0 hc y64 ff3 fs7 fc0 sc0 ls1 ws4">the difference in radioactive counts per minute (cpm)
between tubes incubated at 37˚C and negative controls
kept on ice.
2.4. Adenosine Triphosphate (ATP)
Mycobacterial ATP concentrations were determined us-
ing a sensitive luciferase chemiluminescence procedure
(BacTiterGlo Microbial Cell Viability Assay, Promega,
Madison, WI, USA). Briefly, the bacteria at a concentra-
tion of 1 × 107 cfu/mL in 10 mL K0N0 were incubated for
60 min at 37˚C without and with 7-MJ (0.023 - 1.5 mg/L).
The bacteria were then concentrated by centrifugation
and mixed with an equal volume of BacTiterGlo solu-
tion, which contains the bacteriolytic constituent, for 5
min at room temperature. The lysates were then assayed
for ATP using a 20/20n chemiluminometer (Turner Bio-
systems Inc., Sunnyvale, CA, USA) and the results ex-
pressed as relative light units (rlu).
2.5. Statistical Analysis
The results are expressed as the mean ± standard devia-
tion (SD) for 3 experiments, with at least 3 replicates for
each concentration of the test agents or control systems
in each experiment. Levels of statistical significance were
calculated using the Student’s paired t-test. Differences
were considered significant if the probability value (P)
was less than 0.05.
3. Results
3.1. Effects of 7-MJ on Uptake of 86Rb+ by MTB
Exposure of MTB to 7-MJ resulted in dose-related inha-
bitation of the uptake of 86Rb+ which achieved statistical
significance at a concentration of 0.094 mg/L, with com-
plete inhibition observed at >0.375 mg/L of this agent
(Figure 1).
3.2. Effects of 7-MJ on Mycobacterial ATP
Levels
Exposure of MTB to 7-MJ at the lowest concentration
tested (0.023 mg/L) resulted in a significant (P < 0.05)
increase in microbial ATP levels, followed by a progress-
ive decline, being almost undetectable at 7-MJ concen-
trations of 0.375 mg/L (Figure 2).
4. Discussion
The antimicrobial activity of 1,4-naphthoquinones is well
recognised and appears to result from the electrophilic
addition of these agents to nucleic acids and proteins, as
well as by intracellular redox cycling mechanisms, re-
sulting in the generation of toxic reactive oxygen species
(ROS) such as superoxide and hydrogen peroxide (H2O2)
[6,7]. Although the relative lack of selectivity of these
agents for prokaryotes presents a challenge in respect of
CONTROL
7-MJ [0.023]
7-MJ [0.047]
7-MJ [0.094]
7-MJ [0.188]
7-MJ [0.375]
7-MJ [0.75]
7-MJ [1.5]
0
25
50
75
100
**
*
*
***
86
Rubidium
+
upt ake
(% of control)
Figure 1. The effect of 7-methyljuglone (7-MJ) on the up-
take of K+ by the H37Rv strain of MTB. The results are
from one experiment with five replicates for each concen-
tration and are representative of 3 different experiments
showing similar trends in each. The results are e xpressed as
the mean percentages for uptake of 86Rb+ of the corre-
sponding compound-free control systems SD; the absolute
value of the control system is 100,859 counts per minute. *P
values < 0.05 when compared to the solvent control system.
Copyright © 2012 SciRes. OJRD
V. THAVER ET AL.
Copyright © 2012 SciRes. OJRD
62
CONTROL
7-MJ [0.023]
7-MJ [0.094]
7-MJ [0.375]
7-MJ [1.5]
0
100
200
300
*
*
*
ATP levels
( % of control)
Figure 2. The effect of 7-methyljuglone (7-MJ) on the levels
of ATP in the H37Rv strain of MTB. The data shown are
that of one experiment with five replicates for each concen-
tration and are representative of 3 different experiments
showing similar trends. The results are expressed as the
mean percentages of the corresponding compound-free
control systems SD; the absolute value of the control sys-
tem is 2,306,410 relative light units. *P values < 0.05 when
compared to the solvent control.
clinical development, 7-MJ is a noteworthy exception [4].
The MIC value of this agent for MTB is 0.5 mg/L, which
is significantly lower than its IC50 value (30.0 mg/L) for
eukaryotic cell lines in vitro [4]. Nonetheless, relatively
little is known about either the primary targets or rapidity
of onset of anti-mycobacterial activity of this agent.
In the current study, a relatively brief exposure (30 -
60 min) of MTB to 7-MJ resulted in significant, dose
-related inhibition of both microbial energy metabolism
and uptake of K+, which, in both cases was maximal at
concentrations close to the MIC value. In the case of
ATP levels, exposure of MTB to 7-MJ at a concentration
of 0.0263 mg/L resulted in a significant increase in ATP,
with a progressive, dose-related decrease at higher con-
centrations. The increase may represent a stress response
to moderate oxidative trauma caused by low concentra-
tions of 7-MJ as described for other types of antimicro-
bial agents [6,7], while at higher concentrations, ire-
versible ROS-mediated toxicity predominates. Alterna-
tively, albeit speculatively, 7-MJ may interfere with the
activity of mycobacterial type 2 NADH: quinone oxi-
doreductase, an early step in the mycobacterial respira-
tory chain [8].
Inhibition of mycobacterial K+ transport by 7-MJ
closely paralleled interference with microbial energy
metabolism, and is probably secondary to ATP depletion.
MTB possesses two major K+ uptake systems. These are
the Kdp and Trk A/B systems, driven by ATP and proton
motive force, respectively [5]. The experimental condi-
tions used in the current study (low K+ medium) are
likely to favour preferential utilisation of the inducible,
high-affinity, Kdp system, accounting for the susceptibil-
ity of K+ transport to ATP depletion, which in turn may
lead to inactivation of the Trk A/B system due to dissipa-
tion of the membrane potential.
In conclusion, 7-MJ appears to target mycobacterial
energy metabolism, leading to secondary membrane dys-
function and inhibition of bacterial growth. This agent
may serve as a prototype for the development of novel
naphthoquinone-based anti-tuberculosis agents.
5. Acknowledgements
We thank Dr. Anita Mahapatra for supplying the chemi-
cal compound.
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