NADH dehydrogenase defects confer isoniazid resistance and conditional lethality in Mycobacterium smegmatis

Isoniazid (INH) is a highly effective drug used in the treatment and prophylaxis of Mycobacterium tuberculosis infections. Resistance to INH in clinical isolates has been correlated with mutations in the inhA, katG, and ahpC genes. In this report, we describe a new mechanism for INH resistance in Mycobacterium smegmatis. Mutations that reduce NADH dehydrogenase activity (Ndh; type II) cause multiple phenotypes, including (i) coresistance to INH and a related drug, ethionamide; (ii) thermosensitive lethality; and (iii) auxotrophy. These phenotypes are corrected by expression of one of two enzymes: NADH dehydrogenase and the NADH-dependent malate dehydrogenase of the M. tuberculosis complex. The genetic data presented here indicate that defects in NADH oxidation cause all of the mutant traits and that an increase in the NADH/NAD+ ratio confers INH resistance.     

Inhibition of a Mycobacterium tuberculosis beta-ketoacyl ACP synthase by isoniazid

Although isoniazid (isonicotinic acid hydrazide, INH) is widely used for the treatment of tuberculosis, its molecular target has remained elusive. In response to INH treatment, saturated hexacosanoic acid (C26:0) accumulated on a 12-kilodalton acyl carrier protein (AcpM) that normally carried mycolic acid precursors as long as C50. A protein species purified from INH-treated Mycobacterium tuberculosis was shown to consist of a covalent complex of INH, AcpM, and a beta-ketoacyl acyl carrier protein synthase, KasA. Amino acid-altering mutations in the KasA protein were identified in INH-resistant patient isolates that lacked other mutations associated with resistance to this drug.     

Evidence for differential binding of isoniazid by Mycobacterium tuberculosis KatG and the isoniazid-resistant mutant KatG(S315T)

Isoniazid is a mainstay of antibiotic therapy for the treatment of tuberculosis, but its molecular mechanism of action is unclear. Previous investigators have hypothesized that isoniazid is a prodrug that requires in vivo activation by KatG, the catalase-peroxidase of Mycobacterium tuberculosis, and that resistance to isoniazid strongly correlates with deletions or point mutations in KatG. One such mutation, KatG(S315T), is found in approximately 50% of clinical isolates exhibiting isoniazid resistance. In this work, 1H nuclear magnetic resonance T1 relaxation measurements indicate that KatG and KatG(S315T) each bind isoniazid at a position approximately 12 A from the active site heme iron. Electron paramagnetic resonance spectroscopy revealed heterogeneous populations of high-spin ferric heme in both wild-type KatG and KatG(S315T) with the ratios of each species differing between the two enzymes. Small changes in the proportions of these high-spin species upon addition of isoniazid support the finding that isoniazid binds near the heme periphery of both enzymes. Titration of wild-type KatG with isoniazid resulted in the appearance of a "type I" substrate-induced difference spectrum analogous to those seen upon substrate binding to the cytochromes P450. The difference spectrum may result from an isoniazid-induced change in a portion of the KatG heme iron from 6- to 5-coordinate. Titration of KatG(S315T) with isoniazid failed to produce a measurable difference spectrum indicating an altered active site configuration. These results suggest that KatG(S315T) confers resistance to isoniazid through subtle changes in the isoniazid binding site.     

Cloning and characterization of arylamine N-acetyltransferase genes from Mycobacterium smegmatis and Mycobacterium tuberculosis: increased expression results in isoniazid resistance

Arylamine N-acetyltransferases (NATs) are found in many eukaryotic organisms, including humans, and have previously been identified in the prokaryote Salmonella typhimurium. NATs from many sources acetylate the antitubercular drug isoniazid and so inactivate it. nat genes were cloned from Mycobacterium smegmatis and Mycobacterium tuberculosis, and expressed in Escherichia coli and M. smegmatis. The induced M. smegmatis NAT catalyzes the acetylation of isoniazid. A monospecific antiserum raised against pure NAT from S. typhimurium recognizes NAT from M. smegmatis and cross-reacts with recombinant NAT from M. tuberculosis. Overexpression of mycobacterial nat genes in E. coli results in predominantly insoluble recombinant protein; however, with M. smegmatis as the host using the vector pACE-1, NAT proteins from M. tuberculosis and M. smegmatis are soluble. M. smegmatis transformants induced to express the M. tuberculosis nat gene in culture demonstrated a threefold higher resistance to isoniazid. We propose that NAT in mycobacteria could have a role in acetylating, and hence inactivating, isoniazid.     

Effects of overexpression of the alkyl hydroperoxide reductase AhpC on the virulence and isoniazid resistance of Mycobacterium tuberculosis

Mutations to the regulatory region of the ahpC gene, resulting in overproduction of alkyl hydroperoxide reductase, were encountered frequently in a large collection of isoniazid (INH)-resistant clinical isolates of Mycobacterium tuberculosis but not in INH-susceptible strains. Overexpression of ahpC did not seem to be important for INH resistance, however, as most of these strains were already defective for catalase-peroxidase, KatG, the enzyme required for activation of INH. Transformation of the INH-susceptible reference strain, M. tuberculosis H37Rv, with plasmids bearing the ahpC genes of M. tuberculosis or M. leprae did not result in a significant increase in the MIC. Two highly INH-resistant mutants of H37Rv, BH3 and BH8, were isolated in vitro and shown to produce no or little KatG activity and, in the case of BH3, to overproduce alkyl hydroperoxide reductase as the result of an ahpC regulatory mutation that was also found in some clinical isolates. The virulence of H37Rv, BH3, and BH8 was studied intensively in three mouse models: fully immunocompetent BALB/c and Black 6 mice, BALB/c major histocompatibility complex class II-knockout mice with abnormally low levels of CD4 T cells and athymic mice producing no cellular immune response. The results indicated that M. tuberculosis strains producing catalase-peroxidase were considerably more virulent in immunocompetent mice than the isogenic KatG-deficient mutants but that loss of catalase-peroxidase was less important when immunodeficient mice, unable to produce activated macrophages, were infected. Restoration of virulence was not seen in an INH-resistant M. tuberculosis strain that overexpressed ahpC, and this finding was confirmed by experiments performed with appropriate M. bovis strains in guinea pigs. Thus, in contrast to catalase-peroxidase, alkyl hydroperoxide reductase does not appear to act as a virulence factor in rodent infections or to play a direct role in INH resistance, although it may be important in maintaining peroxide homeostasis of the organism when KatG activity is low or absent.     

A novel antioxidant gene from Mycobacterium tuberculosis

Among the major antimicrobial products of macrophages are reactive intermediates of the oxidation of nitrogen (RNI) and the reduction of oxygen (ROI). Selection of recombinants in acidified nitrite led to the cloning of a novel gene, noxR1, from a pathogenic clinical isolate of Mycobacterium tuberculosis. Expression of noxR1 conferred upon Escherichia coli and Mycobacterium smegmatis enhanced ability to resist RNI and ROI, whether the bacteria were exposed to exogenous compounds in medium or to endogenous products in macrophages. These studies provide the first identification of an RNI resistance mechanism in mycobacteria, point to a new mechanism for resistance to ROI, and raise the possibility that inhibition of the noxR1 pathway might enhance the ability of macrophages to control tuberculosis.     

Characterization of the overproduced NADH dehydrogenase fragment of the NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli

The proton-pumping NADH:ubiquinone oxidoreductase of Escherichia coli is composed of 14 different subunits and contains one FMN and up to nine iron-sulfur clusters as prosthetic groups. By use of salt treatment, the complex can be split into an NADH dehydrogenase fragment, a connecting fragment and a membrane fragment. The water-soluble NADH dehydrogenase fragment has a molecular mass of approximately 170,000 Da and consists of the subunits NuoE, F, and G. The fragment harbors the FMN and probably six iron-sulfur clusters, four of them being observable by EPR spectroscopy. Here, we report that the fully assembled fragment can be overproduced in E. coli when the genes nuoE, F, and G were simultaneously overexpressed with the genes nuoB, C, and D. Furthermore, riboflavin, sodium sulfide, and ferric ammonium citrate have to be added to the culture medium. The fragment was purified from the cytoplasm by means of ammonium sulfate fractionation and chromatographic steps. The preparation contains one noncovalently bound FMN per molecule. Two binuclear (N1b and N1c) and two tetranuclear (N3 and N4) iron-sulfur clusters were detected by EPR in the NADH reduced preparation with spectral characteristics identical with those of the corresponding clusters in complex I. The preparation fulfills all prerequisites for crystallization of the fragment.     

Crystal structure of the iron-dependent regulator (IdeR) from Mycobacterium tuberculosis shows both metal binding sites fully occupied

Iron-dependent regulators are a family of metal-activated DNA binding proteins found in several Gram-positive bacteria. These proteins are negative regulators of virulence factors and of proteins of bacterial iron-uptake systems. In this study we present the crystal structure of the iron-dependent regulator (IdeR) from Mycobacterium tuberculosis, the causative agent of tuberculosis. The protein crystallizes in the hexagonal space group P62 with unit cell dimensions a=b=92.6 A, c=63.2 A. The current model comprises the N-terminal DNA-binding domain (residues 1-73) and the dimerization domain (residues 74-140), while the third domain (residues 141-230) is too disordered to be included. The molecule lies on a crystallographic 2-fold axis that generates the functional dimer. The overall structure of the monomer shares many features with the homologous regulator, diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae. The IdeR structure in complex with Zinc reported here is, however, the first wild-type repressor structure with both metal binding sites fully occupied. This crystal structure reveals that both Met10 and most probably the Sgamma of Cys102 are ligands of the second metal binding site. In addition, there are important changes in the tertiary structure between apo-DtxR and holo-IdeR bringing the putative DNA binding helices closer together in the holo repressor. The mechanism by which metal binding may cause these structural changes between apo and holo wild-type repressor is discussed.     

Mechanisms involved in the intrinsic isoniazid resistance of Mycobacterium avium

Isoniazid (INH), which acts by inhibiting mycolic acid biosynthesis, is very potent against the tuberculous mycobacteria. It is about 100-fold less effective against Mycobacterium avium. This difference has often been attributed to a decreased permeability of the cell wall. We measured the rate of conversion of radiolabelled INH to 4-pyridylmethanol by whole cells and cell-free extracts and estimated the permeability barrier imposed by the cell wall to INH influx in Mycobacterium tuberculosis and M. avium. There was no significant difference in the relative permeability to INH between these two species. However, the total conversion rate in M. tuberculosis was found to be four times greater. Examination of in vitro-generated mutants revealed that the major resistance mechanism for both species is loss of the catalase-peroxidase KatG. Analysis of lipid and protein biosynthetic profiles demonstrated that the molecular target of activated INH was identical for both species. M. avium, however, formed colonies at INH concentrations inhibitory for mycolic acid biosynthesis. These mycolate-deficient M. avium exhibited altered colony morphologies, modified cell wall ultrastructure and were 10-fold more sensitive to treatment with hydrophobic antibiotics, such as rifampin. These findings may significantly impact the design of new therapeutic regimens for the treatment of infections with atypical mycobacteria.     

Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence

Countless millions of people have died from tuberculosis, a chronic infectious disease caused by the tubercle bacillus. The complete genome sequence of the best-characterized strain of Mycobacterium tuberculosis, H37Rv, has been determined and analysed in order to improve our understanding of the biology of this slow-growing pathogen and to help the conception of new prophylactic and therapeutic interventions. The genome comprises 4,411,529 base pairs, contains around 4,000 genes, and has a very high guanine + cytosine content that is reflected in the biased amino-acid content of the proteins. M. tuberculosis differs radically from other bacteria in that a very large portion of its coding capacity is devoted to the production of enzymes involved in lipogenesis and lipolysis, and to two new families of glycine-rich proteins with a repetitive structure that may represent a source of antigenic variation.     

Bactericidal action of ofloxacin, sulbactam-ampicillin, rifampin, and isoniazid on logarithmic- and stationary-phase cultures of Mycobacterium tuberculosis

The bactericidal actions of ofloxacin and sulbactam-ampicillin, alone and in combination with rifampin and isoniazid, on exponential-phase and stationary-phase cultures of a drug-susceptible isolate of Mycobacterium tuberculosis were studied in vitro. In exponential-phase cultures, all drugs were bactericidal, with the higher concentrations of ofloxacin (5 micrograms/ml) and sulbactam-ampicillin (15 micrograms of ampicillin per ml) being as bactericidal as 1 microgram of isoniazid per ml or 1 microgram of rifampin per ml. In two-drug combinations, both drugs increased the levels of activity of isoniazid and rifampin and were almost as bactericidal as isoniazid-rifampin; they also appeared to increase the level of activity of isoniazid-rifampin in three-drug combinations. In contrast, ofloxacin and sulbactam-ampicillin had little bactericidal activity against stationary-phase cultures and were less active than isoniazid or rifampin alone. Furthermore, in two-drug or three-drug combinations, they did not increase the level of activity of isoniazid, rifampin, or isoniazid-rifampin. These findings suggest that ofloxacin and sulbactam-ampicillin are likely to be most useful in the early stages of treatment and in preventing the emergence of resistance to other drugs but are unlikely to be effective as sterilizing drugs helping to kill persisting lesional bacilli.     

Flow cytometric testing of susceptibilities of Mycobacterium tuberculosis isolates to ethambutol, isoniazid, and rifampin in 24 hours

Susceptibility testing of Mycobacterium tuberculosis is seriously limited by the time required to obtain results. We show that susceptibility testing of clinical isolates of M. tuberculosis can be accomplished rapidly with acceptable accuracy by using flow cytometry. The susceptibilities of 35 clinical isolates of M. tuberculosis to various concentrations of isoniazid, rifampin, and ethambutol were tested by the agar proportion method and by flow cytometry. Agreement between the results from the two methods was 95, 92, and 83% for isoniazid, ethambutol, and rifampin, respectively. Only 11 discrepancies were detected among 155 total tests. The results of flow cytometric susceptibility tests were available within 24 h of inoculation of drug-containing medium, while the proportion method required 3 weeks to complete. The flow cytometric method is also simple to perform.     

Mechanisms of isoniazid resistance in Mycobacterium tuberculosis: enzymatic characterization of enoyl reductase mutants identified in isoniazid-resistant clinical isolates

Hepatocyte growth factor (HGF) decreases transforming growth factor beta1 (TGFbeta1) levels in the liver and attenuates hepatic fibrosis caused by dimethylnitrosamine in rats. In the liver, HGF is presumed to act predominantly on parenchymal cells, and TGFbeta1 is produced mainly by mesenchymal cells. In hepatic fibrosis, stellate cells play a central role with undergoing activation, which also occurs when the cells are cultured on plastic. Thus, we wondered if HGF could act directly on stellate cells. c-Met was detected in rat stellate cells activated by culture for 10 days, but not in the cells cultured for 3 days. Specific binding of HGF to the activated cells was determined, and Scatchard analysis indicated an apparent Kd of 1.5 nM. c-Met mRNA was detected in freshly isolated stellate cells from rats treated with carbon tetrachloride for 8 weeks, but not in those cells from normal rats. These results indicate that stellate cells express c-met when activated in vitro and in vivo. HGF enhanced TGFbeta1 production and DNA synthesis in the activated cells.     

Oligonucleotide (GTG)5 as a marker for Mycobacterium tuberculosis strain identification

Culture of Mycobacterium tuberculosis provides no information on the identity of a strain or the distribution of such a strain in the community. Strain identification of M. tuberculosis can help to address important epidemiological questions, e.g., the origin of an infection in a patient's household or community, whether reactivation of infection is endogenous or exogenous in origin, and the spread and early detection of organisms with acquired antibiotic resistance. To research this problem, strain identification must be reliable and accurate. Although genetic identification techniques already exist, it is valuable to have genetic identification techniques based on a number of genetic markers to improve the accurate identification of M. tuberculosis strains. We show that oligonucleotide (GTG)5 can be successfully applied to the identification of M. tuberculosis strains. This technique may be particularly useful in cases in which M. tuberculosis strains have few or no insertion elements (e.g., IS6110) or in identifying other strains of mycobacteria when informative probes are lacking.     

Detection of Mycobacterium tuberculosis DNA in lobular granulomatous panniculitis (erythema induratum-nodular vasculitis)

The antimicrobial spectrum of azithromycin and clarithromycin suggests a number of further uses for these newer macrolides. Favorable clinical and bacteriologic responses have been reported with both antibiotics in children with community-acquired pneumonia. Response rates were high for overall patient populations and for subgroups with infection caused by Mycoplasma pneumoniae and Chlamydia pneumoniae. Treatment with azithromycin or clarithromycin has resulted in a reduction in mycobacteremia and an improvement in clinical symptoms in adult AIDS patients with disseminated Mycobacterium avium-intracellulare complex. Prophylactic treatment with azithromycin may prevent M. avium-intracellulare complex, especially when combined with rifabutin. Preliminary evidence suggests that both azithromycin and clarithromycin in multidrug combinations may effectively eradicate Helicobacter pylori and that azithromycin may be useful in treating bacterial gastritis caused by Campylobacter species. Trachoma and infections caused by Bordetella pertussis and Ureaplasma urealyticum are other possible future indications for the newer macrolides. Limited clinical evidence also suggests that azithromycin may be effective in the prevention and treatment of malaria.     

Mycobacterium tuberculosis and the complement system

Accumulated evidence to date confirms the importance of the C3-CR pathway in the phagocytosis of pathogenic mycobacteria. Detailed receptor-ligand studies for phagocytosis are creating the framework to test the hypothesis that the entry pathway for these bacteria influences the immediate host cell response and their intracellular fate. These types of study are particularly important for improving our understanding of the outcome of primary infection in humans, where the number of bacilli is presumed to be very low.     

Distribution of antibody titres against phenolic glycolipids from Mycobacterium tuberculosis in the sera from tuberculosis patients and healthy controls

We have shown that the cytotoxic response of TNF-sensitive L929 cells and TNF-resistant EMT-6 cells to TNF-alpha can be modulated by ADP-ribosylation inhibitors independently of ADP-ribosylation rates. To explore the possibility that these inhibitors modulate TNF cytotoxicity by interfering with cellular protective mechanisms, we evaluated their effects on general RNA synthesis and on mRNA expression of two proposed protective genes, manganous superoxide dismutase (MnSOD) and heat shock protein 70 (hsp70). We found that ADP-ribosylation inhibitors could inhibit general RNA synthesis in a dose-dependent fashion to a similar extent in both EMT-6 and L929 cells, although these inhibitors increased or decreased the sensitivity of the cells to TNF, respectively. In EMT-6 cells, combination of actinomycin D with these inhibitors further inhibited the RNA synthesis rate, and it actually decreased the TNF sensitivity of the EMT-6 cells. Furthermore, the expression of MnSOD or hsp70 was not regulated by these inhibitors. Thus, TNF resistance must depend on other mechanisms in addition to the expression of these protective genes.     

Serological analysis of C-terminal region of alpha antigen from Mycobacterium avium-intracellulare complex and Mycobacterium tuberculosis

The alpha antigen, which is a 30 kDa protein secreted by mycobacterial species, is an immunodominant antigen. The C-terminal regions of alpha antigens are highly divergent, though there are regions where the amino acid sequence of alpha antigen is conserved. We investigated whether the C-terminal regions of the Mycobacterium avium alpha antigen, M. intracellulare alpha antigen and M. tuberculosis alpha antigen contain sequence-specific B-cell epitopes. The C-terminal regions of M. avium alpha antigen and M. intracelluare alpha antigen reacted to anti-M. avium alpha antigen but not to anti-M. tuberculosis alpha antigen derived from rabbits. Thus, M. avium and M. intracellulare have an antigenic determinant in common with rabbit. The C-terminal region of M. tuberculosis alpha antigen did not react to anti-M. avium alpha antigen or anti-M. tuberculosis alpha antigen. An enzyme-linked immunosorbent assay revealed that only the C-terminal region of M. avium alpha antigen reacted to the sera of two of six patients with M. avium-intracellulare (MAC) but not to the sera of patients with M. tuberculosis. In contrast, the C-terminal regions of M. intracellulare alpha antigen and M. tuberculosis alpha antigen were not recognized by the sera from patients with MAC or M. tuberculosis. This region of M. avium alpha antigen can produce a sequence-specific B-cell epitope in humans.     

PCR-based rapid detection of Mycobacterium tuberculosis in blood from immunocompetent patients with pulmonary tuberculosis

A PCR test based on insertion sequence IS1081 was developed to detect Mycobacterium tuberculosis complex organisms in the peripheral blood. The method was applied to blood samples from immunocompetent individuals with localized pulmonary tuberculosis. Seven of 16 (43.75%) blood samples were found to be positive for the circulating DNA copies of M. tuberculosis complex.