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Clifton E Barry

Publications and source records attributed to Clifton E Barry.

At least 19 recordsLinked to original sources
PubMed2006

Antitubercular nucleosides that inhibit siderophore biosynthesis: SAR of the glycosyl domain.

Tuberculosis is the leading cause of infectious disease mortality in the world by a bacterial pathogen. We previously demonstrated that a bisubstrate inhibitor of the adenylation enzyme MbtA, which is responsible for the second step of mycobactin biosynthesis, exhibited potent antitubercular activity. Here we systematically investigate the structure-activity relationships of the bisubstrate inhibitor glycosyl domain resulting in the identification of a carbocyclic analogue that possesses a KIapp value of 2.3 nM and MIC99 values of 1.56 microM against M. tuberculosis H37Rv. The SAR data suggest the intriguing possibility that the bisubstrate inhibitors utilize a transporter for entry across the mycobacterial cell envelope. Additionally, we report improved conditions for the expression of MbtA and biochemical analysis, demonstrating that MbtA follows a random sequential enzyme mechanism for the adenylation half-reaction.

Adenosine Triphosphate↗
PubMed2006

Design, synthesis, and biological evaluation of beta-ketosulfonamide adenylation inhibitors as potential antitubercular agents.

[reaction: see text] The antitubercular nucleoside antibiotics 1 and 2 were recently described that inhibit the adenylate-forming enzyme MbtA and disrupt biosynthesis of the virulence-conferring siderophore known as mycobactin in Mycobacterium tuberculosis. Herein, we report efforts to refine this inhibitor scaffold by replacing the labile acylsulfamate linkage (highlighted) with the more chemically robust beta-ketosulfonamide linkage of 3 and 4.

Adenosine↗
PubMed2006

Novel route to 5-position vinyl derivatives of thiolactomycin: Olefination vs. deformylation.

Vinyl and diene derivatives of thiolactomycin have been prepared via Horner-Wadsworth-Emmons olefination from protected 5-formyl-3,5-dimethylthiotetronic acid. Several 4-position protecting groups and a variety of phosphonates were evaluated, with MOM protection and beta-ketophosphonates yielding the highest ratio of desired product to deformylated product.

Journal Article↗
PubMed2006

Structure-activity relationships at the 5-position of thiolactomycin: an intact (5R)-isoprene unit is required for activity against the condensing enzymes from Mycobacterium tuberculosis and Escherichia coli.

Thiolactomycin inhibits bacterial cell growth through inhibition of the beta-ketoacyl-ACP synthase activity of type II fatty acid synthases. The effect of modifications of the 5-position isoprenoid side chain on both IC(50) and MIC were determined. Synthesis and screening of a structurally diverse set of 5-position analogues revealed very little tolerance for substitution in purified enzyme assays, but a few analogues retained MIC, presumably through another target. Even subtle modifications such as reducing one or both double bonds of the diene were not tolerated. The only permissible structural modifications were removal of the isoprene methyl group or addition of a methyl group to the terminus. Cocrystallization of these two inhibitors with the condensing enzyme from Escherichia coli revealed that they retained the TLM binding mode at the active site with reduced affinity. These results suggest a strict requirement for a conjugated, planar side chain inserting within the condensing enzyme active site.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed2006

Quantification of small molecule organic acids from Mycobacterium tuberculosis culture supernatant using ion exclusion liquid chromatography/mass spectrometry.

Quantitative analysis of cellular small molecule organic acids of intermediary metabolism can provide critical insight into bacterial metabolic pathways. The concentration of these metabolites in culture supernatant varies at different growth stages or under particular environmental conditions reflecting both the energy and the biosynthetic needs, yielding metabolic information about the microorganism. The method described here utilizes ion exclusion chromatography with formic acid, coupled with a mass-selective detector using selective ion monitoring with negative mode electrospray ionization (SIM ES-), to detect and quantify several small organic acids in culture supernatants. The microM limits of quantitation (LOQs) were found to be 5.5 +/- 0.9 for pyruvate, 7.0 +/- 0.4 for malate, 2.5 +/- 0.5 for succinate, 12.7 +/- 0.8 for lactate, and 6.6 +/- 0.2 for fumarate. The method was used to detect and quantify these acids in the culture supernatants from Mycobacterium tuberculosis. Supernatant samples were spiked with stable-isotope-labeled internal standards, and the organic acids were quantified by isotope ratiometry.

Carboxylic Acids↗
PubMed2006

Mycobacterium leprae is naturally resistant to PA-824.

Leprosy responds very slowly to the current multidrug therapy, and hence there is a need for novel drugs with potent bactericidal activity. PA-824 is a 4-nitroimidazo-oxazine that is currently undergoing phase I clinical trials for the treatment of tuberculosis. The activity of PA-824 against Mycobacterium leprae was tested and compared with that of rifampin in axenic cultures, macrophages, and two different animal models. Our results conclusively demonstrate that PA-824 has no effect on the viability of M. leprae in all three models, consistent with the lack of the nitroimidazo-oxazine-specific nitroreductase, encoded by Rv3547 in the M. leprae genome, which is essential for activation of this molecule.

Animals↗
PubMed2005

Identification of a nitroimidazo-oxazine-specific protein involved in PA-824 resistance in Mycobacterium tuberculosis.

PA-824 is a promising new compound for the treatment of tuberculosis that is currently undergoing human trials. Like its progenitors metronidazole and CGI-17341, PA-824 is a prodrug of the nitroimidazole class, requiring bioreductive activation of an aromatic nitro group to exert an antitubercular effect. We have confirmed that resistance to PA-824 (a nitroimidazo-oxazine) and CGI-17341 (a nitroimidazo-oxazole) is most commonly mediated by loss of a specific glucose-6-phosphate dehydrogenase (FGD1) or its deazaflavin cofactor F420, which together provide electrons for the reductive activation of this class of molecules. Although FGD1 and F420 are necessary for sensitivity to these compounds, they are not sufficient and require additional accessory proteins that directly interact with the nitroimidazole. To understand more proximal events in the reductive activation of PA-824, we examined mutants that were wild-type for both FGD1 and F420 and found that, although these mutants had acquired high-level resistance to PA-824 (and another nitroimidazo-oxazine), they retained sensitivity to CGI-17341 (and a related nitroimidazo-oxazole). Microarray-based comparative genome sequencing of these mutants identified lesions in Rv3547, a conserved hypothetical protein with no known function. Complementation with intact Rv3547 fully restored sensitivity to nitroimidazo-oxazines and restored the ability of Mtb to metabolize PA-824. These results suggest that the sensitivity of Mtb to PA-824 and related compounds is mediated by a protein that is highly specific for subtle structural variations in these bicyclic nitroimidazoles.

Bacterial Proteins↗
PubMed2005

In vivo phenotypic dominance in mouse mixed infections with Mycobacterium tuberculosis clinical isolates.

Clinical isolates of Mycobacterium tuberculosis demonstrate significant heterogeneity in virulence potential in animal models of infection. Isolate CDC1551, for example, has previously been described in mouse survival studies as being hypovirulent, and isolate HN878 has been described as being hypervirulent. Observed differences in this mouse infection experiment have been proposed to reflect differential engagement of the host immune response. To assess whether this is a local or a systemic effect, C57BL/6 mice were infected simultaneously with mixtures of CDC1551 and HN878 in varying ratios and were monitored for mycobacterial growth kinetics, strain proportions during infection, and mouse survival. Strain mixtures that contained primarily HN878 grew more quickly during the first 5 weeks of infection and were more lethal for mice, and HN878 was enriched during in vivo growth. The absolute number of implanted HN878 bacilli at infection correlated inversely with mouse survival and was independent of concomitant infection with CDC1551. In infections of nonactivated mouse macrophages, HN878 grew more quickly. However, phagocyte preactivation reduced and equalized the growth rate of both strains. These results suggest that HN878 exerts a dominant immunosuppressive effect limited to the granuloma in which it is contained.

Animals↗
PubMed2005

Virulence of selected Mycobacterium tuberculosis clinical isolates in the rabbit model of meningitis is dependent on phenolic glycolipid produced by the bacilli.

Infection with Mycobacterium tuberculosis in humans results in active disease in approximately 10% of immune-competent individuals, with the most-severe clinical manifestations observed when the bacilli infect the central nervous system (CNS). Here, we use a rabbit model of tuberculous meningitis to evaluate the severity of disease caused by the M. tuberculosis clinical isolates CDC1551, a highly immunogenic strain, and HN878 or W4, 2 members of the W/Beijing family of strains. Compared with infection with CDC1551, CNS infection with HN878 or W4 resulted in higher bacillary loads in the cerebrospinal fluid and brain, increased dissemination of bacilli to other organs, persistent levels of tumor necrosis factor-alpha , higher leukocytosis, and more-severe clinical manifestations. This pathogenic process is associated with the production by HN878 of a polyketide synthase-derived phenolic glycolipid (PGL), as demonstrated by reduced virulence in rabbits infected with an HN878 mutant disrupted in the pks1-15 gene, which is required for PGL synthesis.

Animals↗
PubMed2005

Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell's endosomal system.

Mycobacterium avium and Mycobacterium tuberculosis are human pathogens that infect and replicate within macrophages. Both organisms live in phagosomes that fail to fuse with lysosomes and have adapted their lifestyle to accommodate the changing environment within the endosomal system. Among the many environmental factors that could influence expression of bacterial genes are the concentrations of single elements within the phagosomes. We used a novel hard x-ray microprobe with suboptical spatial resolution to analyze characteristic x-ray fluorescence of 10 single elements inside phagosomes of macrophages infected with M. tuberculosis and M. avium or with avirulent M. smegmatis. The iron concentration decreased over time in phagosomes of macrophages infected with Mycobacterium smegmatis but increased in those infected with pathogenic mycobacteria. Autoradiography of infected macrophages incubated with (59)Fe-loaded transferrin demonstrated that the bacteria could acquire iron delivered via the endocytic route, confirming the results obtained in the x-ray microscopy. In addition, the concentrations of chlorine, calcium, potassium, manganese, copper, and zinc were shown to differ between the vacuole of pathogenic mycobacteria and M. smegmatis. Differences in the concentration of several elements between M. avium and M. tuberculosis vacuoles were also observed. Activation of macrophages with recombinant IFN-gamma or TNF-alpha before infection altered the concentrations of elements in the phagosome, which was not observed in cells activated following infection. Siderophore knockout M. tuberculosis vacuoles exhibited retarded acquisition of iron compared with phagosomes with wild-type M. tuberculosis. This is a unique approach to define the environmental conditions within the pathogen-containing compartment.

Animals↗
PubMed2005

Tuberculosis - metabolism and respiration in the absence of growth.

Human tuberculosis is a complex disease caused by bacterial populations that are located in discrete lesions (microenvironments) in a single host. Some of these microenvironments are conducive to replication, whereas others restrict bacterial growth without necessarily sterilizing the infecting microorganisms. The physical and biochemical milieu in these lesions is poorly defined. None of the existing animal models for tuberculosis (except perhaps non-human primates) reproduce the diversity of disease progression that is seen in humans. Nonetheless, transcriptomics and studies using bacterial mutants have led to testable hypotheses about metabolic functions that are essential for viability in the absence of replication. A complete picture of bacterial metabolism must balance reducing equivalents while maintaining an energized membrane and basic cellular processes.

Adaptation, Physiological↗
PubMed2005

Contribution of the Mycobacterium tuberculosis MmpL protein family to virulence and drug resistance.

The genome sequence of Mycobacterium tuberculosis revealed the presence of 12 membrane proteins proposed to have a function in the transport of lipids. Insertional inactivation of 11 of these has revealed that only 1 (MmpL3) is apparently essential for viability. Five of these proteins are conserved within the genome of Mycobacterium leprae. The drug susceptibilities of these 11 mutants to a broad spectrum of agents are unaltered, suggesting that unlike their function in other organisms, these proteins do not play a significant role in intrinsic drug resistance. Each of these mutants was assessed for growth kinetics and lethality in a murine low-dose aerosol model of tuberculosis, and four were found to be impaired in one or both measures of virulence. Two of these, with mutations of MmpL4 and the previously characterized MmpL7, which transports phthiocerol dimycocerosate, were found to have both impaired growth kinetics and impaired lethality. Mutants with inactivation of MmpL8, which transports a precursor of the sulfatides, or MmpL11, which transports an unknown substrate, were found to establish infection normally but to be significantly attenuated for lethality in time-to-death studies. These studies support the concept that MmpL-mediated lipid secretion both contributes to the innate ability of the pathogen to survive intracellularly and also contributes directly to the host-pathogen dialogue that determines the ultimate outcome of infection.

Aerosols↗
PubMed2004

A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response.

Fifty million new infections with Mycobacterium tuberculosis occur annually, claiming 2-3 million lives from tuberculosis worldwide. Despite the apparent lack of significant genetic heterogeneity between strains of M. tuberculosis, there is mounting evidence that considerable heterogeneity exists in molecules important in disease pathogenesis. These differences may manifest in the ability of some isolates to modify the host cellular immune response, thereby contributing to the observed diversity of clinical outcomes. Here we describe the identification and functional relevance of a highly biologically active lipid species-a polyketide synthase-derived phenolic glycolipid (PGL) produced by a subset of M. tuberculosis isolates belonging to the W-Beijing family that show 'hyperlethality' in murine disease models. Disruption of PGL synthesis results in loss of this hypervirulent phenotype without significantly affecting bacterial load during disease. Loss of PGL was found to correlate with an increase in the release of the pro-inflammatory cytokines tumour-necrosis factor-alpha and interleukins 6 and 12 in vitro. Furthermore, the overproduction of PGL by M. tuberculosis or the addition of purified PGL to monocyte-derived macrophages was found to inhibit the release of these pro-inflammatory mediators in a dose-dependent manner.

Animals↗
PubMed2004

The transcriptional responses of Mycobacterium tuberculosis to inhibitors of metabolism: novel insights into drug mechanisms of action.

The differential transcriptional response of Mycobacterium tuberculosis to drugs and growth-inhibitory conditions was monitored to generate a data set of 430 microarray profiles. Unbiased grouping of these profiles independently clustered agents of known mechanism of action accurately and was successful at predicting the mechanism of action of several unknown agents. These predictions were validated biochemically for two agents of previously uncategorized mechanism, pyridoacridones and phenothiazines. Analysis of this data set further revealed 150 underlying clusters of coordinately regulated genes offering the first glimpse at the full metabolic potential of this organism. A signature subset of these gene clusters was sufficient to classify all known agents as to mechanism of action. Transcriptional profiling of both crude and purified natural products can provide critical information on both mechanism and detoxification prior to purification that can be used to guide the drug discovery process. Thus, the transcriptional profile generated by a crude marine natural product recapitulated the mechanistic prediction from the pure active component. The underlying gene clusters further provide fundamental insights into the metabolic response of bacteria to drug-induced stress and provide a rational basis for the selection of critical metabolic targets for screening for new agents with improved activity against this important human pathogen.

Antipsychotic Agents↗
PubMed2004

The role of MmpL8 in sulfatide biogenesis and virulence of Mycobacterium tuberculosis.

To study the role of MmpL8-mediated lipid transport in sulfatide biogenesis, we insertionally inactivated the mmpL8 gene in Mycobacterium tuberculosis. Characterization of this strain showed that the synthesis of mature sulfolipid SL-1 was interrupted and that a more polar sulfated molecule, termed SL-N, accumulated within the cell. Purification of SL-N and structural analysis identified this molecule as a family of 2,3-diacyl-alpha,alpha'-D-trehalose-2'-sulfates. This structure suggests that transport and biogenesis of SL-1 are coupled and that the final step in sulfatide biosynthesis may be the extra-cellular esterification of two trehalose 6-positions with hydroxyphthioceranic acids. To assess the effect of the loss of this anionic surface lipid on virulence, we infected mice via aerosol with the MmpL8 mutant and found that, although initial replication rates and containment levels were identical, compared with the wild type, a significant attenuation of the MmpL8 mutant strain in time-to-death was observed. Early in infection, differential expression of cytokines and cytokine receptors revealed that the mutant strain less efficiently suppresses key indicators of a Th1-type immune response, suggesting an immunomodulatory role for sulfatides in the pathogenesis of tuberculosis.

Bacterial Proteins↗