PubMed Health⌕ Search

Biomedical subjects

C E Barry

Publications and source records attributed to C E Barry.

At least 19 recordsLinked to original sources
PubMed2002

Inactivation of the Mycobacterium tuberculosis Nramp orthologue (mntH) does not affect virulence in a mouse model of tuberculosis.

Mycobacterium tuberculosis is an intracellular pathogen which can survive and multiply within the phagosomal compartment of the macrophage, and in doing so has to withstand the various macrophage defense mechanisms, which include limitation of iron and other metals. Analysis of the complete genome sequence of M. tuberculosis revealed an extensive array of cation transporters, including mntH, an orthologue of the eukaryotic Nramp (natural resistance-associated macrophage protein) gene, that encodes a proton-dependent divalent metal transporter. To assess the effect of this transporter on intracellular survival and pathogenesis, an mntH knock-out mutant of M. tuberculosis H37Rv was created and assayed in bone marrow-derived macrophages and in a murine model of tuberculosis. In neither of these systems was any loss of fitness associated with inactivation of mntH, demonstrating that Nramp orthologues are not important determinants of mycobacterial virulence.

Animals↗
PubMed2002

The role of KasA and KasB in the biosynthesis of meromycolic acids and isoniazid resistance in Mycobacterium tuberculosis.

Mycobacterium tuberculosis has two discrete beta-ketoacyl synthases encoded by kasA and kasB that are located in tandem within a five-gene operon that has been implicated in isoniazid-sensitivity and mycolic acid synthesis. We have developed an in vitro meromycolic acid synthase assay to elucidate the anabolic role of these enzymes. Overproduction of KasA and KasB individually and together in M. smegmatis enabled cell-free incorporation of [(14)C]malonyl-CoA into lipids whose chain length was dependent upon the M. tuberculosis elongating enzyme used. KasA specifically elongated palmitoyl-CoA to monounsaturated fatty acids that averaged 40 carbons in length. KasB hyperproduction in the presence of KasA produced longer chain multiunsaturated hydrocarbons averaging 54 carbons in length. These products comigrated with a synthetic standard of meromycolic acid and their production was sensitive to isoniazid, thiolactomycin, and triclosan. KasA mutations associated with isoniazid resistance produced an enzyme that had a diminished overall catalytic activity but conferred enhanced resistance to isoniazid. In vivo analysis confirmed that overexpression of each of the four mutant KasAs enhanced isoniazid resistance when compared to overexpression of wild-type KasA. These results suggest discrete anabolic roles for both KasA and KasB in mycolic acid synthesis and substantiate the involvement of KasA mutations in isoniazid resistance.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed2001

Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-alpha /beta.

To understand how virulent mycobacteria subvert host immunity and establish disease, we examined the differential response of mice to infection with various human outbreak Mycobacterium tuberculosis clinical isolates. One clinical isolate, HN878, was found to be hypervirulent, as demonstrated by unusually early death of infected immune-competent mice, compared with infection with other clinical isolates. The differential effect on survival required lymphocyte function because severe combined immunodeficiency (SCID) mice infected with HN878 or other clinical isolates all died at the same rate. The hypervirulence of HN878 was associated with failure to induce M. tuberculosis-specific proliferation and IFN-gamma production by spleen and lymph node cells from infected mice. In addition, 2- to 4-fold lower levels of tumor necrosis factor-alpha (TNF-alpha), IL-6, IL-12, and IFN-gamma mRNAs were observed in lungs of HN878-infected mice. IL-10, IL-4, and IL-5 mRNA levels were not significantly elevated in lungs of HN878 infected mice. In contrast, IFN-alpha mRNA levels were significantly higher in lungs of these mice. To further investigate the role of Type 1 IFNs, mice infected with HN878 were treated intranasally with purified IFN-alpha/beta. The treatment resulted in increased lung bacillary loads and even further reduced survival. These results suggest that the hypervirulence of HN878 may be due to failure of this strain to stimulate Th1 type immunity. In addition, the lack of development of Th1 immunity in response to HN878 appears to be associated with increased induction of Type 1 IFNs.

Animals↗
PubMed2001

The specificity of methyl transferases involved in trans mycolic acid biosynthesis in Mycobacterium tuberculosis and Mycobacterium smegmatis.

Trans mycolic acid content is directly related to cell wall fluidity and permeability in mycobacteria. Carbon-13 NMR spectroscopy of mycolic acids isolated from Mycobacterium tuberculosis (MTB) and Mycobacterium smegmatis (MSM) fed 13C-labeled precursor molecules was used to probe the biosynthetic pathways that modify mycolic acids. Heteronuclear correlation spectroscopy (HMQC) of ketomycolic acid from MTB allowed assignment of the complete 13C-NMR spectrum. Incorporation patterns from [1-13C]-acetate and [2-13C]-acetate feeding experiments suggested that the mero chain and alpha branch of mycolic acids are both synthesized by standard fatty acid biosynthetic reactions. [13C-methyl]-L-methionine was used to specifically label carbon atoms derived from the action of the methyl transferases involved in meromycolate modification. To enrich for trans mycolic acids a strain of MTB overexpressing the mma1 gene was labeled. Carbon-carbon coupling was observed in mycolate samples doubly labeled with 13C-acetate and [13C-methyl]-L-methionine and this information was used to assess positional specificity of methyl transfer. In MTB such methyl groups were found to occur exclusively on carbons derived from the 2 position of acetate, while in MSM they occurred only on carbons derived from the 1 position. These results suggest that the MSM methyltransferase MMAS-1 operates in an inverted manner to that of MTB.

Acetates↗
PubMed2001

Interpreting cell wall 'virulence factors' of Mycobacterium tuberculosis.

The complex structure of the cell wall of Mycobacterium tuberculosis clearly contributes to the outcome of the dialogue between this pathogen and its host. The effects of mutations in cell wall components are likely to be quite complex, as individual components of the wall could have indirect effects that extend well beyond the physical integrity of the wall itself. Affected processes include the surface exposure or secretion of the many lipid, glycolipid and proteinaceous molecules that can interact directly with components of the host cell.

Cell Wall↗
PubMed2001

Mycobacterium tuberculosis in the post-genomic age.

Since the publication of the complete genome sequence of Mycobacterium tuberculosis in 1998, there has been a marked intensification and diversification of activities in the field of tuberculosis research. Among the areas that have advanced spectacularly are comparative genomics, functional genomics-notably the study of the transcriptome and proteome - and cell envelope biogenesis, especially as it relates to the mechanism of action of antimycobacterial drugs.

Antigenic Variation↗
PubMed2001

Preclinical candidates and targets for tuberculosis therapy.

Like many neglected diseases of the developing world, tuberculosis (TB) has a thin portfolio of new compounds currently in the discovery pipeline with near-term clinical potential. Co-development of broad-spectrum antibacterials for TB indications is superficially attractive but unlikely to result in significant advances in therapy. Genomic information has been useful in the redesign of second-line antituberculars such as ethambutol and such molecules will likely soon enter preclinical development. New targets and lead compounds with activity against the mycobacterial cell wall and non-replicating bacilli are the subject of current discovery programs.

Anaerobiosis↗
PubMed2000

Ethionamide activation and sensitivity in multidrug-resistant Mycobacterium tuberculosis.

Ethionamide (ETA) is an important component of second-line therapy for the treatment of multidrug-resistant tuberculosis. Synthesis of radiolabeled ETA and an examination of drug metabolites formed by whole cells of Mycobacterium tuberculosis (MTb) have allowed us to demonstrate that ETA is activated by S-oxidation before interacting with its cellular target. ETA is metabolized by MTb to a 4-pyridylmethanol product remarkably similar in structure to that formed by the activation of isoniazid by the catalase-peroxidase KatG. We have demonstrated that overproduction of Rv3855 (EtaR), a putative regulatory protein from MTb, confers ETA resistance whereas overproduction of an adjacent, clustered monooxygenase (Rv3854c, EtaA) confers ETA hypersensitivity. Production of EtaA appears to be negatively regulated by EtaR and correlates directly with [(14)C]ETA metabolism, suggesting that EtaA is the activating enzyme responsible for thioamide oxidation and subsequent toxicity. Coding sequence mutations in EtaA were found in 11 of 11 multidrug-resistant MTb patient isolates from Cape Town, South Africa. These isolates showed broad cross-resistance to thiocarbonyl containing drugs including ETA, thiacetazone, and thiocarlide.

Amino Acid Substitution↗
PubMed2000

A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis.

Mycobacterium tuberculosis, which causes tuberculosis, is the greatest single infectious cause of mortality worldwide, killing roughly two million people annually. Estimates indicate that one-third of the world population is infected with latent M. tuberculosis. The synergy between tuberculosis and the AIDS epidemic, and the surge of multidrug-resistant clinical isolates of M. tuberculosis have reaffirmed tuberculosis as a primary public health threat. However, new antitubercular drugs with new mechanisms of action have not been developed in over thirty years. Here we report a series of compounds containing a nitroimidazopyran nucleus that possess antitubercular activity. After activation by a mechanism dependent on M. tuberculosis F420 cofactor, nitroimidazopyrans inhibited the synthesis of protein and cell wall lipid. In contrast to current antitubercular drugs, nitroimidazopyrans exhibited bactericidal activity against both replicating and static M. tuberculosis. Lead compound PA-824 showed potent bactericidal activity against multidrugresistant M. tuberculosis and promising oral activity in animal infection models. We conclude that nitroimidazopyrans offer the practical qualities of a small molecule with the potential for the treatment of tuberculosis.

Animals↗
PubMed2000

Cell wall structure of a mutant of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids.

A mutant strain of Mycobacterium smegmatis defective in the biosynthesis of mycolic acids was recently isolated (Liu, J., and Nikaido, H. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4011-4016). This mutant failed to synthesize full-length mycolic acids and accumulated a series of long chain beta-hydroxymeromycolates. In this work, we provide a detailed characterization of the localization of meromycolates and of the cell wall structure of the mutant. Thin layer chromatography showed that the insoluble cell wall matrix remaining after extraction with chloroform/methanol and SDS still contained a large portion of the total meromycolates. Matrix-assisted laser desorption/ionization and electrospray ionization mass spectroscopy analysis of fragments arising from Smith degradation of the insoluble cell wall matrix revealed that the meromycolates were covalently attached to arabinogalactan at the 5-OH positions of the terminal arabinofuranosyl residues. The arabinogalactan appeared to be normal in the mutant strain, as analyzed by NMR. Analysis of organic phase lipids showed that the mutant cell wall contained some of the extractable lipids but lacked glycopeptidolipids and lipooligosaccharides. Differential scanning calorimetry of the mutant cell wall failed to show the large cooperative thermal transitions typical of intact mycobacterial cell walls. Transmission electron microscopy showed that the mutant cell wall had an abnormal ultrastructure (without the electron-transparent zone associated with the asymmetric mycolate lipid layer). Taken together, these results demonstrate the importance of mycolic acids for the structural and functional integrity of the mycobacterial cell wall. The lack of highly organized lipid domains in the mutant cell wall explains the drug-sensitive and temperature-sensitive phenotypes of the mutant.

Calorimetry, Differential Scanning↗
PubMed2000

Use of genomics and combinatorial chemistry in the development of new antimycobacterial drugs.

With the completion of the genome of Mycobacterium tuberculosis comes the promise of a new generation of potent drugs to combat the emerging epidemic of multiply drug-resistant isolates. Translating this genomic information into realistic assays, valid targets, and preclinical drug candidates represents the next great hope in tuberculosis control. We propose a paradigm for exploiting the genome to inform the development of novel antituberculars, utilizing the techniques of differential gene expression as monitored by DNA microarrays coupled with the emerging discipline of combinatorial chemistry. A comparison of currently used antituberculars with the properties of other pharmaceuticals suggests that such compounds will have a defined range of physiochemical properties. In general, we can expect the next generation of antituberculars to be small, relatively hydrophilic molecules that bind tightly to specific cellular targets. Many current antimycobacterials require some form of cellular activation (e.g. the activation of isoniazid by a catalase-peroxidase). Activation corresponds to the oxidative, reductive, or hydrolytic unmasking of reactive groups, which occurs with many current antimycobacterial prodrugs. Understanding the mechanisms involved in activation of current antimycobacterial therapeutics also may facilitate the development of alternative activation strategies or of analogs that require no such processes.

Animals↗
PubMed2000

The salicylate-derived mycobactin siderophores of Mycobacterium tuberculosis are essential for growth in macrophages.

Mycobacterium tuberculosis is an important pathogen of mammals that relies on 2-hydroxyphenyloxazoline-containing siderophore molecules called mycobactins for the acquisition of iron in the restrictive environment of the mammalian macrophage. These compounds have been proposed to be biosynthesized through the action of a cluster of genes that include both nonribosomal peptide synthase and polyketide synthase components. One of these genes encodes a protein, MbtB, that putatively couples activated salicylic acid with serine or threonine and then cyclizes this precursor to the phenyloxazoline ring system. We have used gene replacement through homologous recombination to delete the mbtB gene and replace this with a hygromycin-resistance cassette in the virulent strain of M. tuberculosis H37Rv. The resulting mutant is restricted for growth in iron-limited media but grows normally in iron-replete media. Analysis of siderophore production by this organism revealed that the biosynthesis of all salicylate-derived siderophores was interrupted. The mutant was found to be impaired for growth in macrophage-like THP-1 cells, suggesting that siderophore production is required for virulence of M. tuberculosis. These results provide conclusive evidence linking this genetic locus to siderophore production.

Bacterial Proteins↗
PubMed2000

The genetics and biochemistry of isoniazid resistance in mycobacterium tuberculosis.

Although the primary targets of activated isoniazid (INH) are proteins involved in the biosynthesis of cell wall mycolic acids, clinical resistance is dominated by specific point mutations in katG. Mutations associated with target mutations contribute to, but still cannot completely explain, resistance to INH. Despite the wealth of genetic information currently available, the molecular mechanism of cell death induced by INH remains elusive.

Antitubercular Agents↗
PubMed2000

Isoniazid affects multiple components of the type II fatty acid synthase system of Mycobacterium tuberculosis.

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.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed2000

A point mutation in the mma3 gene is responsible for impaired methoxymycolic acid production in Mycobacterium bovis BCG strains obtained after 1927.

BCG vaccines are substrains of Mycobacterium bovis derived by attenuation in vitro. After the original attenuation (1908 to 1921), BCG strains were maintained by serial propagation in different BCG laboratories (1921 to 1961). As a result, various BCG substrains developed which are now known to differ in a number of genetic and phenotypic properties. However, to date, none of these differences has permitted a direct phenotype-genotype link. Since BCG strains differ in their abilities to synthesize methoxymycolic acids and since recent work has shown that the mma3 gene is responsible for O-methylation of hydroxymycolate precursors to form methoxymycolic acids, we analyzed methoxymycolate production and mma3 gene sequences for a genetically defined collection of BCG strains. We found that BCG strains obtained from the Pasteur Institute in 1927 and earlier produced methoxymycolates in vitro but that those obtained from the Pasteur Institute in 1931 and later all failed to synthesize methoxymycolates, and furthermore, the mma3 sequence of the latter strains differs from that of Mycobacterium tuberculosis H37Rv by a point mutation at bp 293. Site-specific introduction of this guanine-to-adenine mutation into wild-type mma3 (resulting in the replacement of glycine 98 with aspartic acid) eliminated the ability of this enzyme to produce O-methylated mycolic acids when the mutant was cloned in tandem with mma4 into Mycobacterium smegmatis. These findings indicate that a point mutation in mma3 occurred between 1927 and 1931, and that this mutant population became the dominant clone of BCG at the Pasteur Institute.

Amino Acid Sequence↗
PubMed2000

The stringent response of Mycobacterium tuberculosis is required for long-term survival.

The stringent response utilizes hyperphosphorylated guanine [(p)ppGpp] as a signaling molecule to control bacterial gene expression involved in long-term survival under starvation conditions. In gram-negative bacteria, (p)ppGpp is produced by the activity of the related RelA and SpoT proteins. Mycobacterium tuberculosis contains a single homolog of these proteins (Rel(Mtb)) and responds to nutrient starvation by producing (p)ppGpp. A rel(Mtb) knockout strain was constructed in a virulent strain of M. tuberculosis, H37Rv, by allelic replacement. The rel(Mtb) mutant displayed a significantly slower aerobic growth rate than the wild type in synthetic liquid media, whether rich or minimal. The growth rate of the wild type was equivalent to that of the mutant when citrate or phospholipid was employed as the sole carbon source. These two organisms also showed identical growth rates within a human macrophage-like cell line. These results suggest that the in vivo carbon source does not represent a stressful condition for the bacilli, since it appears to be utilized in a similar Rel(Mtb)-independent manner. In vitro growth in liquid media represents a condition that benefits from Rel(Mtb)-mediated adaptation. Long-term survival of the rel(Mtb) mutant during in vitro starvation or nutrient run out in normal media was significantly impaired compared to that in the wild type. In addition, the mutant was significantly less able to survive extended anaerobic incubation than the wild-type virulent organism. Thus, the Rel(Mtb) protein is required for long-term survival of pathogenic mycobacteria under starvation conditions.

Cell Line↗