Journal of Antimicrobial Chemotherapy (1999) 43, 219-226
© 1999 The British Society for Antimicrobial Chemotherapy
Antimycobacterial activity of cerulenin and its effects on lipid biosynthesis
a Department of Pathology, School of Medicine, Johns Hopkins University, Baltimore, MD b Department of Molecular Microbiology and Immunology, School of Public Health, Johns Hopkins University, Baltimore, MD, USA c Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore, MD
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Abstract |
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Cerulenin is a potent inhibitor of fatty acid synthase (FAS) in a variety of prokaryotic and eukaryotic cells. Using a standardized mycobacterial susceptibility test, we have observed that cerulenin inhibits the growth of several species of mycobacteria, including tuberculous species such as Mycobacterium tuberculosis (H37Rv and clinical isolates) andMycobacterium bovis BCG (hereafter called BCG), as well as several non-tuberculous species: Mycobacterium smegmatis, the Mycobacterium avium-intracellulare complex (MAC), Mycobacterium kansasii and others. All species and strains tested, including multi-drug resistant isolates of M. tuberculosis, were susceptible to cerulenin with MICs ranging from 1.5 to 12.5 mg/L. Two-dimensional thin-layer chromatography revealed different inhibition patterns of lipid synthesis between tuberculous and non-tuberculous mycobacteria. Cerulenin treatment resulted in a relative increase in phospholipids and mycolic acids in MAC and M. smegmatis, whereas in cerulenin-treated BCG, phospholipids and mycolic acids diminished relative to controls. In addition, long-chain extractable lipids (intermediate in polarity), triglycerides and glycopeptidolipids decreased with cerulenin treatment in all three species of mycobacteria tested. Qualitative changes in several of these lipid classes indicate inhibition in the synthesis of intermediate and long-chain fatty acids. Our results suggest that cerulenin's primary effect may be inhibition of intermediate and long-chain lipid synthesis, with little effect on the synthesis of other lipid classes. In addition, the BCG-specific reduction in phospholipids and mycolic acids suggests the presence of a unique cerulenin-sensitive FAS system in tuberculous mycobacteria. Since pathogenic mycobacteria produce novel long-chain fatty acids, inhibition of fatty acid synthesis offers a potential target for the development of antimycobacterial drugs.
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Introduction |
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Mycobacteria produce a large number of complex lipids that constitute its unique cell envelope. Although current advances have been made in our understanding of the structure of the mycobacterial cell wall, many aspects of the biosynthesis of fatty acids and related compounds remain poorly characterized in these organisms.1 ,2,3 Mycobacterial lipids can be divided into two broad categories: extractable lipids, which can be released simply by using an appropriate organic solvent, and saponifiable lipids, whose release requires chemical agents capable of breaking covalent bonds. Extractable lipids include those of the non-polar variety such as phthiocerol and its derivatives, unesterified fatty acids, trehalose monomycolate and dimycolate as well as other acylated trehaloses, sulpholipids and triglycerides. Polar extractable lipids include various phospholipids, glycopeptidolipids and some uncharacterized glycolipids. Lipids requiring saponification for release from the cell wall include mycolic acids and other long-chain fatty acids. Mycolic acids are high molecular weight,
-alkyl, ß-hydroxy
fatty acids of 70-90 carbons and comprise a large portion of the mycobacterial cell wall. Current
models propose that the arrangement of the mycolic acids in the cell wall produces an irregular
monolayer which is intercalated with other compounds including mycocerosates, acylglycerols
and various glycolipids.4,5 These lipids are thought to associate with the distal portions of the mycolic acids by
hydrophobic interactions. Fatty acid synthases (FASs) have been classified into two types. Type I FAS, present in eukaryotes, consists of seven enzymatic functions located on one or two polypeptides; type II FASs in prokaryotes employ separate proteins for each enzyme function. In both prokaryotes and eukaryotes, the products of FAS are long-chain fatty acids, used predominantly for storage or structural lipids. Mycobacteria are known to possess both type I and type II FASs1, 1,6,7 as well as mycocerosic acid synthase which produces long-chain, multimethyl branched fatty acids.3 Recent evidence suggests the presence of an additional synthase which may be involved in the synthesis of mycolic acids. 7 Thus, mycobacteria contain multiple complex fatty acid synthetic pathways, which may provide potential therapeutic targets.
The complex lipid composition of the mycobacteria led us to evaluate the in-vitroactivity and biochemical effects of a well characterized FAS inhibitor, cerulenin (2R,3S-epoxy-4-oxo-7,10-trans,trans-dodecanoic acid amide). 8 Cerulenin inhibits both type I and type II FASs through irreversible inhibition of the ß-ketoacyl synthase, the condensing enzyme required for the biosynthesis of fatty acids.8,9,10,11 Although cerulenin is not a candidate for antimicrobial therapy, owing to its instability in mammalian systems, in this study we use it to evaluate the potential feasibility of fatty acid synthesis as a target pathway for antibiotic development in mycobacteria. We report the in-vitroantimicrobial activity of cerulenin against a variety of mycobacteria, including multi-drug resistant isolates. In addition, we demonstrate the differential effect of this inhibitor on lipid synthesis in three species of mycobacteria, Mycobacterium bovis BCG, the Mycobacterium avium-intracellularecomplex (MAC) and Mycobacterium smegmatis. The data suggest a significant difference in mycolic acid synthesis or regulation between tuberculous and non-tuberculous mycobacteria and demonstrate the plausibility of fatty acid synthesis as a potential antibiotic target pathway in mycobacteria.
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Materials and methods |
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Mycobacteria
A control strain of Mycobacterium tuberculosis, H37Rv, was used throughout this study, as well as M. bovis BCG (Pasteur strain, ATCC 35734) and M. smegmatis (mc2 6 1-2c).12 Additional isolates from our collection were speciated by standard methods13 and included the following: MAC, M. bovis, Mycobacterium chelonei, Mycobacterium gordonae, Mycobacterium fortuitum, Mycobacterium kansasii and 11 clinical isolates of M. tuberculosis.
Susceptibility testing and determination of MICs
Susceptibility testing and determination of MICs were performed using the BACTEC
radiometric
growth system (Becton Dickinson, Sparks, MD, USA).
14 A 1 mg/mL initial stock solution of cerulenin (Sigma, St
Louis, MO, USA) was prepared and diluted in dimethylsulphoxide (DMSO; Sigma). Stock
concentrations (0.1 mL) were then added to individual 4.0 mL BACTEC bottles resulting in
final concentrations of 25, 12.5, 6.25, 3.0 and 1.5 mg/L. For each strain tested a 1.0 McFarland
suspension was prepared and 0.1 mL was added to each of the following bottles: each
concentration of drug, a direct control (bottle containing diluent, DMSO, but no antibiotic) and a
control containing a 1/100 organism dilution (also without antibiotic). All bottles were incubated
at 37°C, and the growth index (GI) of each bottle was recorded until the GI of the 1/100
control reached 30. The MIC for the isolate was determined using the following criterion: once
the GI of the 1/100 control bottle had reached a value of 30, the change in GI (
) was
calculated for a 1 day period at each concentration tested. The MIC was defined as the lowest
cerulenin concentration that yielded a growth index change less than that of the 1/100 control
bottle. A modification of this protocol adopted by The National Jewish Center for Immunology
and Respiratory Medicine was used to determine MICs for MAC.
15 The susceptibilities of isolates of M. tuberculosis
and determination of their MICs of isoniazid, streptomycin, ethambutol, rifampicin and
pyrazinamide were performed using standard BACTEC methods.
14 All primary drugs were purchased from Becton
Dickinson.
Lipid pulse-labelling and treatment of cultures with cerulenin
BCG and MAC were grown in Middlebrook 7H9-ADC-Tween (Difco, Detroit, MI, USA) to early log phase. From this a 1.0 McFarland suspension was prepared and diluted to yield a final concentration of 3 x 107 cells/mL in 50 mL total volume of M7H9-ADC-Tween. Cultures were aerated and incubated at 37°C for 24 h (approximately one generation time) with 5% CO2. Cerulenin was added to give final concentrations of 3.0 mg/L for BCG and M. smegmatis, and 6.25 mg/L for MAC, and the cultures were incubated under the same conditions for 24 h. Subsequently, 1 µCi/mL of [1,2-14C]acetate (Amersham, Arlington Heights, IL, USA) was added and the cultures were incubated as before for an additional 24 h. For M. smegmatis a 0.5 McFarland suspension was used with an initial incubation time of 10 h before addition of drug and subsequent incubations of 5 h each (based on a doubling time of 3–5 h) following the addition of drug and label, respectively. All assays were performed in duplicate.
Preparation of extractable mycobacterial lipids
Extractions were performed as previously described. 16,17 ,18 Briefly, extractable non-polar and polar lipids were separated from saponifiable lipids as follows: 50 mL cultures of M. smegmatis, BCG or MAC were harvested by centrifugation at 3000g for 10 min. Equal volumes of cells (100-150 mg wet weight) were suspended in 2 mL of methanolic saline (methanol–0.3% aqueous NaCl, 100:10 (v/v)) and extracted three times with 1 mL of petroleum ether (boiling point (bp) 60-80°C)) to yield extractable non-polar lipids. The remaining cells and residual aqueous phase were then boiled for 5 min, cooled for 5 min at 37°C and extracted once with a mixture of mono-phasic chloroform–methanol–0.3% NaCl (90:100:30 by volume) and twice using chloroform–methanol–0.3% NaCl (50:100:40 by volume) with mixing for 30 min. The residue containing the defatted cells and saponifiable lipids was saved. Extractable polar lipids were obtained by adding chloroform–0.3% NaCl (1:1 v/v) to the aqueous supernatants, mixing for 5 min and then centrifuging. The lower phase, containing polar extractable lipids, was dried under nitrogen (20°C).
Preparation of mycolic acids and other saponifiable lipids
Saponifiable lipids were released from defatted cells by alkaline hydrolysis in methanol (1 mL), 30% KOH (1 mL) and toluene (0.1 mL) at 75°C overnight and subsequently cooled to room temperature.16,18 The mixture was acidified to pH 1 with 3.6% HCl and extracted three times with diethyl ether. Combined extracts were dried under nitrogen. Fatty acid methyl esters of mycolic acids and other long-chain compounds were prepared by mixing 1 mL of dichloromethane, 1 mL of a catalyst solution 19 and 25 mL of iodomethane for 30 min, centrifuging and discarding the upper phase. The lower phase was dried under nitrogen.
Analysis of mycobacterial lipids
Incorporation of [14C]acetate into lipid fractions was determined by scintillation counting (Beckman LS6500 multi-purpose scintillation counter). Extracted samples were dissolved in chloroform and dried under nitrogen, then equal counts of each of the three lipid fractions were loaded on to thin-layer chromatography (TLC) plates (20 cm x 20 cm x 250 µm silica gel G analytical plates; Analtech, Newark, DE, USA). Extractable non-polar and polar fractions were subjected to the following two-dimensional solvent systems in order of increasing polarity (first dimension; second dimension). System 1 (for the isolation of triglycerides): (i) petroleum ether (bp 60-80°C)-ethyl acetate (98:2 v/v, three times); (ii) petroleum ether (bp 60-80°C)-acetone (98:2 v/v, once). System 2 (for the separation of non-polar extractable lipids): (i) petroleum ether (bp 60-80°C)-acetone (92:8 v/v, three times); (ii) toluene–acetone (95:5 v/v, once). System 3 (for glycopeptidolipids): (i) chloroform–methanol–water (100:14:0.8 by volume); (ii) chloroform–acetone–methanol–water (50:60:2.5:3 by volume). System 4 (for phospholipids, glycolipids and other polar lipids): (i) chloroform–methanol–water (60:30:6 by volume); (ii) chloroform–acetic acid–methanol–water (40:25:3:6 by volume). Saponifiable lipids were examined using both one- and two-dimensional TLC systems. Extracted samples were applied to 20 cm x 20 cm silica gel G TLC plates (250 µm analytical plates, Analtech) and developed one-dimensionally in petroleum ether (bp 60- 80°C)-acetone (95:5 v/v, three times). Rf; values were recorded for each spot and were compared. Two-dimensional TLC was done using petroleum ether (bp 60-80°C)-acetone (95:5 v/v, three times) in the first dimension and toluene–acetone (97:3 v/v, once) in the second dimension.
Determination of spot identities in this study were based on those assigned by Dobson et al.16 and Minnikin et al. 17 using the identical extraction and two-dimensional TLC systems already described. 16,17
Visualization and comparison of thin-layer chromatograms were performed using either a Molecular Dynamics 425E (Sunnyvale, CA, USA) or a Fujix BAS 1000 (Fuji) phosphorimager. Spot intensities were quantified using either Imagequant (Molecular Dynamics version 3.3) or NIH Image (version 1.57, National Institutes of Health, Bethesda, MD, USA) software programs and were expressed as a percentage of the total phosphorimage signal for the particular TLC plate; relative intensities were then calculated by normalizing cerulenin treatment values to controls.
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Results |
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MICs of cerulenin for several species of mycobacteria
As may be seen in Table I, all species of mycobacteria tested were susceptible to cerulenin with MICs ranging from 1.5 to 12.5 mg/L. Resistance to other antimycobacterial agents did not lead to cross-resistance to cerulenin. Isolates of M. tuberculosis resistant to one or more of the following drugs: isoniazid, rifampicin, ethambutol, streptomycin and pyrazinamide, were found to be susceptible to cerulenin, including one strain that was resistant to all five of the first-line antimycobacterial drugs.
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Overall effects of cerulenin on mycobacterial lipids
In all three species of mycobacteria tested, cerulenin treatment resulted in overall inhibition of [ 14C]acetate incorporation into total lipids. However, not all species were equally susceptible to the effects of cerulenin. Inhibition in M. smegmatis and M. bovis BCG was 82% and 83% respectively, while for MAC only 60% inhibition of [14C]acetate incorporation was observed. Furthermore, cerulenin treatment resulted in a differential effect on individual lipid fractions in all three species of mycobacteria with the greatest amount of inhibition in extractable non-polar lipids followed by extractable polar lipids and saponifiable fractions, respectively, in M. bovis BCG and M. smegmatis as is shown in Figure 1. However, in MAC, the greatest inhibition was seen in the extractable polar lipids followed by extractable non-polar and saponifiable lipids (Figure 1).
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),
extractable polar (
) and saponifiable (
) fractions. Inhibition is expressed as
% reduction in cpm between treated and control groups.Effects of cerulenin on extractable non-polar and polar lipids
The qualitative and quantitative effects of cerulenin on the synthesis of non-polar long-and intermediate-chain lipids, triglycerides, phospholipids and glycopeptidolipids are shown in Table II. For each species tested, a panel of four, two-dimensional thin-layer chromatograms were prepared in order to assess the effects of cerulenin on particular lipid classes.
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Extractable non-polar lipids of intermediate polarity, consistent with certain types of glycolipids (e.g. trehalose mycolipinates), were reduced significantly with cerulenin treatment in all three mycobacterial species tested (Table II). Virtually complete inhibition of this subset of extractable non-polar lipids was observed in MAC and BCG, but inhibition was not so extensive in M. smegmatis. Long-chain compounds in this fraction decreased in M. smegmatis and MAC by 38%, while in BCG this same class showed a relative increase of 2000%. Since these compounds migrate close to the solvent front in both directions (first dimension: petroleum ether (bp 60-80°C)-acetone (92:8 v/v, three times); second dimension: toluene–acetone (95:5 v/v, once)), these TLC spots may represent either long-chain fatty acid esters of the mycobacterial outer wall or unesterified longer-chain fatty acids of varying lengths. Triglycerides were uniformly reduced in cerulenin-treated M. smegmatis, BCG and MAC.
Of the extractable polar lipids, glycopeptidolipids differed quantitatively in cerulenin-treated groups compared with controls (Table II). Inhibition of labelled acetate incorporation into glycopeptidolipids ranged from 18% in MAC, through 23% in BCG, to 69% in M. smegmatis. Qualitative changes in this class may reflect an alteration in the character of the acyl chains attached to the glycopeptides. Relative production of phospholipids, specifically, phosphatidyl inositol mannosides, (PIMs), was increased in cerulenin-treated M. smegmatis (609%) and, to a lesser extent, in MAC (4.3%). BCG showed a 43% decrease in this same lipid class relative to controls.
Effects of cerulenin on saponifiable lipids
Mycobacterial saponifiable lipids include mycolic acids and other long-chain fatty acids. The
effect of cerulenin treatment on the mycolates of BCG differed significantly from that observed
for M. smegmatis and MAC, as shown in Figure 2. In BCG,
both the -mycolates and ketomycolates were reduced (by 13% and 35%,
respectively), while in M. smegmatis and MAC all types of mycolate showed relative
increases in levels. In M. smegmatis, the -mycolate levels increased
906% relative to controls, as did the '-mycolates (172%) and the
epoxymycolates (682%). Similar relative increases were observed in all three of the major
mycolic acid classes of MAC: -mycolates (80%), methoxymycolates (21% )
and wax ester mycolates (66%).
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-carboxymycolate; x, unknown. Equivalent cpm
of the saponifiable lipids were spotted at each origin.Of particular interest is the increase or decrease of an unknown lipid(s) in this fraction (labelled `x' in Figure 2). This molecular species may represent a series of non-hydroxylated fatty acid methyl esters16 which appear to be inversely related to the amount of completed mycolate present. Thus, in BCG, the unknown showed a relative increase (569%), while the individual mycolic acids are decreased, and in MAC the unknown is decreased (72% ) while the individual mycolates are relatively increased. This latter finding was also observed in M. smegmatis (data not shown). Moreover, pattern differences between the unknown of BCG, M. smegmatis and MAC are accentuated in side-by-side analysis using one-dimensional TLC. As seen in Figure 3 the unknown lipids appear as discrete bands (identified by Rf values) in the one-dimensional system. Controls in all three species of mycobacteria tested show bands with Rf values of 0.79. With cerulenin treatment, the band at 0.79 diminishes significantly in MAC and M. smegmatis and is completely absent in BCG. In addition, while no other major band alterations appear to occur in MAC as a result of cerulenin treatment, additional bands appear in BCG and M. smegmatis. Of these, one is present in both BCG and M. smegmatis (Rf = 0.83) while another at 0.86 is found only in BCG. Interestingly, a series of unique lipids running from Rf values of 0.61-0.74 appear in cerulenin-treated BCG and are absent in treated MAC and M. smegmatis.
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Discussion |
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In this study we demonstrate that cerulenin, a FAS inhibitor, is inhibitory for a broad range of mycobacterial species including multi-drug resistant strains of M. tuberculosis. There was no cross-resistance to cerulenin by isolates resistant to rifampicin, ethambutol, streptomycin, pyrazinamide and isoniazid, suggesting a novel and unexploited mechanism of action of cerulenin even against multi-drug resistant M. tuberculosis. Early work by Omura11 demonstrated the activity of cerulenin against a variety of fungi and bacteria including some mycobacteria using a broth dilution susceptibility test system. In contrast to other mycobacteria tested, the strain of M. tuberculosisreported had an MIC of 100 mg/L. In our study all species of mycobacteria tested, including M. tuberculosis, were susceptible to cerulenin with MICs
12.5 mg/L using the rapid
radiometric system. The discrepancy between our findings and this earlier work is likely to have
resulted from degradation of cerulenin during the 4 week incubation in Omura's broth
dilution system. More recently, Barrow and co-workers
20 have observed the inhibitory activity of cerulenin against
MAC using a rapid radiometric susceptibility test method. Although cerulenin is not a
candidate drug, as a result of its lack of stability, the growth inhibitory effect of cerulenin against
multi-drug resistant mycobacteria suggests that fatty acid synthesis is a novel target pathway for
antibiotic drug development. The overall effects of cerulenin on lipid synthesis suggest inhibition of a variety of FAS systems. The primary fatty acid products of the mycobacterial type I FAS are either C16 ,18 or C24,26, depending on the availability of mycobacterial carbohydrate and other reaction conditions.6 The former are commonly incorporated into phospholipids and triglycerides. Thus, incorporation of fatty acid into these lipids serves as an indirect indicator of FAS I activity. In mammalian cells, cerulenin inhibits the type I FAS with profound inhibition of fatty acid incorporation into phospholipids.21 Although there was a uniform decrease in triglycerides among the three species, cerulenin treatment resulted in a different phospholipid response in M. smegmatis and MAC compared with M. bovis BCG. This observation is consistent with previous work done with M. smegmatis.22 The investigators suggested that cerulenin may affect both the synthesis and degradation of phospholipids, and, as a result, quantitative changes in phospholipids would be affected in a time-dependent manner. Other possible explanations for the quantitative differences observed in phospholipid profiles between mycobacterial species include the following: (i) FAS I enzymes may differ between species, leading to cerulenin inhibition in BCG but not in MAC or M. smegmatis; 23 (ii) an alternative pathway may exist in BCG, capable of shunting fatty acids from the pool of precursors necessary for phospholipid synthesis, indirectly diminishing the levels of phospholipids, and (iii) inherent differences in phospholipid metabolic regulation may exist between mycobacterial species.
Overall inhibition of extractable non-polar lipids following cerulenin occurred in all three species. The most profound reduction was noted in lipids of intermediate polarity consistent with glycolipids such as mycolipenates of trehalose.1,4,5,16 The cerulenin effect on particular glycolipids is probably due to inhibition of fatty acid elongation. Mycolipenates of trehalose commonly contain fatty acids up to but not beyond C24 in length.1,3,6 The cerulenin effect on these implies the presence of a cerulenin-sensitive type II elongation system in mycobacteria. Inhibition of this class of fatty acids (C30) may result in the substitution of abnormally short acyl chains on commonly occurring sugar moieties of the outer wall, such as trehalose, resulting in an overall shift in the glycolipid pattern as demonstrated by TLC (data not shown). Similar changes were observed with glycopeptidolipids. Previous studies have shown similar alterations in mobility following deacetylation using mild alkali hydrolysis.16,24,25
The most dramatic differential effect of cerulenin treatment on BCG, MAC and M.
smegmatis was observed in the non-polar long-chain lipids and saponifiable fraction. The
differences observed occurred between the tuberculous species (BCG) and the non-tuberculous
species (MAC and M. smegmatis) examined in this study. In both MAC and M.
smegmatis, increases were observed in non-polar long chain lipids, -mycolates and
species-specific mycolates following cerulenin treatment, while the opposite was true in BCG.
Additionally, the amount of uncharacterized lipids in the saponifiable fraction appeared to be
inversely related to the amount of completed mycolates present. Previous studies have tentatively
identified these lipids as non-hydroxylated fatty acid methyl esters.
16,18 These
observations imply significant differences in mycolic acid synthesis between tuberculous and
non-tuberculous mycobacteria, a finding consistent with that of other investigators in attempting
to determine the target of isoniazid.
7 Although considerable further investigation is required it
is tempting to speculate that these differences in mycolic acid synthesis might be significant in
defining the pathogenic versus non-pathogenic mycobacteria.
In summary, our data demonstrate that inhibition of fatty acid synthesis is a target for novel drug development in mycobacteria. Using cerulenin as a prototypical inhibitor, significant antimycobactericidal effects were seen with BCG, MAC and M. smegmatis. While the cytotoxic effects were similar, we observed significant differences in the patterns of lipid accumulation following FAS inhibition among these species. While the differential effects may be due to varying affinities of the drug for homologous FAS systems, it is also possible that additional FAS or FAS-like systems are present in particular species such as BCG and that expression of novel FAS enzymes explains the different lipid profiles in response to cerulenin. Further use of pharmacological FAS inhibitors such as cerulenin, thiolactomycin 26 and other compounds in development will be valuable in dissecting the individual components of fatty acid biosynthesis in mycobacteria. Elucidation of these enzymatic pathways may provide novel targets for drug development.
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Acknowledgments |
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We thank Mark Romagnoli and Krista Sturm for their assistance with the initial susceptibility testing of mycobacteria with cerulenin and other first-line drugs. The technical advice of Dr Craig Townsend is also greatly appreciated. This work was supported in part by grants AI36973 and AI37856 from the National Institutes of Health.
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Notes |
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* Corresponding author. Johns Hopkins Medical Institutions, Department of Pathology/Division of Medical Microbiology, 600 N. Wolfe Street, Baltimore, MD 21287, USA. Tel:+1410-955-5077; Fax: 1 +410-614-8087; E-mail: jdick{at}pathlan.path.jhu.edu
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Received 10 March 1998; returned 27 April 1998; revised 5 June 1998; accepted 17 July 1998
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