E. coli and plant plastids. Additionally, M. tuberculosis has an associative type 1 fatty acid synthase (FASI) system (5), which includes several enzymatic activities within a multifunctional homohexamer and resembles the cytosolic synthase in eukaryotes (47). The two systems combine to produce mycolic acids, which are very-long-chain (C54 to C63) α-branched, β-hydroxylated fatty acids that act with other factors to form the protective layer around the pathogen, thereby adding to its persistence despite lengthy treatment, and are also associated with its virulence (48). Based on its fully sequenced genome (13), it has been proposed that M. tuberculosis contains the entire complement of FASII components (48). The 2-trans-enoyl-acyl carrier protein (2-trans-enoyl-ACP) reductase of FASII is represented by INHA, which carries out the final step of the fatty acid elongation process. The M. tuberculosis genome harbors genes for four additional proteins, Rv0927c, Rv3485c, Rv3530c, and Rv3559c, that are all similar to INHA (48) and exhibit about 24 to 26% sequence identity to the latter protein (Fig. (Fig.1).1). It has been proposed that in M. tuberculosis, INHA catalyzes the reduction of 2-trans-enoyl-ACPs with a chain length greater than C12, whereas INHA in Mycobacterium smegmatis acts on C16 thioesters (35). FIG. 1. Comparison of M. tuberculosis INHA with four proteins most similar to it: Genedoc-based comparison of the deduced amino acid sequences of INHA (Rv1484) and Rv3485c, Rv0927c, Rv3530c, and Rv3559c. Dashes were added to the sequences to obtain the best fit. ... Yeast (Saccharomyces cerevisiae), mammals, and other higher eukaryotes have traditionally been considered organisms that are capable of synthesizing fatty acids only through FASI. This view has very recently been completely overhauled, since an additional mitochondrial FASII has been discovered in both yeast and mammals (2, 28, 32, 37, 50, 55, 56). In the first committed step of S. cerevisiae FASII activity, Hfa1p, representing mitochondrial acetyl coenzyme A (acetyl-CoA) carboxylase (29), converts acetyl-CoA to malonyl-CoA. A malonyl-CoA transferase, mitochondrial Mct1p (45), transfers the C3 moiety to ACP, a mitochondrial protein encoded by ACP1 (46). Chain elongation begins with the condensation of acetyl-ACP and malonyl-ACP by Cem1p (23), which acts as a mitochondrial 3-oxoacyl-ACP synthase. Mitochondrial Oar1p (45), a 3-oxoacyl-ACP reductase, produces the 3-hydroxyacyl-ACP intermediate, which is then dehydrated by mitochondrial Htd2p (32), a 3-hydroxyacyl-thioester dehydratase, to generate the 2-trans-enoyl-ACP species. The last step in each round of elongation is catalyzed by mitochondrial Etr1p representing 2-trans-enoyl-thioester reductase (50). A yeast mutant that lacks Etr1p contains abnormally small mitochondria, does not assemble respiratory complexes, and is exclusively fermentative (50, 54). This phenotype can be rescued by supplying the mutant with the gene for fungal or human mitochondrial 2-trans-enoyl-ACP reductase (37, 50). In addition, the etr1Δ mutant phenotype can also be rescued with a mitochondrially targeted E. coli FabI protein (50), representing a structurally unrelated FASII enoyl-ACP reductase (4). Whereas it has been proposed that mitochondrial FASII is involved in de novo production of the C8 precursor of lipoic acid (20), mycobacterial FASII is thought to be incapable of de novo synthesis (5). Here, we used S. cerevisiae as a surrogate for hosting M. tuberculosis protein genes (17). To examine whether INHA could physiologically metabolize short-chain enoyl-ACP substrates and to determine whether there are functional INHA homologues in M. tuberculosis, INHA and the four similar proteins were expressed in the yeast etr1Δ mutant, and transformed mutant cells were compared to cells of an otherwise isogenic strain expressing the corresponding native enzyme in terms of growth on glycerol, lipoic acid production, assembly of cytochrome complexes, respiration, and the presence of 2-trans-enoyl-thioester reductase activity. The implications of our finding that INHA can participate in de novo lipoic acid synthesis in yeast mitochondria for fatty acid biosynthesis in M. tuberculosis are briefly discussed below.