Characterization of the katG gene encoding a catalase-peroxidase required for the isoniazid susceptibility of Mycobacterium tuberculosis.

The isoniazid susceptibility of Mycobacterium tuberculosis is mediated by the product of the katG gene which encodes the heme-containing enzyme catalase-peroxidase. In this study, the chromosomal location of katG has been established and its nucleotide sequence has been determined so that the primary structure of catalase-peroxidase could be predicted. The M. tuberculosis enzyme is an 80,000-dalton protein containing several motifs characteristic of peroxidases and shows strong similarity to other bacterial catalase-peroxidases. Expression of the katG gene in M. tuberculosis, M. smegmatis, and Escherichia coli was demonstrated by Western blotting (immunoblotting). Homologous genes were detected in other mycobacteria, even those which are naturally insensitive to isoniazid.     

Crystal structure of Mycobacterium tuberculosis catalase-peroxidase

The Mycobacterium tuberculosis catalase-peroxidase is a multifunctional heme-dependent enzyme that activates the core anti-tuberculosis drug isoniazid. Numerous studies have been undertaken to elucidate the enzyme-dependent mechanism of isoniazid activation, and it is well documented that mutations that reduce activity or inactivate the catalase-peroxidase lead to increased levels of isoniazid resistance in M. tuberculosis. Interpretation of the catalytic activities and the effects of mutations upon the action of the enzyme to date have been limited due to the lack of a three-dimensional structure for this enzyme. In order to provide a more accurate model of the three-dimensional structure of the M. tuberculosis catalase-peroxidase, we have crystallized the enzyme and now report its crystal structure refined to 2.4-A resolution. The structure reveals new information about dimer assembly and provides information about the location of residues that may play a role in catalysis including candidates for protein-based radical formation. Modeling and computational studies suggest that the binding site for isoniazid is located near the delta-meso heme edge rather than in a surface loop structure as currently proposed. The availability of a crystal structure for the M. tuberculosis catalase-peroxidase also permits structural and functional effects of mutations implicated in causing elevated levels of isoniazid resistance in clinical isolates to be interpreted with improved confidence.     

Missense mutations in the catalase-peroxidase gene, katG, are associated with isoniazid resistance in Mycobacterium tuberculosis

The toxicity of the powerful anti-tuberculosis drug isoniazid (INH) is believed to be mediated by the haem-containing enzyme catalase-peroxidase, encoded by the katG gene of Mycobacterium tuberculosis. Compelling evidence for this was obtained by studying a panel of INH-resistant clinical isolates using a novel strategy based on the polymerase chain reaction and single-strand-conformation polymorphism analysis (PCR-SSCP) to detect mutations in katG. In most cases INH resistance was associated with missense mutations while in a small number of strains the gene had been completely, or partially, deleted. The missense mutations fell into two groups, the larger of which contained several independent mutations that affected the N-terminal peroxidase domain of the protein, resulting in the production of a catalase peroxidase with strongly reduced enzyme activity and increased heat lability. The effects of these substitutions could be interpreted by means of molecular modelling using the crystal structure of the related enzyme cytochrome c peroxidase from yeast as a template. The second group comprises a frequently occurring amino acid substitution and a single mutation that are both located in the C-terminal domain but do not noticeably alter either enzyme activity or heat stability.     

Hydrogen peroxide-mediated isoniazid activation catalyzed by Mycobacterium tuberculosis catalase-peroxidase (KatG) and its S315T mutant

Inhibition of the enzyme Mycobacterium tuberculosis InhA (enoyl-acyl carrier protein reductase) due to formation of an isonicotinoyl-NAD adduct (IN-NAD) from isoniazid (INH) and nicotinamide adenine dinucleotide cofactor is considered central to the mode of action of INH, a first-line treatment for tuberculosis infection. INH action against mycobacteria requires catalase-peroxidase (KatG) function, and IN-NAD adduct formation is catalyzed in vitro by M. tuberculosis KatG under a variety of conditions, yet a physiologically relevant approach to the process has not emerged that allows scrutiny of the mechanism and the origins of INH resistance in the most prevalent drug-resistant strain bearing KatG[S315T]. In this report, we describe how hydrogen peroxide, delivered at very low concentrations to ferric KatG, leads to efficient inhibition of InhA due to formation of the IN-NAD adduct. The rate of adduct formation mediated by wild-type KatG was about 20-fold greater than by the isoniazid-resistant KatG[S315T] mutant under optimal conditions (H2O2 supplied along with NAD+ and INH). Slow adduct formation also occurs starting with NADH and INH, in the presence of KatG even in the absence of added peroxide, due to endogenous peroxide. The poor efficiency of the KatG[S315T] mutant can be enhanced merely by increasing the concentration of INH, consistent with this enzyme's reduced affinity for INH binding to the resting enzyme and the catalytically competent enzyme intermediate (Compound I). Origins of drug resistance in the KatG[S315T] mutant enzyme are analyzed at the structural level through examination of the three-dimensional X-ray crystal structure of the mutant enzyme.     

Impact of distal side water and residue 315 on ligand binding to ferric Mycobacterium tuberculosis catalase-peroxidase (KatG)

The catalase-peroxidase (KatG) of Mycobacterium tuberculosis (Mtb) is important for the virulence of this pathogen and also is responsible for activation of isoniazid (INH), an antibiotic in use for over 50 years in the first line treatment against tuberculosis infection. Overexpressed Mtb KatG contains a heterogeneous population of heme species that present distinct spectroscopic properties and, as described here, functional properties. A six-coordinate (6-c) heme species that accumulates in the resting enzyme after purification is defined as a unique structure containing weakly associated water on the heme distal side. We present the unexpected finding that this form of the enzyme, generally present as a minority species along with five-coordinate (5-c) enzyme, is the favored reactant for ligand binding. The use of resting enzyme samples with different proportional composition of 5-c and 6-c forms, as well as the use of KatG mutants with replacements at residue 315 that have different tendencies to stabilize the 6-c form, allowed demonstration of more rapid cyanide binding and preferred peroxide binding to enzyme containing 6-c heme. Optical-stopped flow and equilibrium titrations of ferric KatG with potassium cyanide reveal complex behavior that depends in part on the amount of 6-c heme in the resting enzymes. Resonance Raman and low-temperature EPR spectroscopy clearly demonstrate favored ligand (cyanide or peroxide) binding to 6-c heme. The 5-c and 6-c enzyme forms are not in equilibrium on the time scale of the experiments. The results provide evidence for the likely participation of specific water molecule(s) in the first phases of the reaction mechanism of catalase-peroxidase enzymes.     

Characterization of mutations causing rifampicin and isoniazid resistance of Mycobacterium tuberculosis in Syria

In order to characterize mutations causing rifampicin and isoniazid resistance of M. tuberculosis in Syria, 69 rifampicin resistant (Rif(r)) and 72 isoniazid resistant (Inh(r)) isolates were screened for point mutations in hot spots of the rpoB, katG and inhA genes by DNA sequencing and real time PCR. Of 69 Rif(r) isolates, 62 (90%) had mutations in the rifampin resistance determining region (RRDR) of the rpoB gene, with codons 531 (61%), 526 (13%), and 516 (8.7%) being the most commonly mutated. We found two new mutations (Asp516Thr and Ser531Gly) described for the first time in the rpoB-RRDR in association with rifampicin resistance. Only one mutation (Ile572Phe) was found outside the rpoB-RRDR. Of 72 Inh(r) strains, 30 (41.6%) had a mutation in katGcodon315 (with Ser315Thr being the predominant alteration), and 23 (32%) harbored the inhA(-15C-->T) mutation. While the general pattern of rpoB-RRDR and katG mutations reflected those found worldwide, the prevalence of the inhA(-15C-->T mutation was above the value found in most other countries, emphasizing the great importance of testing the inhA(-15C-->T) mutation for prediction of isoniazid resistance in Syria. Sensitivity of a rapid test using real time PCR and 3'-Minor groove binder (MGB) probes in detecting Rif(r) and Inh(r) isolates was 90% and 69.4%, respectively. This demonstrates that a small set of MGB-probes can be used in real time PCR in order to detect most mutations causing resistance to rifampicin and isoniazid.     

结核分枝杆菌对异烟肼耐药的分子机制

抗结核一线药物异烟肼是应用最广泛的抗结核药物之一,自1952年应用于临床以来,异烟肼就成了治疗结核和潜在感染的基础药物.有报道,我国异烟肼耐药已排在首位.结核分枝杆菌对异烟肼耐药的分子机制十分复杂,涉及katG、inhA、kasA、ndh、axyR等多种基因,本研究仅就此方面的研究作一综述.     

Resonance Raman spectroscopy of Compound II and its decay in Mycobacterium tuberculosis catalase-peroxidase KatG and its isoniazid resistant mutant S315T

The reaction of Mycobacterium tuberculosis KatG and the mutant KatG(S315T) with two different organic peroxides is studied using resonance Raman spectroscopy. For the first time, an intermediate is observed in a catalase-peroxidase with vibrations that are characteristic of Compound II. The observation of this intermediate is consistent with photoreduction of Compound I and is in agreement with the formation of Compound I during the catalytic cycle of KatG. The same intermediate is detected in KatG(S315T), a mutant associated with resistance to isoniazid (INH), but with a lower yield, indicating that the organic peroxides cannot react with the heme iron in KatG(S315T) as efficiently as in wild-type KatG. Our results are consistent with catalytic competence of the S315T mutant and support the model that the S315T mutation confers antibiotic resistance by modifying the interaction between the enzyme and the drug.     

Characterization of ndh gene of isoniazid resistant and susceptible Mycobacterium tuberculosis isolates from Brazil

Resistance in Mycobacterium tuberculosis to isoniazid (INH) is caused by mutations in the catalase-peroxidase gene (katG), and within the inhA promoter and/or in structural gene. A small percentage (approximately 10%) of INH-resistant strains do not present mutations in both of these loci. Other genes have been associated with INH resistance including the gene encoding for NADH dehydrogenase (ndh). Here we report the detection of two ndh locus mutations (CGT to TGT change in codon 13 and GTG to GCG change in codon 18) by analyzing 23 INH-resistant and in none of 13 susceptible isolates from Brazilian tuberculosis patients. We also detected two isolates without a mutation in ndh, or any of the other INH resistance-associated loci examined, suggesting the existence of additional, as yet to be described, INH resistance mechanisms.     

Spin trapping investigation of peroxide- and isoniazid-induced radicals in Mycobacterium tuberculosis catalase-peroxidase

A new approach, the immuno-spin trapping assay, used a novel rabbit polyclonal anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antiserum to detect protein radical-derived DMPO nitrone adducts in the hemoprotein Mycobacterium tuberculosis catalase-peroxidase (KatG). This work demonstrates that the formation of protein nitrone adducts is dependent on the concentrations of tert-BuOOH and DMPO as shown by Western blotting and an enzyme-linked immunosorbent assay (ELISA). We have also detected protein-protein cross-links formed during turnover of Mtb KatG driven by tert-butyl peroxide ( tert-BuOOH) or enzymatic generation of hydrogen peroxide. DMPO inhibits this dimerization due to its ability to trap the amino acid radicals responsible for the cross-linkage. Chemical modifications by tyrosine and tryptophan blockage suggest that tyrosine residues are one site of formation of the dimers. The presence of the tuberculosis drug isoniazid (INH) also prevented cross-linking as a result of its competition for the protein radical. Protein-DMPO nitrone adducts were also generated by a continuous flux of hydrogen peroxide. These findings demonstrated that the protein-based radicals were formed not only during Mtb KatG turnover with alkyl peroxides but also in the presence of hydrogen peroxide. Furthermore, the formation of protein-DMPO nitrone adducts was accelerated in the presence of isoniazid.     

Using cryo-EM to understand antimycobacterial resistance in the catalase-peroxidase (KatG) from Mycobacterium tuberculosis

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Proteome-wide profiling of isoniazid targets in Mycobacterium tuberculosis

Isoniazid (INH) is an essential drug used to treat tuberculosis. The mycobactericidal agents are INH adducts [INH-NAD(P)] of the pyridine nucleotide coenzymes, which are generated in vivo after INH activation and which bind to, and inhibit, essential enzymes. The NADH-dependent enoyl-ACP reductase (InhA) and the NADPH-dependent dihydrofolate reductase (DfrA) have both been shown to be inhibited by INH-NAD(P) adducts with nanomolar affinity. In this paper, we profiled the Mycobacterium tuberculosis proteome using both the INH-NAD and INH-NADP adducts coupled to solid supports and identified, in addition to InhA and DfrA, 16 other proteins that bind these adducts with high affinity. The majority of these are predicted to be pyridine nucleotide-dependent dehydrogenases/reductases. They are involved in many cellular processes, including S-adenosylmethionine-dependent methyl transfer reactions, pyrimidine and valine catabolism, the arginine degradative pathway, proton and potassium transport, stress response, lipid metabolism, and riboflavin biosynthesis. The targeting of multiple enzymes could, thus, account for the pleiotropic effects of, and powerful mycobactericidal properties of, INH.     

Contribution of efflux to the emergence of isoniazid and multidrug resistance in Mycobacterium tuberculosis

Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment.     

Analysis of heme structural heterogeneity in Mycobacterium tuberculosis catalase-peroxidase (KatG)

Mycobacterium tuberculosis catalase-peroxidase (KatG) is a heme enzyme considered important for virulence, which is also responsible for activation of the anti-tuberculosis pro-drug isoniazid. Here, we present an analysis of heterogeneity in KatG heme structure using optical, resonance Raman, and EPR spectroscopy. Examination of ferric KatG under a variety of conditions, including enzyme in the presence of fluoride, chloride, or isoniazid, and at different stages during purification in different buffers allowed for assignment of spectral features to both five- and six-coordinate heme. Five-coordinate heme is suggested to be representative of "native" enzyme, since this species was predominant in the enzyme examined immediately after one chromatographic protocol. Quantum mechanically mixed spin heme is the most abundant form in such partially purified enzyme. Reduction and reoxidation of six-coordinate KatG or the addition of glycerol or isoniazid restored five-coordinate heme iron, consistent with displacement of a weakly bound distal water molecule. The rate of formation of KatG Compound I is not retarded by the presence of six-coordinate heme either in wild-type KatG or in a mutant (KatG[Y155S]) associated with isoniazid resistance, which contains abundant six-coordinate heme. These results reveal a number of similarities and differences between KatG and other Class I peroxidases.     

Identification and characterization of tyrosyl radical formation in Mycobacterium tuberculosis catalase-peroxidase (KatG)

The catalytic function of Mycobacterium tuberculosis catalase-peroxidase (KatG) and its role in activation of the anti-tuberculosis antibiotic isoniazid were investigated using rapid freeze-quench electron paramagnetic resonance (RFQ-EPR) experiments. The reaction of KatG with peroxyacetic acid was followed as a function of time using x-band EPR at 77 K. A doublet EPR signal appears within 6.4 ms after mixing and at time points through hundreds of milliseconds. Thereafter, a singlet signal develops and finally predominates after 1 s, with a total yield of radical approximately 0.5 spin/heme. Simulation of the spectra provided EPR parameters consistent with those for tyrosyl radicals. Changes in the hyperfine splitting and/or line width in spectra for l-3,3-[2H2]tyrosine-labeled, but not l-2,4,5,6,7-[2H5]tryptophan-labeled KatG confirmed this assignment. The initial rate of radical formation was unchanged using a 3-fold or 10-fold excess of peroxyacetic acid, consistent with a rate-determining step involving an intermediate. Although Compound I is likely to be the precursor of tyrosyl radical in KatG, neither its EPR signal nor its reduction to Compound II during formation of the radical(s) could be observed. The tyrosyl radical doublet signal was rapidly quenched by addition of isoniazid and benzoic hydrazide, but not by iproniazid, which binds poorly to KatG.     

Mechanism for the pyridoxal neutralization of isoniazid action of Mycobacterium tuberculosis

In Sauton's synthetic liquid medium, 10 mug of pyridoxal per ml completely protected Mycobacterium tuberculosis (H37R(a)) from the effects of a minimal inhibitory concentration of isoniazid (0.01 mug/ml). (14)C-labeled isoniazid was employed to study the nature of this protective effect. Uptake of the drug by cells in a Sauton environment containing 0.01 mug of (14)C-isoniazid per ml was inhibited 20 to 40% by 10 mug of pyridoxal per ml during the early hours of drug exposure. A stronger inhibition of uptake resulted when labeled isoniazid and pyridoxal were increased to 0.1 mug/ml and 50 to 100 mug/ml, respectively. Further studies revealed that certain Sauton nutrients are required to achieve this effect. When l-asparagine or salts (MgSO(4) and ferric ammonium citrate) or both were deleted from the menstruum, pyridoxal did not inhibit isoniazid incorporation by the tubercle bacilli. Pyridoxal also failed to inhibit uptake when (NH(4))(2)SO(4) was substituted for l-asparagine. Growth experiments in Sauton's medium modified to contain (NH(4))(2)SO(4) instead of l-asparagine were consistent with the latter finding. Pyridoxal did not prevent isoniazid growth inhibition in this medium. It is postulated that a large excess of pyridoxal in Sauton's medium protects tubercle bacilli from the effects of isoniazid through formation of an extracellular complex involving drug, vitamin, and certain medium constituents, thereby reducing the level of isoniazid available to the cells.     

The effects of isoniazid on the carbohydrates of Mycobacterium tuberculosis BCG

1. Mycobacterium tuberculosis BCG was usually grown in glycerol-asparagine-casein hydrolysate medium. A soluble fraction was obtained from the cells with aq. 50% ethanol; unbound lipids were then removed and the cells were treated with dilute alkali to give, after acidification, an alkali-extractable fraction and an insoluble fraction. On occasion, lipopolysaccharides were obtained by extracting with phenol or dimethyl sulphoxide instead of alkali. The soluble fraction contained, particularly after long extraction, polysaccharide containing mainly glucose, in addition to trehalose and monosaccharides and their derivatives. The alkali-extractable fraction contained polysaccharides containing mannose, glucose, arabinose, galactose and 6-O-methylglucose. These could be resolved into three fractions of markedly different molecular size. It is argued that the high-molecular-weight materials originated from the outside of the cell envelope and the medium-molecular-weight materials from a middle layer of the envelope. 2. Exposure of the growing cells to isoniazid, usually at 1 or 10mug/ml for 6-12h, increased the total cell carbohydrate, mainly due to an increase in trehalose and in insoluble glucan. It also facilitated the extraction of polysaccharide into the medium and the soluble fraction. This produced about a 25% decrease in the amount of carbohydrate in the alkaline-extractable fraction, mainly due to a fall in glucose, arabinose and 6-O-methylglucose. The decrease was confined to polysaccharides of large and medium molecular weight. When intact lipopolysaccharides were extracted, their amount was also decreased by isoniazid. 3. Substitution of ammonium sulphate for asparagine and casein hydrolysate in the medium, so that glycerol was the sole carbon source, decreased the carbohydrate accumulation brought about by isoniazid but did not alter its effect on polysaccharide extraction. 4. Growth with (14)C-labelled substrates showed that glycerol provided two to four times as much of the cell carbon as did asparagine, when both were present. Under these conditions isoniazid inhibited the incorporation of carbon atoms from asparagine into the cells, but had little effect on the total incorporation from glycerol. These experiments also showed that the effect of isoniazid on alkali-extractable polysaccharides was due to their loss to the soluble fraction and external medium. 5. It is suggested that isoniazid inhibits a pathway (probably the synthesis of mycolic acid) involved in the formation of the cell envelope, and that this inhibition results in some re-channelling of intermediates into carbohydrate synthesis and in some loss of polysaccharides through damage to the envelope.