In vitro activity of a novel antimycobacterial compound, N-octanesulfonylacetamide, and its effects on lipid and mycolic acid synthesis

Article abstract of DOI:10.1128/AAC.45.4.1143-1150.2001

β-Sulfonyl carboxamides have been proposed to serve as transition-state analogues of the β-ketoacyl synthase reaction involved in fatty acid elongation. We tested the efficacy of N-octanesulfonylacetamide (OSA) as an inhibitor of fatty acid and mycolic acid biosynthesis in mycobacteria. Using the BACTEC radiometric growth system, we observed that OSA inhibits the growth of several species of slow-growing mycobacteria, including Mycobacterium tuberculosis (H37Rv and clinical isolates), the Mycobacterium avium complex (MAC), Mycobacterium bovis BCG, Mycobacterium kansasii, and others. Nearly all species and strains tested, including isoniazid and multidrug resistant isolates of M. tuberculosis, were susceptible to OSA, with MICs ranging from 6.25 to 12.5 μg/ml. Only three clinical isolates of M. tuberculosis (CSU93, OT2724, and 401296), MAC, and Mycobacterium paratuberculosis required an OSA MIC higher than 25.0 μg/ml. Rapid-growing mycobacterial species, such as Mycobacterium smegmatis, Mycobacterium fortuitum, and others, were not susceptible at concentrations of up to 100 μg/ml. A 2-dimensional thin-layer chromatography system showed that OSA treatment resulted in a significant decrease in all species of mycolic acids present in BCG. In contrast, mycolic acids in M. smegmatis were relatively unaffected following exposure to OSA. Other lipids, including polar and nonpolar extractable classes, were unchanged following exposure to OSA in both BCG and M. smegmatis. Transmission electron microscopy of OSA-treated BCG cells revealed a disruption in cell wall synthesis and incomplete septum formation. Our results indicate that OSA inhibits the growth of several species of mycobacteria, including both isoniazid-resistant and multidrug resistant strains of M. tuberculosis. This inhibition may be the result of OSA-mediated effects on mycolic acid synthesis in slow-growing mycobacteria or inhibition via an undescribed mechanism. Our results indicate that OSA may serve as a promising lead compound for future antituberculous drug development.

Full text of DOI:10.1128/AAC.45.4.1143-1150.2001

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2001, p. 1143–1150
0066-4804/01/$04.00ϩ0 DOI: 10.1128/AAC.45.4.1143–1150.2001
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Vol. 45, No. 4
In Vitro Activity of a Novel Antimycobacterial Compound,
N-Octanesulfonylacetamide, and Its Effects on
Lipid and Mycolic Acid Synthesis
NIKKI M. PARRISH,1 TODD HOUSTON,2† PAUL B. JONES,2‡ CRAIG TOWNSEND,2
AND JAMES D. DICK1,3
*
Department of Pathology, School of Medicine,1 Department of Molecular Microbiology and Immunology,
School of Public Health,3 and Department of Chemistry, Johns Hopkins School of
Arts and Sciences,2 Johns Hopkins University, Baltimore, Maryland
Received 29 September 2000/Returned for modification 8 November 2000/Accepted 22 January 2001
␤-Sulfonyl carboxamides have been proposed to serve as transition-state analogues of the ␤-ketoacyl
synthase reaction involved in fatty acid elongation. We tested the efficacy of N-octanesulfonylacetamide (OSA)
as an inhibitor of fatty acid and mycolic acid biosynthesis in mycobacteria. Using the BACTEC radiometric
growth system, we observed that OSA inhibits the growth of several species of slow-growing mycobacteria,
including Mycobacterium tuberculosis (H37Rv and clinical isolates), the Mycobacterium avium complex (MAC),
Mycobacterium bovis BCG, Mycobacterium kansasii, and others. Nearly all species and strains tested, including
isoniazid and multidrug resistant isolates of M. tuberculosis, were susceptible to OSA, with MICs ranging from
6.25 to 12.5 ␮g/ml. Only three clinical isolates of M. tuberculosis (CSU93, OT2724, and 401296), MAC, and
Mycobacterium paratuberculosis required an OSA MIC higher than 25.0 ␮g/ml. Rapid-growing mycobacterial
species, such as Mycobacterium smegmatis, Mycobacterium fortuitum, and others, were not susceptible at con-
centrations of up to 100 ␮g/ml. A 2-dimensional thin-layer chromatography system showed that OSA treatment
resulted in a significant decrease in all species of mycolic acids present in BCG. In contrast, mycolic acids in
M. smegmatis were relatively unaffected following exposure to OSA. Other lipids, including polar and nonpolar
extractable classes, were unchanged following exposure to OSA in both BCG and M. smegmatis. Transmission
electron microscopy of OSA-treated BCG cells revealed a disruption in cell wall synthesis and incomplete
septum formation. Our results indicate that OSA inhibits the growth of several species of mycobacteria,
including both isoniazid-resistant and multidrug resistant strains of M. tuberculosis. This inhibition may be the
result of OSA-mediated effects on mycolic acid synthesis in slow-growing mycobacteria or inhibition via an
undescribed mechanism. Our results indicate that OSA may serve as a promising lead compound for future
antituberculous drug development.
Tuberculosis continues to be the leading cause of death
worldwide due to an infectious agent (8). Approximately 8
million new active cases arise each year, with about 3 million
deaths (8). Of equivalent concern has been the emergence of
multidrug-resistant Mycobacterium tuberculosis. As a result,
newly infected individuals no longer have the assurance that
prophylaxis with isoniazid (INH) will eliminate infection or
that active disease will be treatable with our current arsenal of
drugs. In addition, therapies for the treatment of atypical my-
cobacterial infections in immunocompromised patients are
limited (24). Thus, the development of new drugs is essential in
combating both drug resistant M. tuberculosis and opportunis-
tic infections with atypical mycobacteria, such as the Mycobac-
terium avium complex (MAC).
the unique lipids produced by mycobacteria, such as mycolic
acids. These high-molecular-weight, ␣-alkyl, ␤-hydroxy fatty
acids comprise the single largest component of the mycobac-
terial cell envelope (3, 4, 9, 10, 29, 30, 37). They are found in
free lipids as trehalose mono- and dimycolate and esterified to
the arabinogalactan matrix of the mycobacterial cell wall (5,
10). They are vital for the growth and survival of mycobacteria,
as evidenced by the bactericidal properties of mycolic acid
inhibitory drugs, such as isoniazid and ethionamide (1, 2, 32,
33, 43, 44, 47, 48, 51, 53–58).
Synthesis of mycolic acids and other mycobacterial lipids
requires a variety of fatty acid synthase and elongation en-
zymes (7, 10, 23). Although the synthesis of fatty acids is
essentially the same at the primary chemical level, fatty acid
synthases (FAS) are organized into two types. In Type I FAS
(FAS I), most often found in eukaryotes, the individual enzy-
matic reactions are contained in one multienzyme complex. In
Type II FAS (FAS II), commonly found in prokaryotes, the
enzyme functions are carried out by seven individual proteins.
Mycobacteria are known to possess both FAS I and II (6, 7,
23). Thus, inhibition of these enzymes, especially those in-
volved in chain elongation of unique mycobacterial fatty acids,
may provide novel targets for drug design.
Potential new targets for antimycobacterial drug develop-
ment may exist among the synthetic enzymes needed to make
* Corresponding author. Mailing address: Johns Hopkins Medical
Institutions, Department of Pathology/Division of Medical Microbiol-
ogy, 600 N. Wolfe St., Baltimore, MD 21287. Phone: (410) 955-5077.
Fax: (410) 614-8087. E-mail: jdick@jhmi.edu.
† Present address: Department of Chemistry, Virginia Common-
wealth University, Richmond, Va.
In the past, characterization of FAS has been aided through
the use of two natural product inhibitors of FAS components,
‡ Present address: Department of Chemistry, Wake Forest Univer-
sity, Winston-Salem, N.C.
1143

1144
PARRISH ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
thiolactomycin, 25.0 ␮g/ml [BCG] and 75.0 ␮g/ml [M. smegmatis]; OSA, 6.25
␮g/ml [BCG], 25.0 ␮g/ml [MAC], and 100 ␮g/ml [M. smegmatis]), and cultures
were incubated under the same conditions for approximately 1 generation time
(BCG and MAC, approximately 24 h; M. smegmatis, approximately 5 h). Subse-
quently, 1 ␮Ci of [1,2-14C]acetic acid (Amersham, Arlington Heights, Ill.)/ml was
added and the cultures were incubated as before for an additional 24 h. In order
to demonstrate a concentration-dependent effect of OSA on mycolic acid syn-
thesis in BCG, lipid pulse labeling was also performed at OSA concentrations of
12.5 and 25.0 ␮g/ml. A slight variation of this protocol was used for labeling in
M. smegmatis cells. Since this species of mycobacteria was not susceptible to
OSA, the highest concentration tested (100 ␮g/ml) in this study was used for
labeling purposes. Additionally, a 0.5 McFarland suspension was done with an
initial incubation time of 10 h prior to the addition of compound and subsequent
incubations of 5 h each (based on a doubling time of 3 to 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 (13, 34, 42). Briefly, 100 to 150 mg (wet weight) of cells
was suspended in methanolic saline (methanol–0.3% aqueous NaCl [100:10,
vol/vol] [2 ml]) and extracted three times with petroleum ether, yielding nonpolar
extractable lipids. The remaining cells and residual aqueous phase were boiled
for 5 min, cooled for 5 min at 37°C, and extracted with monophasic chloroform–
methanol–0.3% NaCl (90:100:30, vol/vol; used once) and chloroform–methanol–
0.3% NaCl (50:100:40, vol/vol; used twice). All extracts were subsequently dried
under N2 at room temperature. The defatted cells containing saponifiable lipids
were saved.
Mycolic acid extraction and preparation of MAMES and FAMES. Extractions
of mycobacterial mycolic acids were performed as previously described (13, 35,
42). Briefly, 50-ml cultures of M. smegmatis, BCG, or MAC cells were harvested
by centrifugation at 3,000 ϫ g for 10 min. Equal volumes of cells (100 to 150 mg
[wet weight]) were extracted to remove polar and nonpolar extractable lipids (13,
42). The resulting defatted cells containing bound mycolic acids and other sa-
ponifiable lipids were subjected to 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 (13, 42). The mixture was then acidified to pH 1 with 3.6%
HCl and extracted three times with diethyl ether. Combined extracts were dried
under N2. Mycolic acid methyl esters (MAMES) and other long-chain fatty acids
(fatty acid methyl esters [FAMES]) were prepared by mixing dichloromethane (1
ml), a catalyst solution (1 ml) (14), and iodomethane (25 ml) for 30 min;
centrifuging; and discarding the upper phase. The lower phase was dried under
N2.
FIG. 1. Structure of OSA.
cerulenin and thiolactomycin (15, 16, 36, 39–42, 46). Cerulenin
is a potent inhibitor of both FAS I and FAS II systems while
thiolactomycin inhibits only synthases of the FAS II variety.
Activity of both of these inhibitors on the mycolic acids of
mycobacteria has recently been described (25, 42, 50). Al-
though cerulenin and thiolactomycin are structurally different,
both compounds inhibit the two-carbon homologation cata-
lyzed by the ␤-ketoacyl synthase, the condensing enzyme re-
quired for fatty acid biosynthesis. Specifically, cerulenin irre-
versibly inhibits the ␤-ketoacyl synthase (20, 39, 40), while
thiolactomycin inhibits both the ␤-ketoacyl–acyl carrier pro-
tein (ACP) synthase and acetyl coenzyme A:ACP transacylase
(15). ␤-Sulfonyl carboxamides were designed to mimic the
transition state of the reaction catalyzed by the ␤-ketoacyl
synthase. In the following study we evaluated the in vitro ac-
tivity of one of these compounds, N-octanesulfonylacetamide
(OSA), on a variety of mycobacteria and specifically evaluated
its effects on lipid and mycolic acid synthesis in Mycobacterium
bovis BCG and Mycobacterium smegmatis.
MATERIALS AND METHODS
Synthesis of OSA. The synthesis of alkyl sulfoxides and sulfones has been
described previously (22). Briefly, OSA was synthesized in three steps from
commercially available materials. Octyl bromide and methyl thioglycolate were
reacted together to yield methyl 3-thioundecanoate. This sulfide was then oxi-
dized to the sulfoxide by using 3-chloroperoxybenzoic acid. OSA was obtained
from the ammonylysis of the methyl ester. Overall yield was 70% following
crystallization of the final product (Fig. 1).
Mycobacteria. M. tuberculosis strains H37Rv and CSU93 (52), M. bovis (ATCC
35734), M. bovis BCG (Pasteur strain, ATCC 35734), Mycobacterium kansasii
(ATCC 12478), Mycobacterium paratuberculosis (ATCC 19698), and M. smegma-
tis (mc2 6 1-2c) (53) were utilized as reference strains. Clinical and other isolates
were speciated using standard methods (38) and included MAC, Mycobacterium
fortuitum, Mycobacterium chelonei, Mycobacterium abscessus, and both INH- and
multidrug-resistant clinical isolates of M. tuberculosis.
Susceptibility testing. Susceptibility testing and determination of MICs for M.
tuberculosis, M. bovis, M. kansasii, and M. bovis BCG were done using the
BACTEC radiometric growth system (Becton Dickinson, Sparks, Md.) and a
standardized method (42, 49). Initial stock solutions (1 mg/ml) and subsequent
dilutions of OSA, cerulenin (Sigma, St. Louis, Mo.), and thiolactomycin (gener-
ously provided by T. Yoshida) were prepared in dimethyl sulfoxide (Sigma). A
modification of this procedure adopted by The National Jewish Center for
Immunology and Respiratory Medicine was used to determine MICs for MAC
(17). Susceptibility testing of M. paratuberculosis was accomplished by varying the
standard BACTEC protocol to include the addition of mycobactin J (Allied
Monitor, Fayette, Mo.) to commercially prepared 12B media (Becton Dickin-
son). Initial mycobactin J solutions (2 mg/ml) were brought up in 95% ethanol
and diluted in sterile distilled water to a concentration of 40 ␮g/ml. Mycobactin
J was then added to each BACTEC vial (final concentration ϭ 1.0 ␮g/ml) along
with OSA. All primary drugs were purchased from Becton Dickinson. Suscepti-
bilities and MIC determinations of specific inhibitors for M. smegmatis, M.
fortuitum, M. chelonei, and M. abscessus were established by broth dilution using
Middlebrook 7H9-ADC incubated at 37°C for 4 days.
[1,2-14C]acetate incorporation into mycobacterial lipids. Incorporation of
[1,2-14C]acetate into polar and nonpolar extractable and saponifiable lipid frac-
tions was determined by scintillation counting and expressed in counts per
minute (cpm) (Beckman LS6500 multi-purpose scintillation counter).
Analysis of MAMES and FAMES. Mycobacterial saponifiable extracts con-
taining MAMES and FAMES were dissolved in chloroform and equal counts (in
counts per minute) of each sample were loaded onto thin-layer chromatography
(TLC) plates (20- by 20-cm silica gel G, 250-␮m-diamter analytical plates; An-
altech, Newark, Del.). Samples were subsequently subjected to a 2-dimensional
solvent system (petroleum ether [bp 60 to 80°C]–acetone [95:5, vol/vol]) in the
first dimension [three times] and toluene-acetone [97:3, vol/vol] in the second
dimension [one time]).
Data analysis. Mycolic acids of each species of mycobacteria were identified
according to methods described by Dobson et al. (13, 42). Visualization and
comparison of thin-layer chromatograms were done using a Fuji Systems (Fujix
BAS 1000) phosphorimager. Spots were quantified using NIH Image (version
1.57; National Institutes of Health, Bethesda, Md.) software programs. Due to
the nonequivalency of the counts per minute as determined by scintillation
counting and phosphor counts as determined by phosphorimaging, relative in-
tensities of chromatographed compounds were calculated for each TLC plate on
the basis of total number of phosphor counts per plate. Phosphor counts for
MAMES and FAMES were normalized for each TLC plate pair (control and
inhibitor treated). The difference in normalized phosphor counts between each
control and inhibitor treated pair represents the percent change.
Electron microscopy. All chemicals and reagents for electron microscopy were
obtained from Electron Microscopy Sciences, Ft. Washington, Pa. Cultures (50
ml) of BCG were grown to early log phase (optical density at 600 nm, ϳ0.2), at
which time OSA (100 ␮g/ml) or diluent (dimethyl sulfoxide) was added to
treated and control cultures, followed by additional aeration and incubation at
37°C for 24 h. Cells were harvested by low-speed centrifugation and washed in
0.1 M cacodylate (CACO) buffer (pH 7.3). Washed cells were then fixed in 0.1
M CACO buffer (pH 7.3) containing 2.0% glutaraldehyde–osmium tetroxide
(1:1, vol/vol) for 45 min at 4°C (11, 28, 45). Secondary fixation was done at 4°C
Treatment of cultures with OSA and lipid pulse labeling. BCG and MAC cells
were grown in M7H9-ADC-Tween (Difco, Detroit, Mich.) to early log phase.
From this, a 1.0 McFarland suspension was prepared and diluted to yield a final
concentration of 3 ϫ 107 cells/ml in a total volume of 50 ml in M7H9-ADC-
Tween. Cultures were aerated and incubated at 37°C for 24 h (approximately 1
generation time). Each inhibitor was added at its MIC (final concentrations:

VOL. 45, 2001
Organism
EFFECT OF OSA ON LIPID AND MYCOLIC ACID SYNTHESIS
1145
TABLE 1. Susceptibility of different species of mycobacteria
amide (19), were found to be susceptible to OSA, with MICs of
12.5 and 6.25 ␮g/ml, respectively. Similar results were obtained
for cerulenin as previously reported (42). BCG and M. smeg-
matis were susceptible to thiolactomycin at a MIC of 25 and 75
␮g/ml, respectively.
to OSA
MIC (␮g/ml) of OSA
M. bovis .................................................................................
M. bovis BCG.......................................................................
6.25
6.25
M. kansasii ............................................................................ 12.5
M. avium complex ................................................................ 25.0
M. paratuberculosis............................................................... 25.0
M. smegmatis ........................................................................Ͼ100
M. fortuitum..........................................................................Ͼ100
M. chelonei............................................................................Ͼ100
M. abscessus..........................................................................Ͼ100
Overall effects of OSA on mycobacterial lipids. Labeling
assays were conducted at the calculated MIC (6.25 ␮g/ml) of
OSA for BCG and at the highest concentration of compound
tested for M. smegmatis. This particular concentration of OSA
is not equivalent to the lethal concentration of compound in
mycobacteria, as evidenced by continued 14CO2 production in
the radiometric susceptibility test system, which indicated con-
tinued metabolism, albeit at a reduced level relative to those of
controls. OSA had no significant effect on [14C]acetate incor-
poration into nonpolar extractable or polar extractable lipids at
a concentration of 6.25 ␮g/ml, the calculated MIC for M. bovis
BCG, or 100 ␮g/ml for M. smegmatis (Fig. 2). A moderate
decrease in the incorporation of label was observed in sapon-
ifiable lipids in OSA-treated BCG. However, this change was
not statistically significant. Label incorporation into the same
lipid fraction in M. smegmatis was unaltered by exposure to
OSA (Fig. 2). In order to demonstrate a concentration-depen-
dent effect of OSA on lipid metabolism, studies were run at
12.5 ␮g/ml (two times the MIC) and 25.0 ␮g/ml (four times the
MIC). At these higher concentrations, there was a dose-de-
pendent decrease of label incorporation in the saponifiable
lipid fraction with a concomitant increase of label in the non-
polar extractable fraction (data not shown).
Effect of OSA on mycolic acid synthesis as compared with
thiolactomycin. Qualitative and quantitative analysis of myco-
bacterial saponifiable lipids containing MAMES was per-
formed using [14C]acetate pulse-labeling with 2-dimensional
TLC and phosphorimaging. Differences in the effects of OSA
and thiolactomycin were found between BCG and M. smegma-
tis. OSA treatment in BCG resulted in inhibition of all mycolic
acids commonly found in this mycobacterial species (Fig. 3;
Table 3). This decrease or percent change in both ␣- and
keto-mycolates reached greater than 90% with an OSA con-
centration of four times the MIC. Similar results were demon-
overnight in 4.0% formaldehyde–1.0% glutaraldehyde. Samples were post-fixed
at room temperature for 1 h in 0.1 M CACO buffer containing 1% tannic acid
and dehydrated through a graded ethanol series of 50, 70, 95 (twice), and 100%
(three times). Subsequently, samples were infiltrated at room temperature with
a series of Spurrs resins (Spurrs-ethanol [1:1, vol/vol; 2 h], Spurrs-ethanol [2:1,
vol/vol; 2 h], and pure Spurrs [overnight]), and blocks were polymerized at 60°C
for 48 h. Sections were cut on a Sorvall MT2B microtome, and 80-nm-thick
sections were picked up on 200-mesh copper grids and stained with uranyl
acetate and lead citrate. Prepared samples were then analyzed on a Hitachi
HU12A electron microscope.
RESULTS
In vitro susceptibilities. Tables 1 and 2 show the suscepti-
bility of various mycobacterial species and strains to OSA using
the BACTEC radiometric growth system. Nearly all strains of
M. tuberculosis and other slow-growing mycobacterial species,
such as M. bovis, M. bovis BCG, and M. kansasii, were suscep-
tible to OSA, with MICs ranging from 6.25 to 12.5 ␮g/ml. Only
three clinical isolates of M. tuberculosis (CSU 93, 42-1C9383,
and 10129-3), the MAC, and M. paratuberculosis required a
higher OSA MIC of 25 ␮g/ml. None of the rapid-growing
mycobacterial species tested, M. smegmatis, M. fortuitum, M.
chelonei, and M. abscessus, were susceptible to OSA at con-
centrations up to 100 ␮g/ml. Resistance to known antimyco-
bacterial agents did not give cross-resistance to OSA. Three
clinical isolates of M. tuberculosis, resistant to either INH alone
or INH plus rifampin, ethambutol, streptomycin, and pyrazin-
TABLE 2. Activities of OSA and first-line antimycobacterial drugs against various strains of M. tuberculosisa
INH
OSA
RIF
(2.0 ␮g/ml)
EMB
(2.0 ␮g/ml)
STR
(2.0 ␮g/ml)
PZA
(100 ␮g/ml)
M. tuberculosis strain
0.1
␮g/ml
0.4
␮g/ml
6.25
␮g/ml
12.5
␮g/ml
25.0
␮g/ml
H37Rv
S
S
S
S
S
S
S
R
S
S
R
R
S
S
S
S
S
S
S
S
S
S
R
S
S
R
R
S
S
S
S
S
S
S
S
S
S
R
S
S
S
S
S
S
S
S
S
S
S
S
S
S
R
S
S
S
S
S
S
S
S
S
S
S
S
S
S
R
S
S
S
S
S
S
S
S
S
S
S
S
S
S
R
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
R
R
R
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
5D5178
1D4924
1H1337
42401315
6T2709
S
S
S
S
S
1T2768
S
TBL54 EP066
7D5245
OT2769
42-1C9383
10129-3
CSU93
S
S
R
R
R
R
R
R
OT2724
401296
a S, susceptible; R, resistant; RIF, rifampin; EMB, ethambutol; STR, streptomycin; PZA, pyrazinamide.

1146
PARRISH ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 2. Effects of OSA on the synthesis of various lipid fractions in M. bovis BCG and M. smegmatis. Incorporation of [1,2-14C]acetate into
extractable polar, nonpolar, and saponifiable lipids in the presence and absence of OSA. Abbreviations: NPF, nonpolar extractable lipids; PLF,
polar extractable lipids; SF, saponifiable lipids, including mycolic acids. Concentrations of OSA used: for M. smegmatis, 100 ␮g/ml; for M. bovis
BCG, 12.5 ␮g/ml. Differences were not statistically significant.
strated for MAC (data not shown). However, in M. smegmatis,
individual mycolate classes were only slightly inhibited follow-
ing exposure to OSA (Fig. 4; Table 3). Other lipids present in
the saponifiable fraction included FAMES. No appreciable
change was observed in this lipid class following OSA treat-
ment in either of the two mycobacterial species characterized
in this study (Fig. 3 and 4; Table 3).
Control organisms exhibited an intact cell wall and clearly
defined septum, whereas in the presence of OSA, cell wall
synthesis was disrupted with incomplete septum formation. In
addition, outer-wall-associated lipids appeared to be dispersed
from the electron transparent zone of the mycolic acids.
DISCUSSION
Thiolactomycin caused a decrease in all mycolate species in
BCG (Fig. 3; Table 3) and M. smegmatis. However, while
thiolactomycin uniformly inhibited all mycolate species in
BCG, in M. smegmatis, a differential effect was observed be-
tween individual mycolic acid classes. Both ␣-mycolates and
epoxymycolates were nearly completely diminished (95 and
87%, respectively), whereas ␣Ј-mycolates were less affected
(57%) (Fig. 4; Table 3). In contrast to OSA, FAMES accumu-
lated in both BCG and M. smegmatis following treatment with
thiolactomycin.
In this study, we demonstrate that OSA, a compound de-
signed to inhibit fatty acid synthesis by mimicking the transi-
tion state of the ␤-ketoacyl synthase, is inhibitory for a broad
range of slow-growing mycobacterial species, including multi-
drug-resistant M. tuberculosis. OSA treatment reduced mycolic
acid accumulation in BCG and MAC cells, presumably by its
effect on FAS systems in these mycobacteria. Cross-resistance
was not observed for isolates resistant to isoniazid, rifampin,
ethambutol, streptomycin, and pyrazinamide. A comparison of
isoniazid, a known inhibitor of mycolic acid synthesis, and OSA
revealed pertinent information. Isoniazid has been shown to
inhibit both InhA, an enoyl-ACP reductase involved in fatty
acid elongation, and KasA, a ␤-ketoacyl–ACP synthase (2, 12,
Transmission electron microscopy of OSA-treated BCG. In-
hibition of mycolic acid synthesis is known to disrupt the my-
cobacterial cell wall. Figure 5 shows transmission electron mi-
crographs of OSA-treated BCG cells during cell division.

VOL. 45, 2001
EFFECT OF OSA ON LIPID AND MYCOLIC ACID SYNTHESIS
1147
FIG. 4. Two-dimensional TLC showing the comparative effects of
OSA and thiolactomycin on the mycolic acids of M. smegmatis. First
dimension, petroleum ether (bp 60 to 80°C)-acetone (95:5, vol/vol;
three times); second dimension, toluene-acetone (97:3, vol/vol; one
time). Abbreviations: ori, origin; ␣, ␣-mycolate; ␣Ј, ␣Ј-mycolate; e,
epoxymycolate. Equivalent counts per minute of the saponifiable lipid
fraction were spotted at each origin.
FIG. 3. Two-dimensional TLC showing the comparative effects of
OSA and thiolactomycin on the mycolic acids of M. bovis BCG. First
dimension, petroleum ether (bp 60 to 80°C)–acetone (95:5, vol/vol;
three times); second dimension, toluene-acetone (97:3, vol/vol; one
time). Abbreviations: ori, origin; ␣, ␣-mycolate; k, keto-mycolate.
Equivalent counts per minute of the saponifiable lipid fraction were
spotted at each origin.
it has not been tested as an inhibitor of isolated FAS. In an
earlier, related study conducted in our laboratory, structurally
related sulfones and sulfoxides were found to inhibit FAS I
isolated from M. smegmatis (41). Several of these compounds
were both FAS I inhibitors and active against M. tuberculosis
H37Rv in the radiometric growth assay. However, the corre-
lation was not exact and issues of cell wall permeability and
solubility prevented direct comparison of the data. OSA was
selected for further study based on its solubility and its perfor-
mance in whole-cell assays.
The differential susceptibility to OSA observed between
slow- and rapid-growing mycobacterial species argues for the
presence of unique targets in BCG and MAC. M. smegmatis
may contain the same OSA target as BCG and MAC but
possesses alternate cell wall compounds that permit survival in
spite of OSA inhibition. Another potential possibility is the
requirement for alteration, i.e., activation, of OSA prior to its
interaction with the target protein(s) which may occur in slow-
growing mycobacteria but not in rapid-growing species. How-
ever, the most probable interpretation is that differences in the
mycolic acid biosynthetic pathway may exist between mycobac-
terial species. This possibility is further strengthened when the
work of other investigators is considered in conjunction with
our own. For example, InhA, a long chain, enoyl-ACP-depen-
dent reductase involved in fatty acid elongation, is present in
both M. smegmatis and M. tuberculosis (31). Several studies
have revealed compelling evidence that InhA is the target for
KatG-activated INH in M. smegmatis (12, 44). However, addi-
tional investigations suggest that this particular enzyme is not
the principal target for KatG-activated INH in M. tuberculosis
28, 31, 32). INH resistance has been associated with mutations
in both of these genes, as well as the KatG gene, which encodes
the mycobacterial catalase-peroxidase (54, 55, 57, 58). In this
study, the mechanism of INH resistance for the M. tuberculosis
isolates used was not determined. Of relevance is the finding
that OSA inhibited the growth of INH-resistant M. tuberculosis
(Ͼ0.04 ␮g/ml) as well as MAC, typically resistant to INH (Ͼ2.5
␮g/ml) (19). This observation suggests that the inhibition of
mycolic acid synthesis by OSA may be due to interaction with
an enzymatic target different from that of INH, indicating a
novel and possibly unexploited mechanism of action of OSA in
M. tuberculosis.
Although OSA was designed to inhibit ␤-ketoacyl synthases,
TABLE 3. Effects of OSA and thiolactomycin on the mycolic acids
of M. bovis BCG and M. smegmatis
Inhibition
(% change)
Organism
Mycolic acid species
OSA
TLM
M. bovis BCG
␣
Ϫ56
Ϫ52
Ϫ69
Ϫ79
ϩ147
keto
FAMES
a
—
M. smegmatis
␣
␣Ј
epoxy
FAMES
Ϫ19
Ϫ18
Ϫ8
Ϫ95
Ϫ57
Ϫ87
—
ϩ134
a —, no appreciable change was observed.

1148
PARRISH ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 5. Transmission electron microscopy of OSA-treated M. bovis BCG (6.25 ␮g/ml). (Top) Intact cell wall ultrastructure (left) (magnification,
ϫ117,000) and completed septum (right) (magnification, ϫ108,000). (Bottom) Disrupted cell wall ultrastructure (left) (magnification, ϫ130,000)
and incomplete septum formation (right) (magnification, ϫ78,000).
(31, 32). This discrepancy may reflect inherent differences in
mycolate biosynthesis between the two organisms (31, 32, 42).
Additionally, previous studies in our laboratory examining the
effect of cerulenin on mycolate synthesis in M. smegmatis and
BCG revealed clear differences in mycolic acid profiles be-
tween these two mycobacterial species following inhibitor ex-
posure. Not only did the changes in mycolate synthesis differ
between BCG and M. smegmatis, they were in direct opposi-
tion. For instance, completed mycolates decreased in BCG
following exposure to cerulenin, whereas in M. smegmatis, all
mycolate species increased, again suggesting that inherent dif-
ferences in the mycolate biosynthetic pathway between the two
species are responsible for these disparate responses (42).
This possibility is further strengthened by the differential
effect of thiolactomycin on individual mycolic acid classes in M.
smegmatis. In both the present study and that of other inves-
tigators (50), exposure to thiolactomycin resulted in substan-
tially decreased amounts of ␣-mycolates and epoxymycolates,
with a minor decrease in ␣Ј-mycolates (49). The authors of the
previous study suggested that potential targets for thiolacto-
mycin in this mycobacterial species include an elongation en-
zyme leading from either the C24:1⌬5 intermediate or from the
shorter-chain ␣Ј-mycolates to the longer ␣-mycolates and ox-
ygenated mycolates (50). In view of the current experimental
evidence, the latter seems to be the more likely possibility. It
should be noted that ␣Ј-mycolates, commonly found in rapid-
growing mycobacteria, are not present in BCG and other slow-
growing mycobacteria characterized in this study (18). Thus,

VOL. 45, 2001
EFFECT OF OSA ON LIPID AND MYCOLIC ACID SYNTHESIS
1149
while thiolactomycin treatment of BCG and M. smegmatis re-
sulted in inhibition of both ␣-mycolates and oxygenated my-
colates, the presence of ␣Ј-mycolic acids in the latter case may
partially explain the differences observed in MICs between the
two mycobacterial species (for BCG, 25.0 ␮g/ml; for M. smeg-
matis, 75 ␮g/ml) and indicate that disparities in mycolate bio-
synthesis may exist between BCG and M. smegmatis. One could
speculate that such differences may extend to other slow- ver-
sus rapid-growing mycobacterial species.
saturated alkyl intermediate(s) in mycolic acid synthesis (31).
While 2-dimensional TLC of OSA-treated BCG revealed that
mycolic acid synthesis was inhibited, no apparent effect was
seen in the FAMES present in this fraction when the com-
pound was used at the MIC (6.25 ␮g/ml). A similar observation
was noted in OSA-treated M. smegmatis. However, at four
times the MIC, OSA treatment of BCG resulted in an increase
in label incorporation into extractable nonpolar lipids. This
may suggest that a noncovalently bound, extractable interme-
diate in mycolate synthesis accumulates following OSA treat-
ment, an effect intensified with higher concentrations (four
times the MIC) of compound. In contrast, FAMES increased
in thiolactomycin-treated BCG, while mycolic acids decreased,
a finding consistent with that of earlier studies using cerulenin
(42). Thus, in BCG, while completed mycolates decreased with
OSA, thiolactomycin, and cerulenin (42), the changes in
FAMES were clearly not the same, suggesting that inhibition
of mycolic acid synthesis in BCG may occur prior to synthesis
of the saturated alkyl intermediate with OSA, but between this
intermediate and completed mycolates with cerulenin and
thiolactomycin. Alternatively, OSA-mediated inhibition of my-
colate synthesis in BCG and MAC may involve an as-yet-
unidentified enzyme or enzyme system. In summary, the effects
of OSA, cerulenin, and thiolactomycin are mycobacterial spe-
cies specific and compound specific and inherent differences in
the mycolic acid biosynthetic pathway may exist between rapid-
and slow-growing mycobacteria.
Differences in mycolic acid profiles following OSA treat-
ment in BCG and M. smegmatis were also noted. In the present
study, all mycolic acids were significantly and uniformly inhib-
ited in OSA-treated BCG (␣- and keto-mycolates). This inhi-
bition increased with an increasing concentration of OSA. In
contrast, the effect of OSA on the mycolates of M. smegmatis
(␣Ј, ␣, and epoxy) was negligible and not inhibitory to growth.
Previous studies have suggested the existence of multiple ACP-
dependent FAS II systems in mycobacteria, responsible for not
only fatty acid biosynthesis but also mycolic acid biosynthesis
(2, 32, 42, 44). Such systems could be envisioned to interact
with separate and distinct ␤-ketoacyl–ACP synthases as well as
other enzymes involved in biosynthetic reactions of this type,
including ␤-ketoacyl–ACP reductases, ␤-hydroxyacyl–ACP de-
hydratases, and ␤-enoyl–ACP reductases. A biological prece-
dent for the existence of such enzymes has been described for
Esherichia coli, in which multiple ␤-ketoacyl–ACP synthases
have been found (21, 26, 27). Although thiolactomycin was
originally thought to inhibit all three ␤-ketoacyl–ACP syn-
thases in E. coli, recent evidence suggests that the principal
target of thiolactomycin in this particular organism may be
only ␤-ketoacyl–ACP I (25). Since OSA was designed to inhibit
the ␤-ketoacyl synthase by mimicking the transition state of the
reaction catalyzed by this enzyme, this compound could in
theory inhibit both the multifunctional FAS I and monofunc-
tional FAS II mycobacterial systems, as in the case of cerule-
nin. However, in this study, additional assays were performed
which were designed to indirectly determine FAS I activity in
the presence of each inhibitor by measuring phospholipid pro-
duction. Only cerulenin, known to inhibit both FAS I and FAS
II systems, interfered with phospholipid synthesis in either
BCG or M. smegmatis (42). Neither thiolactomycin, active only
against FAS II systems, nor OSA inhibited phospholipid syn-
thesis in either of the two mycobacterial species tested (data
not shown), suggesting that the principal target of OSA may lie
in an ACP-dependent FAS II system. In addition, previous
work in our laboratory and others has demonstrated that ceru-
lenin and thiolactomycin strongly inhibited [14C]acetate incor-
poration into other extractable mycobacterial lipids, a finding
consistent with the known mechanism of action of both inhib-
itors. In contrast, OSA inhibited only mycolic acids, with no
appreciable change in label incorporation in any of the other
mycobacterial lipid classes tested. This distinction suggests the
presence of a unique and highly specific target for this com-
pound in slow-growing mycobacteria. Such a target may in-
volve an as-yet-unidentified enzyme or enzyme system present
in slow-growing mycobacteria which is not present or inactive
in rapid-growing species.
ACKNOWLEDGMENTS
We thank Janet Folmer for performance of electron microscopy and
William Bishai for providing equipment and laboratory space for some
portions of this study.
This study was financially supported in part by NIH grant AI43846,
the Raynam Research Fund, and the Becton Dickinson Centennial
Fellowship in Clinical Microbiology (N.M.P.).
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