Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UMLS:C0026918 (Mycobacterium)
52,428 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID: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. 1086 86

Structural and genetic studies indicate that the antibacterial compound triclosan, an additive in many personal care products, is an inhibitor of EnvM, the enoyl reductase from Escherichia coli. Here we show that triclosan specifically inhibits InhA, the enoyl reductase from Mycobacterium tuberculosis and a target for the antitubercular drug isoniazid. Binding of triclosan to wild-type InhA is uncompetitive with respect to both NADH and trans-2-dodecenoyl-CoA, with K(i)' values of 0.22+/-0.02 and 0.21+/-0.01 microM, respectively. Replacement of Y158, the catalytic tyrosine residue, with Phe, reduces the affinity of triclosan for the enzyme and results in noncompetitive inhibition, with K(i) and K(i)' values of 36+/-5 and 47+/-5 microM, respectively. Consequently, the Y158 hydroxyl group is important for triclosan binding, suggesting that triclosan binds in similar ways to both InhA and EnvM. In addition, the M161V and A124V InhA mutants, which result in resistance of Mycobacterium smegmatis to triclosan, show significantly reduced affinity for triclosan. Inhibition of M161V is noncompetitive with K(i)' = 4.3+/-0.5 microM and K(i) = 4.4+/-0.9 microM, while inhibition of A124V is uncompetitive with K(i)' = 0. 81 +/- 0.11 microM. These data support the hypothesis that the mycobacterial enoyl reductases are targets for triclosan. The M161V and A124V enzymes are also much less sensitive to isoniazid compared to the wild-type enzyme, indicating that triclosan can stimulate the emergence of isoniazid-resistant enoyl reductases. In contrast, I47T and I21V, two InhA mutations that occur in isoniazid-resistant clinical isolates of M. tuberculosis, show unimpaired inhibition by triclosan, with uncompetitive inhibition constants (K(i)') of 0.18+/-0.01 and 0.12+/- 0.01 microM, respectively. The latter result indicates that InhA inhibitors targeted at the enoyl substrate binding site may be effective against existing isoniazid-resistant strains of M. tuberculosis.
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PMID:Inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis, by triclosan and isoniazid. 1086 70

The anti-tuberculosis drug ethionamide (ETH), which is a structural analog of isoniazid (INH), is known to strongly inhibit mycolic acid synthesis in Mycobacterium tuberculosis. Although several targets have been identified for INH, only speculative information is available concerning ETH. Mutations within the promoter and the coding region of enoyl-acyl carrier protein reductase (InhA) were found to confer resistance to both drugs, thus leading to the impression that INH and ETH may share a common mode of action. However, a notable distinction between the two drugs lies in the lack of cross-resistance in clinical isolates. This may be attributed in part to the fact that the pro-drug INH must be activated via KatG, and no activation step for ETH has yet been described. Here we report the identification of an activator for ETH. The ETH activator (Rv3854c), which we have termed EthA, was found to be homologous to various monooxygenases and induced ETH sensitivity when overexpressed in mycobacteria. Interestingly, the neighboring open reading frame (Rv3855), which was found homologous to transcriptional repressors of the tetR family, led to ETH resistance when overexpressed. In addition, chromosomal inactivation of this gene by transposition led to ETH hypersensitivity. These data strongly suggest that Rv3855, which we have termed EthR, regulates the production of EthA, which subsequently activates the pro-drug ETH. This study opens up new avenues of research relating to ETH activation in mycobacteria, possibly leading to an improved efficacy of ETH and to the generation of new anti-mycobacterial agents.
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PMID:Activation of the pro-drug ethionamide is regulated in mycobacteria. 1086 56

Mycobacteria are intracellular pathogens that survive and grow in host macrophages. Following phagocytosis, sustained intracellular bacterial growth depends on its ability to avoid destruction by macrophage-mediated host defences such as lysosomal enzymes, reactive oxygen and the reactive nitrogen intermediates. This suggests that the interaction between host cell and microbe is delicately balanced, and can be tipped in favour of either organism. The identification of Mycobacterium tuberculosis H37Rv (MTB) genes expressed within host cells would contribute greatly to the development of new strategies to fight tuberculosis. In the present study, we compared MTB gene expression in the course of intra- (human macrophages) and extracellular growth (Sauton's medium) to ascertain whether differences might occur between gene-expression patterns in the two habitats of replication. Using reverse-transcriptase polymerase chain reaction (RT-PCR) on a group of 14 MTB-Complex-specific genes, we found that MT10Sa (a small stable RNA), 35 kDa (unknown), ahpC (alkyl hydroperoxide reductase, AhpC), sigF (alternative RNA Polymerase sigma factor), and katG (catalase-peroxidase, HPI) genes are expressed in both the environments, while Ag85B, Ag85C (members of the Antigen 85 Complex), rpoV (RNA Polymerase sigma factor) and ESAT6 (early secretory antigen, 6 kDa) are expressed only in the in vitro culture; on the other hand, Ag85A (Antigen 85 Complex), rpoB (RNA Polymerase beta sub-unit), pab (Protein antigen b), invA and invB genes (encoding proteins that show homologies with p60 of Listeria monocytogenes) are expressed only inside the macrophage. Positive RT-PCR products on cDNAs for these genomic regions were not obtained from approximately 1000-fold more bacteria grown in Laboratory Broth. Identification of M. tuberculosis genes expressed in response to phagocytosis by human macrophages increases our basic understanding of the host-pathogen interaction, and helps to identify bacterial factors necessary for in vivo survival and growth.
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PMID:Mycobacterium tuberculosis H37Rv comparative gene-expression analysis in synthetic medium and human macrophage. 1094 May 66

Nitric oxide (NO) is present in soil and air, and is produced by bacteria, animals and plants. Superoxide (O2-) arises in all organisms inhabiting aerobic environments. Thus, many organisms are likely to encounter peroxynitrite (OONO-), a product of NO and O2- that forms at near diffusion-limited rates, and rapidly decomposes upon protonation through isomerization to nitrate (NO3-; ref. 1) while generating hydroxyl radical (*OH) and nitrogen dioxide radical (*NO2) (refs 2, 3), both more reactive than peroxynitrite's precursors. The oxidative, inflammatory, mutagenic and cytotoxic potential (ref. 4) of peroxynitrite contrasts with the antioxidant, anti-inflammatory and tissue-protective properties ascribed to NO itself. Thus, the ability of cells to cope with peroxynitrite is central in determining the biological consequences of NO production. We considered whether cells might be equipped with enzymes to detoxify peroxynitrite. Peroxiredoxins have been identified in most genomes sequenced, but their functions are only partly understood. Here we show that the peroxiredoxin alkylhydroperoxide reductase subunit C (AhpC) from Salmonella typhimurium catalytically detoxifies peroxynitrite to nitrite fast enough to forestall the oxidation of bystander molecules such as DNA. Results are similar with peroxiredoxins from Mycobacterium tuberculosis and Helicobacter pylori. Thus, peroxynitrite reductase activity may be widespread among bacterial genera.
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PMID:Peroxynitrite reductase activity of bacterial peroxiredoxins. 1100 Oct 62

Genetic and biochemical evidence has implicated two different target enzymes for isoniazid (INH) within the unique type II fatty acid synthase (FAS) system involved in the production of mycolic acids. These two components are an enoyl acyl carrier protein (ACP) reductase, InhA, and a beta-ketoacyl-ACP synthase, KasA. We compared the consequences of INH treatment of Mycobacterium tuberculosis (MTB) with two inhibitors having well-defined targets: triclosan (TRC), which inhibits InhA; and thiolactomycin (TLM), which inhibits KasA. INH and TLM, but not TRC, upregulate the expression of an operon containing five FAS II components, including kasA and acpM. Although all three compounds inhibit mycolic acid synthesis, treatment with INH and TLM, but not with TRC, results in the accumulation of ACP-bound lipid precursors to mycolic acids that were 26 carbons long and fully saturated. TLM-resistant mutants of MTB were more cross-resistant to INH than TRC-resistant mutants. Overexpression of KasA conferred more resistance to TLM and INH than to TRC. Overexpression of InhA conferred more resistance to TRC than to INH and TLM. Co-overexpression of both InhA and KasA resulted in strongly enhanced levels of INH resistance, in addition to cross-resistance to both TLM and TRC. These results suggest that these components of the FAS II complex are not independently regulated and that alterations in the expression level of InhA affect expression levels of KasA. Nonetheless, INH appeared to resemble TLM more closely in overall mode of action, and KasA levels appeared to be tightly correlated with INH sensitivity.
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PMID:Isoniazid affects multiple components of the type II fatty acid synthase system of Mycobacterium tuberculosis. 1106 75

T cells are prominent components of both early and late atherosclerotic lesions and the role of Th1/Th2 cells subsets in the evolution and rupture of the plaque is currently under investigation. Statins, which are inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, exert actions beyond that of simply lowering cholesterol levels, and some effects on immune function have been reported. We studied in vitro the effects of fluvastatin on Th1/Th2 cytokine release in relation to caspase-1 activation, in human peripheral-blood mononuclear cells (PBMC) stimulated or not with Mycobacterium tuberculosis. Fluvastatin treatment resulted in the activation of caspase-1 and in a small secretion of interleukin (IL)-1beta, IL-18, and IFNgamma (Th1). In the presence of bacteria, the release of these cytokines was highly increased by the statin in a synergistic way. By contrast, production of IL-12, IL-10 and IL-4 were unaffected by the statin. Not only did mevalonate abolish the effects of the statin but it also prevented the caspase-1 activation induced by the bacteria, suggesting the involvement of isoprenoids in the response to M. tuberculosis. It is proposed that inhibition of HMG-CoA reductase may be immunoprotective by enhancing the Th1 response, which has therapeutical potential not only in atherosclerosis but also in infectious diseases.
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PMID:Hydroxymethylglutaryl-coenzyme A reductase inhibition stimulates caspase-1 activity and Th1-cytokine release in peripheral blood mononuclear cells. 1116 19

Patients with Crohn's disease (CD) (n = 10) and ulcerative colitis (UC) (n = 10) were tested for immune responses against various antigens from Mycobacterium avium subsp. paratuberculosis; alkyl hydroperoxide reductase C (AhpC) and alkyl hydroperoxide reductase D (AhpD), which are constitutively expressed in this species as opposed to other mycobacteria, a 14-kDa secreted antigen and PPD-J. The CD patients had significantly elevated antibody levels against the 14 kDa protein (P < 0.05) that were negatively correlated with the duration of the disease (r(s) = - 0.85). They also seemed to have increased antibody levels against AhpC and AhpD, but the differences between the two groups were not significant. However, taken together, the antibody responses to three individual mycobacterial antigens in CD patients strengthen the possibility that the observed responses are caused by mycobacterial infection. No significant differences in the interferon (IFN)-gamma production, the interleukin (IL)-10 production and the ability to proliferate upon stimulation with these antigens were observed. These results show that measuring antibody responses against purified specific antigens is a suitable and simple approach when assessing the connection between CD and mycobacteria in patients with clinical CD. Another important aspect in such studies is to have well defined patient groups tested at the onset of clinical symptoms.
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PMID:Elevated antibody responses in patients with Crohn's disease against a 14-kDa secreted protein purified from Mycobacterium avium subsp. paratuberculosis. 1116 25

Mycobacterial genomics has uncovered a novel regulatory gene, oxyS, belonging to the LysR family. There is extensive similarity in the DNA-binding domain of OxyS with that of OxyR, the oxidative stress response protein of many bacteria. Since the oxyR gene of Mycobacterium tuberculosis has been multiply inactivated during evolution it was conceivable that some of its functions could be effected by OxyS. It is shown here that OxyS is produced at low levels and that there are at least three different oxyS alleles present in clinical isolates of M. tuberculosis that are susceptible or resistant to isoniazid. Overproduction or depletion of OxyS did not affect susceptibility to isoniazid but increasing the concentration of the regulator lowered levels of the alkyl hydroperoxide reductase, AhpC, and rendered the tubercle bacillus more susceptible to organic hydroperoxides.
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PMID:Role of OxyS of Mycobacterium tuberculosis in oxidative stress: overexpression confers increased sensitivity to organic hydroperoxides. 1148 19

Intracellular pathogens such as Mycobacterium tuberculosis are able to survive in the face of antimicrobial products generated by the host cell in response to infection. The product of the alkyl hydroperoxide reductase gene (ahpC) of M. tuberculosis is thought to be involved in protecting the organism against both oxidative and nitrosative stress encountered within the infected macrophage. Here we report that, contrary to expectations, ahpC expression in virulent strains of M. tuberculosis and Mycobacterium bovis grown in vitro is repressed, often below the level of detection, whereas expression in the avirulent vaccine strain M. bovis BCG is constitutively high. The repression of the ahpC gene of the virulent strains is independent of the naturally occurring lesions of central regulator oxyR. Using a green fluorescence protein vector (gfp)-ahpC reporter construct we present data showing that repression of ahpC of virulent M. tuberculosis also occurred during growth inside macrophages, whereas derepression in BCG was again seen under identical conditions. Inactivation of ahpC on the chromosome of M. tuberculosis by homologous recombination had no effect on its growth during acute infection in mice and did not affect in vitro sensitivity to H2O2. However, consistent with AhpC function in detoxifying organic peroxides, sensitivity to cumene hydroperoxide exposure was increased in the ahpC::Km(r) mutant strain. The preservation of a functional ahpC gene in M. tuberculosis in spite of its repression under normal growth conditions suggests that, while AhpC does not play a significant role in establishing infection, it is likely to be important under certain, as yet undefined conditions. This is supported by the observation that repression of ahpC expression in vitro was lifted under conditions of static growth.
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PMID:Silencing of oxidative stress response in Mycobacterium tuberculosis: expression patterns of ahpC in virulent and avirulent strains and effect of ahpC inactivation. 1155 32


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