Chemical Characterization of Ethambutol Binding to
            <i>Mycobacterium smegmatis</i>

Beggs, William H.; Andrews, Fred A.

doi:10.1128/aac.5.3.234

Cited by 22 publications

(10 citation statements)

References 9 publications

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“…However, in contrast to EMB-treated M. smegmatis, there was no initial accumulation of lipids in this organism before their secretion. Beggs and Andrews showed that EMB binds almost instantaneously near the cell surface of M. smegmatis (2). Based upon these observations, it appears that the target site(s) for EMB is in the region of the cell surface.…”

Section: Discussionmentioning

confidence: 96%

Effects of ethambutol on accumulation and secretion of trehalose mycolates and free mycolic acid in Mycobacterium smegmatis

Kilburn¹,

Takayama²

1981

Antimicrob Agents Chemother

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We examined the early effects of ethambutol on the synthesis of trehalose monomycolate, trehalose dimycolate, and free mycolic acid in actively growing cells of Mycobacterium smegmatis. At about 1 min after the addition of 3.0 ,ig of ethambutol per ml, the cellular level of trehalose monomycolate began to increase over the control culture. This was followed 8 to 12 min later by the cellular increases in free mycolic acid and trehalose dimycolate over the control culture and the inhibition of incorporation of mycolic acid into the cell wall. Exposure of M. smegmatis to ethambutol for more than 30 min caused all of these lipids to leak out of the cells more rapidly than in the control cells. The mechanism by which ethambutol initiates these events is unknown, but these early imbalances in lipid synthesis may be responsible for the lethal action of this drug.Ethambutol (EMB) is a primary drug used in treating tuberculosis. However, the mechanism of action of this valuable chemotherapeutic agent is not known (1). We have recently found that EMB affects the synthesis of cardiolipin and phosphatidylinositol mannosides and causes the leakage of phosphatidylethanolamine into the culture medium (5). We have also shown that the drug inhibits the incorporation of mycolic acid into the cell wall (7). We now report that EMB induces cellular accumulation and subsequent leakage of trehalose monomycolate (TM), trehalose dimycolate (TD), and free mycolic acid in Mycobacterium smegmatis. MATERIALS AND METHODSBacteria and growth conditions. The strain of M. smegmatis used in this study was CDC 8 (derived from ATCC 607); it was grown in Middlebrook 7H-9 broth (Difco Laboratories, Detroit, Mich.). The albumin-dextrose enrichment was omitted, and 0.2% glycerol and 0.05% Tween 80 (Atlas Powder Co., Wilmington, Del.) were substituted. The cultures were grown at 37°C in a shaking water bath to an absorbance at 650 nm of 0.025 (approximately 2.5 x 10 colony-forming units per ml) before use.Radioactive labeling and lipid extraction. To a culture grown to the desired absorbance was added 1.0 ,LCi of [1-_4C]acetate (58.7 mCi/mmol; Amersham Corp., Arlington Heights, Ill.) per ml. The culture was divided into two equal volumes, and EMB was added to one to a final concentration of 3.0 ,g/ml (minimum 401 bactericidal concentration) and incubated at 37°C. In one experiment, cells were prelabeled with 1.0 ,uCi of [1-'4C]acetate per ml for 30 min, at which time a 100-fold excess of unlabeled acetate was added. The culture was divided, 3.0 ,tg of EMB per ml was added to one volume, and incubation was continued. At various times, 20-ml samples were transferred into tubes containing 1.0 ml of 1.0 M potassium cyanide to stop further incorporation of label into the lipids. The tubes were centrifuged at 27,000 x g for 15 min, and the upper 10-ml portions of the supernatants (growth medium) were carefully removed. The cell pellets were recovered separately, and the lipids were extracted by blending in a Vortex mixer in 4 ml of chloroformmethanol (2:1, vo...

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Section: Discussionmentioning

confidence: 96%

Effects of ethambutol on accumulation and secretion of trehalose mycolates and free mycolic acid in Mycobacterium smegmatis

Kilburn¹,

Takayama²

1981

Antimicrob Agents Chemother

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“…At selected time intervals, the cells contained in 8.0-ml samples were collected on bacteriological filter membranes (diameter, 2.54 cm; pore size, 0.8 j,m) under negative pressure. The cell-containing filter membranes were then washed three times with 2-ml volumes of either deionized water or drugfree Sauton medium, dried, and assayed for radioactivity as previously described (2). In the 37 C experiments, optical densities at 600 nm were determined at appropriate sampling times and converted to milligrams (dry weight) of cells per milliliter from a standard curve.…”

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confidence: 99%

Inhibition of Dihydrostreptomycin Binding to Mycobacterium smegmatis by Monovalent and Divalent Cation Salts

Beggs

Andrews

1976

Antimicrob Agents Chemother

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To better understand salt antagonism ofdihydrostreptomycin (DSM) action on Mycobacterium smegmatis, the effects of monovalent and divalent cation salts on drug uptake were studied in relation to the lethal activity of DSM. In Sauton liquid medium NaCl, MgCl2, and SrCl2 inhibited the initial instantaneous binding of [3H]DSM to the organism and suppressed secondary uptake. These data correlated well with the capacity of each salt to prevent the lethal activity of DSM. It was concluded that monovalent and divalent cation salt antagonism of DSM action on M. smegmatis involves nonspecific interference with drug uptake.Previous studies in our laboratory showed that both monovalent and divalent cation salts can antagonize the inhibitory and bactericidal effects of dihydrostreptomycin (DSM) on Mycobacterium smegmatis (3, 4). The mechanisms involved, however, appeared to be different for the two classes of salts (3). Results suggested that antagonism by monovalent cation salts is a nonspecific ionic strength effect. In contrast, neutralization of DSM action by MgSO4 and MgCl2 appeared to involve a divalent cation concentration-dependent mechanism. Since chloride salts of the nutritionally nonessential cations Sr2+ and Ba2+ inhibited DSM action as effectively as the chlorides of Mg2+ and Ca2+ (4), it appeared that divalent cation antagonism is dependent more upon a double positive charge than on a particular metal ion species. We postulated, therefore, that the divalent cation effect, like the ionic strength effect, probably involves nonspecific interactions that suppress cellular uptake of the aminoglycoside. Streptomycin uptake by susceptible organisms appears to be a diphasic process: an initial instantaneous binding followed by a secondary period of time-dependent uptake (1,7,11,14). In the present study, evidence is presented that monovalent and divalent cation salts can interfere with both of these events. Uptake data correlated well with viability studies, showing that each salt prevented or retarded the bactericidal action of DSM.MATERIALS AND METHODS General. The source of M. smegmatis, the composition of Sauton medium, growth conditions, preparation of DSM solutions, additions of test salts, and methods used in viability studies were previously described in detail (3, 5). It should be stated again, however, that Sauton medium was supplemented with test salts on the basis of ionic strength (,I). From the equation ,u = JMZ2/2, where m is the ion molarity and Z is the ion charge, molarities were calculated that gave desired u values.[3H]DSM binding and uptake experiments. Labeled drug ([3H]DSM sesquisulfate) was purchased from Amersham/Searle, Arlington Heights, Ill. The material had a specific activity of 3.0 Ci/mmol (2.1 mCi/mg). Aqueous solutions of labeled and unlabeled DSM sesquisulfate were mixed to give a working stock solution that contained 2.0 ,ug of [3H]DSMfree base plus 18 ,ug of DSM-free base per ml (total of 20 ,ug of DSM, and 5.0 ACi/ml). Overnight 16-to 18-h Sauton cultures ofM. smegmatis grown at 3...

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“…Consequently, these species are regarded as being of minor significance in these titrations. With the exception of ethambutol protonation constants [28] no data for ethambutol or EDBA is available for comparison in the literature. However, neither exhibit any unexpected trends.…”

Section: Results and Dlseusslonmentioning

confidence: 97%

Metal binding by pharmaceuticals. Part 1. Copper(II) and zinc(II) interactions following ethambutol administration

1981

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Formation constants for copper(II) and zinc(II) complexes of dextro-2,2'-(ethylenediimino)-di-1-butanol (ethambutol) and its metabolic oxidation product, 2,2'-(ethylenediimino)-dibutyric acid (EDBA) have been measured potentiometrically at 37 degrees C, I = 0.15 mol dm-3 [NaCl]. The constants are used in computer models to assess the extent of the formation of these complexes in vivo. These simulations indicate that whereas ethambutol forms metal complexes only to a limited extent in vivo, EDBA competes effectively under physiological conditions for copper(II) and zinc(II). This study suggests that zinc(II) binding by EDBA may account for a number of side effects of ethambutol treatment.

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Chemical Characterization of Ethambutol Binding to Mycobacterium smegmatis

Cited by 22 publications

References 9 publications

Effects of ethambutol on accumulation and secretion of trehalose mycolates and free mycolic acid in Mycobacterium smegmatis

Effects of ethambutol on accumulation and secretion of trehalose mycolates and free mycolic acid in Mycobacterium smegmatis

Inhibition of Dihydrostreptomycin Binding to Mycobacterium smegmatis by Monovalent and Divalent Cation Salts

Metal binding by pharmaceuticals. Part 1. Copper(II) and zinc(II) interactions following ethambutol administration

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