DNA microarrays and combinatorial chemical libraries: tools for the drug discovery pipeline.
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Antitubercular Agents↗
Biomedical subjects
C E Barry
Publications and source records attributed to C E Barry.
Mycobacterium tuberculosis CDC1551 induces a more vigorous host response in vivo and in vitro, but is not more virulent than other clinical isolates.
Mycobacterium tuberculosis CDC1551, a clinical isolate reported to be hypervirulent and to grow faster than other isolates, was compared with two other clinical isolates (HN60 and HN878) and two laboratory strains (H37Rv and Erdman). The initial (1-14 days) growth of CDC1551, HN60, HN878, and H37Rv was similar in the lungs of aerosol-infected mice, but growth of Erdman was slower. Thereafter, the growth rate of CDC1551 decreased relative to the other strains which continued to grow at comparable rates up to day 21. In the lungs of CDC1551-infected mice, small well-organized granulomas with high levels of TNF-alpha, IL-6, IL-10, IL-12, and IFN-gamma mRNA were apparent sooner than in lungs of mice infected with the other strains. CDC1551-infected mice survived significantly longer. These findings were confirmed in vitro. The growth rates of H37Rv and CDC1551 in human monocytes were the same, but higher levels of TNF-alpha, IL-10, IL-6, and IL-12 were induced in monocytes after infection with CDC1551 or by exposure of monocytes to lipid fractions from CDC1551. CD14 expression on the surface of the monocytes was up-regulated to a greater extent by exposure to the lipids of CDC1551. Thus, CDC1551 is not more virulent than other M. tuberculosis isolates in terms of growth in vivo and in vitro, but it induces a more rapid and robust host response.
Aerosols↗
AhpC, oxidative stress and drug resistance in Mycobacterium tuberculosis.
The Mycobacterium tuberculosis AhpC is similar to a family of bacterial and eukaryotic antioxidant proteins with alkylhydroperoxidase (Ahp) and thioredoxin-dependent peroxidase (TPx) activities. AhpC expression is associated with resistance to the front-line antitubercular drug isoniazid in the naturally resistant organisms E. coli and M. smegmatis. We identified several isoniazid-resistant M. tuberculosis isolates with ahpC promoter mutations resulting in AhpC overexpression. These strains were more resistant to cumene hydroperoxide than were wild-type strains. However, these strains were unchanged in their sensitivity to isoniazid, refuting a role for AhpC in detoxification of this drug. All the isoniazid-resistant, AhpC-overexpressing strains were also deficient in activity of the mycobacterial catalase-peroxidase KatG. KatG, the only known catalase in M. tuberculosis, is required for activation of isoniazid. We propose that compensatory ahpC promoter mutations are selected from KatG-deficient, isoniazid-resistant M. tuberculosis during infections, to mitigate the added burden imposed by organic peroxides on these strains.
Amino Acid Sequence↗
Mycobacterium tuberculosis catalase and peroxidase activities and resistance to oxidative killing in human monocytes in vitro.
Mycobacterium tuberculosis has a relatively high resistance to killing by hydrogen peroxide and organic peroxides. Resistance may be mediated by mycobacterial catalase-peroxidase (KatG) and possibly by alkyl hydroperoxide reductase (AhpC). To determine the interrelationship between sensitivity to H2O2, catalase and peroxidase activities, and bacillary growth rates measured both intracellularly in human monocytes and in culture medium, we examined one laboratory strain, two clinical isolates, and three recombinant strains of M. tuberculosis with differing levels of KatG and AhpC. Five of the mycobacterial strains had intracellular doubling times of 27 to 32 h, while one KatG-deficient clinical isolate (ATCC 35825) doubled in approximately 76 h. Killing of mycobacteria by exogenously added H2O2 was more pronounced for intracellular bacilli than for those bacilli derived from disrupted monocytes. Strains with no detectable KatG expression or catalase activity were relatively sensitive to killing (43 to 67% killing) by exogenous H2O2. However, once even minimal catalase activity was present, mycobacterial catalase activity over a 10-fold range (0.56 to 6.2 U/mg) was associated with survival of 85% of the bacilli. Peroxidase activity levels correlated significantly with resistance of the mycobacterial strains to H2O2-mediated killing. An endogenous oxidative burst induction by 4beta-phorbol 12beta-myristate 13alpha-acetate treatment of infected monocytes reduced the viability of the KatG null strain (H37Rv Inhr) but not the KatG-overexpressing strain [H37Rv(pMH59)]. These results suggest that mycobacterial resistance to oxidative metabolites (including H2O2 and other peroxides) may be an important mechanism of bacillary survival within the host phagocyte.
Bacterial Proteins↗
Iron acquisition and metabolism by mycobacteria.
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Humans↗
The biosynthesis of mycolic acids in Mycobacterium tuberculosis. Enzymatic methyl(ene) transfer to acyl carrier protein bound meromycolic acid in vitro.
A closely related family of enzymes from Mycobacterium tuberculosis has been shown by heterologous expression to catalyze the modification of mycolic acids through the addition of a methyl (or methylene) group derived from S-adenosyl-L-methionine (SAM). Overproduction of all six of these enzymes in Escherichia coli and subsequent in vitro reactions with heat-inactivated acceptor fractions derived from Mycobacterium smegmatis in the presence of [methyl-3H]SAM demonstrated that the immediate substrate to which methyl group addition occurs was a family of very long-chain fatty acids. Inhibitors of methyl transfer, such as S-adenosyl-L-homocysteine and sinefungin, were shown to inhibit this reaction but had no effect on whole cells of either M. smegmatis or M. tuberculosis. Purified mycolic acids from M. tuberculosis were pyrolyzed, and the resulting meroaldehyde was oxidized and methylated to produce full-length methyl meromycolates. These esters were shown to comigrate with a fraction of the acceptor from the in vitro reactions, suggesting that methyl group addition occurs up to the level of the meromycolate. Protease and other treatments destroyed the activity of the acceptor fraction, which was also found to be extremely sensitive to basic pH. Antibody to the acyl carrier protein AcpM, which has recently been shown to be the carrier of full-length meromycolate produced by a unique type II fatty acid synthase system, inhibited the cell-free methyl(en)ation of these acids. These results suggest that mycolate modification reactions occur parallel with the synthesis of the AcpM-bound meromycolate chain.
Acyl Carrier Protein↗
The 16-kDa alpha-crystallin (Acr) protein of Mycobacterium tuberculosis is required for growth in macrophages.
Although the 16-kDa alpha-crystallin homologue of Mycobacterium tuberculosis (MTB) is the dominant protein produced by stationary phase cultures in vitro, it is undetectable in logarithmically growing cultures. By growing bacilli at defined oxygen concentrations, acr transcription was shown to be strongly induced by mildly hypoxic conditions. Acr expression also was found to be induced during the course of in vitro infection of macrophages. The acr gene was replaced with a hygromycin resistance cassette by allelic exchange in MTB H37Rv. The resulting Deltaacr::hpt strain was shown to be equivalent to wild-type H37Rv in in vitro growth rate and infectivity but was significantly impaired for growth in both mouse bone marrow derived macrophages and THP-1 cells. In addition to its proposed role in maintenance of long-term viability during latent, asymptomatic infections, these results establish a role for the Acr protein in replication during initial MTB infection.
Animals↗
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.
Chromosome Mapping↗
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.
3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
Mechanisms of isoniazid resistance in Mycobacterium tuberculosis.
Isoniazid (INH) is a widely used front-line antituberculous agent with bacteriocidal activity at concentrations as low as 150 nM against Mycobacterium tuberculosis. INH is a prodrug and requires activation by an endogenous mycobacterial enzyme, the catalase-peroxidase KatG, before exerting toxic effects on cellular targets. Resistance to INH develops primarily through failure to activate the prodrug due to point mutations in the katG gene. In addition to mutations in katG, mutations in several other loci, such as the alkylhydroperoxidase AhpC and the enoylreductase InhA, may contribute to INH resistance. Although these markers can be used to accurately predict clinical INH resistance in a large number of cases, the molecular mechanisms involved remain largely speculative and incomplete.
Journal Article↗
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.
Bacterial Proteins↗
The effect of oxygenated mycolic acid composition on cell wall function and macrophage growth in Mycobacterium tuberculosis.
There are three major structural classes of mycolic acids in the cell envelope of Mycobacterium tuberculosis (MTB): alpha-, methoxy- and ketomycolate. The two oxygen-containing classes are biosynthetically related through a common alpha-methyl hydroxymycolate intermediate. BCG strains that fail to produce methoxymycolate and instead produce only keto- and alpha-mycolic acids show apparent defects in the O-methyltransferase MMAS-3. Overproduction of MMAS-3 from MTB resulted in a complete replacement of ketomycolate by methoxymycolate in both BCG and MTB. In vitro growth of these recombinant strains lacking ketomycolate was impaired at reduced temperatures but appeared to be normal at 37 degrees C. Glucose uptake was significantly decreased in such strains, but uptake of chenodeoxycholate and glycine was unaffected. Although sensitivity to INH remained unchanged, these cells were found to be hypersensitive to ampicillin and rifampicin. Infectivity of BCG and H37Rv wild type or MMAS-3 overproducers in THP-1 cells was somewhat affected, but the ability of the strains lacking ketomycolate to grow within this macrophage-like cell line was severely compromised. In vivo labelling of mycolic acids during growth of H37Rv within THP-1 cells revealed a substantial increase in ketomycolate and alphamycolate synthesized by intracellularly grown mycobacteria. These results establish a critical role for mycolate composition in proper cell wall function during the growth of MTB in vivo.
Amino Acid Sequence↗
New horizons in the treatment of tuberculosis.
The development of new chemotherapy for the treatment of tuberculosis has three major objectives: first, the development of faster-acting drugs to shorten the duration of treatment; second, the development of novel antimicrobials to counter the emergence of bacteria resistant to current therapies; and, third, the development of chemotherapeutics that specifically target dormant bacilli to treat the one-third of the world's population latently infected with tubercle bacilli. Strategies based upon optimizing the inhibition of known targets require an extensive knowledge of the detailed mechanism of action of current antimycobacterial agents. For many agents such as isoniazid, ethambutol, rifampin, and pyrazinamide such knowledge is now available. Strategies based upon the identification of novel targets will necessitate the identification of biochemical pathways specific to mycobacteria and related organisms. Many unique metabolic processes occur during the biosynthesis of mycobacterial cell wall components, and some attractive new targets have emerged. The development of targets specific to latency will require a detailed picture of the metabolism and biochemical pathways occurring in dormant bacilli. Recent evidence suggests that anaerobic metabolic pathways may operate in dormant bacilli, and the enzymes involved in such pathways may also provide significant new targets for intervention. The combination of the mycobacterial genome sequence that is anticipated to become available this year with an improved understanding of the unique metabolic processes that define mycobacteria as a genus offers the greatest hope for the elimination of one of mankind's oldest enemies.
Animals↗
MMAS-1, the branch point between cis- and trans-cyclopropane-containing oxygenated mycolates in Mycobacterium tuberculosis.
The proportion of mycolic acid containing trans-substituents at the proximal position of the meromycolate chain is an important determinant of fluidity of the mycobacterial cell wall and is directly related to the sensitivity of mycobacterial species to hydrophobic antibiotics. MMAS-1, an enzyme encoded in the gene cluster responsible for the biosynthesis of methoxymycolates, was overexpressed in Mycobacterium tuberculosis and shown to result in the overproduction of trans-cyclopropane and trans-olefin-containing oxygenated mycolic acids. MMAS-1 converted a cis-olefin into a trans-olefin with concomitant introduction of an allylic methyl branch in a precursor to both the methoxy and ketone-containing mycolic acids. In addition to an increase in the amount of trans-mycolate, MMAS-1 expression resulted in a substantial increase in the amount of ketomycolate produced relative to methoxymycolate. Thus MMAS-1 may act at a complex branch point where expression of this enzyme directly affects the cis- to trans-ratio and indirectly affects the keto to methoxy ratio. Overexpression of MMAS-1 resulted in a substantially slower growth rate at moderately elevated temperature, decreased thermal stability of the cell wall as measured by differential scanning calorimetry, and an increased permeability to chenodeoxycholate. These results provide experimental evidence for the intermediacy of trans-olefinic mycolate precursors in trans-cyclopropane formation and suggest that increasing the proportion of the polar ketomycolate subclass may exert a significant fluidizing effect on the cell wall.
Bacterial Proteins↗
Mycolic acid structure determines the fluidity of the mycobacterial cell wall.
The low permeability of the mycobacterial cell wall is thought to contribute to the well known resistance of mycobacteria to antibiotics and chemotherapeutic agents. We have used differential scanning calorimetry to demonstrate that the high temperature phase transition observed in purified cell walls, usually in the 60-70 degrees C range, suggestive of a lipid environment of extremely low fluidity, can also be observed in whole organisms and in cell walls from which much of the free lipids was removed by extraction with Triton X-114. A survey of seven mycobacterial species demonstrated that this high temperature transition was a general property of these organisms. Cell walls isolated from two Corynebacterium species, which contain much shorter corynemycolic acids, displayed a much lower temperature transition, suggesting that the transition temperature was directly correlated to the length of mycolic acid. Methyl esters of mycolic acids were found to have a phase transition temperature that was linearly related to the amount of trans-mycolate. Both Mycobacterium avium and M. smegmatis responded to increasing growth temperature by increasing the proportion of trans-mycolate and displaying a correspondingly higher melting temperature. Whole cells of M. smegmatis grown at higher temperature allowed a less rapid influx of two lipophilic agents, norfloxacin and chenodeoxycholate. These results provide strong evidence that the nature of mycolic acid plays a crucial role in determining the fluidity and permeability of mycobacterial cell wall.
Calorimetry, Differential Scanning↗
A common mechanism for the biosynthesis of methoxy and cyclopropyl mycolic acids in Mycobacterium tuberculosis.
Mycobacterium tuberculosis produces three classes of mycolic acids that differ primarily in the presence and nature of oxygen-containing substituents in the distal portion of the meromycolate branch. The methoxymycolate series has a methoxy group adjacent to a methyl branch, in addition to a cyclopropane in the proximal position. Using the gene for the enzyme that introduces the distal cyclopropane (cma1) as a probe, we have cloned and sequenced a cluster of genes coding for four highly homologous methyl transferases (mma1-4). When introduced into Mycobacterium smegmatis, this gene cluster conferred the ability to synthesize methoxymycolates. By determining the structure of the mycolic acids produced following expression of each of these genes individually and in combination, we have elucidated the biosynthetic steps responsible for the production of the major series of methoxymycolates. The mma4 gene product (MMAS-4) catalyzes an unusual S-adenosyl-L-methionine-dependent transformation of the distal cis-olefin into a secondary alcohol with an adjacent methyl branch. MMAS-3 O-methylates this secondary alcohol to form the corresponding methyl ether, and MMAS-2 introduces a cis-cyclopropane in the proximal position of the methoxy series. The similarity of these reactions and the enzymes that catalyze them suggests that some of the structural diversity of mycolic acids results from different chemical fates of a common cationic intermediate, which in turn results from methyl group addition to an olefinic mycolate precursor.
Amino Acid Sequence↗
Compensatory ahpC gene expression in isoniazid-resistant Mycobacterium tuberculosis.
Mutations that eliminate KatG catalase-peroxidase activity prevent activation of isoniazid and are a major mechanism of resistance to this principal drug for the treatment of Mycobacterium tuberculosis infections. However, the loss of KatG activity in clinical isolates seemed paradoxical because KatG is considered an important factor for the survival of the organism. Expression of either KatG or the recently identified alkyl hydroperoxidase AhpC was sufficient to protect bacilli against the toxic effects of organic peroxides. To survive during infection, isoniazid-resistant KatG mutants have apparently compensated for the loss of KatG catalase-peroxidase activity by a second mutation, resulting in hyperexpression of AhpC.
Amino Acid Sequence↗
Isolation and characterization of the versicolorin B synthase gene from Aspergillus parasiticus. Expansion of the aflatoxin b1 biosynthetic gene cluster.
Versicolorin B synthase catalyzes the side chain cyclization of racemic versiconal hemiacetal to the bisfuran ring system of(-)-versicolorin B, an essential transformation in the aflatoxin biosynthetic pathway of Aspergillus parasiticus. The dihydrobisfuran is key to the mutagenic nature of aflatoxin B1. The protein, which shows 58% similarity and 38% identity with glucose oxidase from Aspergillus niger, possesses an amino-terminal sequence homologous to the ADP-binding region of other flavoenzymes. However, this enzyme does not require flavin or nicotinamide cofactors for its cyclase activity. The 643-amino acid native enzyme contains three potential sites for N-linked glycosylation, Asn-Xaa-Thr or Asn-Xaa-Ser. The cDNA and genomic clones of versicolorin B synthase were isolated by screening the respective libraries with random-primed DNA probes generated from an exact copy of an internal vbs sequence. This probe was created through polymerase chain reaction by using nondegenerate polymerase chain reaction primers derived from the amino acid sequences of peptide fragments of the enzyme. The 1985-base genomic vbs DNA sequence is interrupted by one intron of 53 nucleotides. Southern blotting, nucleotide sequencing, and detailed restriction mapping of the vbs-containing genomic clones revealed the presence of omtA, a methyltransferase active in the biosynthesis, 3.3 kilobases upstream of vbs and oriented in the opposite direction from vbs. The presence of omtA in close proximity to vbs supports the theory that the genes encoding the aflatoxin biosynthetic enzymes in A. parasiticus are clustered.
Aflatoxin B1↗