Investigation of the Anti-Mycobacterial Mechanism of Action of 7-Methyljuglone

doi:10.4236/ojrd.2012.22009

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Open Journal of Respiratory Diseases, 2012, 2, 60-62

http://dx.doi.org/10.4236/ojrd.2012.22009 Published Online May 2012 (http://www.SciRP.org/journal/ojrd)

Investigation of the Anti-Mycobacterial Mechanism of

Action of 7-Methyljuglo n e *

Veneesha Thaver1,2,3,4, J. J. M. Meyer 3, Riana Cockeran2#, Moloko C. Cholo2,

Ronald Anderson2, Namrita Lall3

1Department of Physiology, University of Limpopo, Medical University of South Africa Campus, Pretoria, South Africa

2MRC Unit for Inflammation and Immunity, Department of Immunology, Faculty of Health Sciences, University of Pretoria

and Tshwane Academic Division of the National Health Laboratory Service, Pretoria, South Africa

3Department of Plant Science, Faculty of Natural and Agricultural Sciences, University of Pretoria, Pretoria, South Africa

4Department of Physiology, School of Laboratory Medicine and Medical Sciences, University of KwaZulu Natal, Westville

Campus, Durban, South Africa

Email: #[email protected]

Received February 20, 2012; revised March 19, 2012; accepted March 26, 2012

ABSTRACT

Objectives: Although the naphthoquinone, 7-methyljuglone (7-MJ), is active against Mycobacterium tuberculosis

(MTB) in vitro, neither the cellular site nor mechanism of anti-mycobacterial action of this agent has been identified.

The primary objective of the current study was to investigate the mycobacterial outer membrane as a potential target of

7-MJ by measuring the effects of this agent (0.023 - 1.5 mg/L) on microbial ATP levels and uptake of K+. Methods:

Bioluminescence and radiometric (uptake of 86Rb+) procedures were used to assay microbial ATP levels and K+ trans-

port respectively. Results: Exposure of MTB (strain H37Rv) to 7-MJ for 60 min resulted in dose-related decreases in

both microbial ATP levels and uptake of 86Rb+ which achieved statistical significance (P < 0.05) at concentrations of

0.4 and 0.1 mg/L respectively. Conclusions: These observations are compatible with the mycobacterial membrane as

being the putative site of action of 7-MJ, targeting microbial energy metabolism and K+ transport.

Keywords: Adenosine Triphosphate; Cell Membrane; Energy Metabolism; Potassium

1. Introduction

Tuberculosis (TB) remains a major world health problem,

in particular since the incidence of multi-drug-resistant

tuberculosis has increased in many countries. Conse-

quently, discovery of new drugs with novel modes of

action represents a major challenge [1].

Euclea natalensis is a shrub or small to medium size

tree, which occurs in a variety of habitats, including

coastal and inland forests, as well as bushveld in southern

Africa. The roots of E. natalensis are used by indigenous

people of southern Africa to treat various bacterial infec-

tions. Powdered root bark of this species is used as an

ingredient in medicines to treat urinary tract infections,

venereal diseases and dysmenorrhoea [2]. The Zulus, a

South African tribe, use the root bark to treat respiratory

diseases such as TB, bronchitis, pleurisy and asthma [3].

Several secondary metabolites such asterpenoids, naph-

thoquinones etc. have been isolated from E. natalensis

[3]. According to previous studies, the minimum inhibit-

tory concentration (MIC) of a naphthoquinone isolated

from the roots of the plant, methyljuglone (7-MJ),

against a drug-sensitive strain of Mycobacterium tuber-

culosis (MTB) was very significant and comparable to

some of the existing antituberculosis drugs (MIC: 0.5

mg/L) [4].

The primary objective of the current study was to in-

vestigate the mycobacterial outer membrane as a poten-

tial target of 7-MJ by measuring the effects of this agent

on microbial ATP levels and uptake of potassium (K+).

2. Materials and Methods

2.1. 7-Methyljuglon e (7-MJ)

7-MJ was dissolved in dimethyl sulphoxide (DMSO) and

used at a final concentration range of 0.023 - 1.5 mg/L in

the assays described below. Appropriate solvent controls

(0.15% DMSO) were included in all experiments.

*Funding: This study was supported by National Research Foundation

and the Medical Research Council, Pretoria, South Africa.

Transparency declarations: Nothing to declare.

#Corresponding author.

2.2. Mycobacterium tuberculosis

MTB strain H37Rv, ATCC 26518 was provided by the

V. THAVER ET AL. 61

Inflammation and Immunity Research Unit of the South

African Medical Research Council (Pretoria, South Af-

rica). MTB was grown for 7 days at 37˚C in OADC (oleic

acid, albumin dextrose, catalase)-supplemented Middle-

brook 7H9 nutrient broth (Difco, Detroit, MI, USA) con-

taining 0.05% Tween 80.

2.3. Potassium (K+) Uptake

86Rb+ (rubidium-86 chloride, 37 MBq, PerkinElmer Ra-

diochemicals, Boston, MA, USA) was used as a surrogate

tracer for measurement of uptake of K+ by MTB as pre-

viously described [5]. Briefly, following 7 days of cul-

ture, MTB was harvested from the bacteriological culture

medium by centrifugation (2500 g/15 min), washed and

resuspended in glucose- and K+-free minimal medium

(K0N0 buffer, pH 7.4) to deplete intracellular K+ [5]. The

bacterial suspension was adjusted turbidometrically to a

concentration of 1 × 107 colony forming units (cfu)/mL.

Approximately 2 × 106 cfu/mL of MTB were then treated

with 7-MJ (0.023 - 1.5 mg/L) and incubated at 37˚C for

30 min, then pelleted by centrifugation, and resuspended

in 2 mL K0N0 containing 86Rb+ (2 mCi/L) and glucose

(22 mM) and a further incubation period of 90 min at

37˚C. The reaction was terminated by the addition of

ice-cold phosphate buffered saline (PBS, 0.15 mM, pH

7.4, 100 mM cold K+), and washed twice with ice-cold

PBS. The pellets were disrupted by adding 0.4 mL warm

5% trichloroacetic acid (Merck, Darmstadt, Germany).

Radioactivity was assayed using a liquid scintillation

spectrometer (Tri-Carb 2100 TR, Packard Instrument Co,

Meriden, CT, USA) and net uptake of 86Rb+ expressed as

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]

100

***

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.

V. THAVER ET AL.

CONTROL

7-MJ [0.023]

7-MJ [0.094]

7-MJ [0.375]

7-MJ [1.5]

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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