Inhibition of potassium transport and growth of mycobacteria exposed to clofazimine and B669 is associated with a calcium-independent increase in microbial phospholipase A2 activity

H. C. Steel*, N. M. Matlola and R. Anderson

Medical Research Council Unit for Inflammation and Immunity, Department of Immunology, Institute for Pathology, University of Pretoria, Pretoria, South Africa


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Altered phospholipase A2 (PLA2) activity and its relationship to cation (K+, Ca2+) uptake and growth were investigated in mycobacteria exposed to the riminophenazine antimicrobial agents, clofazimine and B669 (0.15–2.5 mg/L). Microbial PLA2 activity was measured using a radiometric thin-layer chromatography procedure, whereas K+ and Ca2+ transport were measured using 86Rb+ or 42K+ and 45Ca2+, respectively. Short-term exposure (15–30 min) of Mycobacterium aurum A+ or the virulent and avirulent isolates of Mycobacterium tuberculosis H37R to the riminophenazines resulted in dose-related enhancement of microbial PLA2 activity, which was associated with inhibition of K+ influx and growth. Uptake of Ca2+ by mycobacteria was unaffected, or minimally affected, by the riminophenazines at concentrations of <=0.6 mg/L, whereas higher concentrations resulted in increased uptake of the cation in the setting of decreased microbial ATP concentrations. The results of kinetic studies using a fixed concentration (2.5 mg/L) of B669 demonstrated that riminophenazine-mediated enhancement of PLA2 activity and inhibition of K+ uptake in mycobacteria are rapid and probably related events that precede, by several minutes, any detectable effects on microbial ATP concentrations and uptake of Ca2+. Inclusion of the extracellular and intracellular Ca2+-chelating agents EGTA (0.2–7.2 g/L) and BAPTA/FURA-2 (0.2–9.5 mg/L), individually or in combination, did not prevent the effects of B669 on mycobacterial PLA2 activity or K+ transport, whereas {alpha}-tocopherol, which neutralizes PLA2 primary hydrolysis products, antagonized the inhibitory effects of the riminophenazines on microbial K+ uptake and growth. These results demonstrate that the antimycobacterial activities of clofazimine and B669 are related to a Ca2+-independent increase in mycobacterial PLA2, leading to interference with microbial K+ transport.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
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Although the antimycobacterial riminophenazine agent clofazimine has been in clinical use for several decades, primarily as an antileprosy agent, 1,2 the biochemical–molecular basis of its antimicrobial action remains to be conclusively established.

We have previously reported that clofazimine and its derivative, B669, are broadly active against Gram-positive bacteria, whereas Gram-negative bacteria are uniformly resistant to these agents. 3 In these previous studies we observed that brief exposure to the riminophenazines was accompanied by increased activity of microbial PLA2 in both Gram-positive and Gram-negative bacteria. 3 The relationship between riminophenazine-mediated enhancement of PLA2 activity and inhibition of the growth of Gram-positive bacteria was strengthened by the observation that {alpha}-tocopherol and lysophospholipase, both of which neutralize lysophospholipids, 4 antagonized the antimicrobial action of both clofazimine and B669. 3 Lysophospholipids are generated during the cleavage of membrane phospholipids by PLA2, and have potent membrane-destabilizing effects on eukaryotic cells, as well as inhibitory effects on Na+K+-ATPase, the primary K+ transporter in these cells. 5,6 More recently, we have reported that the riminophenazines inhibit K+ transport in Gram-positive, but not in Gram-negative bacteria. 7 The resistance of Gram-negative bacteria to the riminophenazines appeared to be related to the range and diversity of their K+ transporters, 8,9 only one of which, the Kup system, was affected by clofazimine and B669. 7 This is in contrast to Gram-positive bacteria, which possess only a single K+-uptake system operative under normal culture conditions. 10

In the present study we have addressed important, unresolved aspects of the antimicrobial mechanism of action of the riminophenazines, using non-pathogenic and pathogenic mycobacteria, the primary chemotherapeutic targets of these agents. Most importantly, the time-courses of altered PLA2 activity and K+ transport in clofazimine- and B669-treated mycobacteria have been examined and compared. The involvement of extracellular and intracellular Ca2+ in riminophenazine-mediated dysregulation of PLA2 and uptake of K+ in mycobacteria was also investigated.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antimicrobial agents

Clofazimine (3-(p-chloroanilino)-10-(p-chlorophenyl)-2,10-dihydro-2-(isopropylimino)-phenazine) and B669 (3-anilino-10-phenyl-2,10-dihydro-2-(cyclohexylimino)-phenazine) were synthesized by Dr J. F. O'Sullivan (Department of Chemistry, University College, Dublin, Republic of Ireland). Clofazimine, also known as B663, and B669 were dissolved in dimethyl sulphoxide (DMSO) to give a stock concentration of 2 g/L. Subsequent dilutions were made in DMSO and both riminophenazines were used at final concentrations ranging from 0.15 to 2.5 mg/L. Appropriate solvent controls were included in the various assays described.

Chemicals and reagents

Unless otherwise indicated, all chemicals used were obtained from the Sigma Chemical Co. (St Louis, MO, USA). Radiochemicals were purchased from Du Pont–NEN Research Products (Boston, MA, USA).

Bacteria

The mycobacterial reference isolates used in this study were Mycobacterium aurum A+, provided by the Glaxo–Wellcome Medicines Research Centre (Stevenage, UK), and the virulent and avirulent isolates of Mycobacterium tuberculosisH37R, which were cultured and provided by the Tuberculosis Research Institute of the South African Medical Research Council (Pretoria, South Africa). Only a limited number of confirmatory experiments were performed using Mycobacterium tuberculosisH37Rv.

Measurement of bacterial growth

The effects of the riminophenazines on the growth of the test mycobacterial isolates were investigated using the BACTEC TB system (Becton Dickinson Diagnostic Instrument Systems, Towson, MD, USA). Following culture of M. aurum A+ for 4 days and M. tuberculosis H37R isolates for 14 days in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI, USA), the bacterial cells were resuspended in 0.15 M phosphate-buffered saline (PBS), pH 7.4, to obtain a McFarland Standard Number 1 (3 x 108 cfu/mL). These suspensions were diluted 10-fold and five-fold in the case of M. aurumA+ and the M. tuberculosisH37R isolates, respectively. One hundred microlitres of the diluted suspensions were inoculated into vials containing 12B TB medium (Becton Dickinson & Co., Cockeysville, MD, USA) with and without the riminophenazines (0.15–2.5 mg/L).

The effects of ouabain (0.07–0.15 g/L), a potent inhibitor of the Na+K+-ATPase of eukaryotic cells, 11 on the growth of the mycobacteria were also investigated.

K+ transport studies

86Rb+ (rubidium-86 chloride, 37 MBq) and 42K+ (potassium-42 carbonate, 74 MBq, South African Atomic Energy Corporation, Pretoria, South Africa) were used as tracers for measuring K+ uptake. 86Rb+ has been described in several previous studies as being a useful tracer for the measurement of microbial transport of K+.7,1214 In the present study, the suitability of using 86Rb+ for the various test bacteria was investigated in a series of preliminary experiments (results not shown) in which it was established that the kinetics of uptake of 86Rb+ and 42K+ were comparable and that the uptake of 86Rb+ was inhibited in a dose-related manner by the addition of increasing amounts (0.1–10 mM) of cold potassium chloride (KCl). Moreover, data on the effects of the test antimicrobial agents obtained with 86Rb+ as tracer were confirmed using 42K+ in a limited number of experiments.

With the exception of the bacterial concentrations used (0.5 x 107 and 1 x 107 cfu/mL for M. aurum A+ and the M. tuberculosis H37R isolates, respectively), the uptake of K+ was determined as described previously. 7 The effects of the riminophenazines (0.15–2.5 mg/L) on bacterial K+ transport were assessed using 45 min and 90 min incubation times for M. aurum A+ and the M. tuberculosis H37R isolates, respectively.

In an additional series of experiments, the effects of several other commonly used antimycobacterial agents, namely, streptomycin, rifampicin, ethambutol, pyrazinamide and isoniazid at two to five times their MIC (5, 5, 5, 20 and 0.25 mg/L, respectively), as well as those of ouabain (0.07–0.15 g/L) were investigated.

Kinetics of K+ influx and efflux

Because apparent inhibition of K+ transport may be due to decreased influx and/or accelerated efflux of the cation, the effects of B663 and B669 (0.6 mg/L) on the kinetics of influx and efflux of K+ were investigated in the mycobacteria using 42K+ as tracer. The influx and efflux of K+ in M. aurum A+ and the M. tuberculosisH37R isolates were determined as described previously. 7 The kinetics of K+ transport in control and riminophenazine-treated bacteria was measured after 0, 5, 10, 20, 30, 45, 60 and 90 min at 37°C.

In an additional set of influx kinetics experiments, the effect of B669 (2.5 mg/L) on the uptake of 86Rb+ by M. aurumA+ was measured after short exposure times (0, 10 and 30 s, and 1, 3 and 5 min).

Ca2+ uptake studies

The M. aurum A+ and M. tuberculosis H37R isolates were cultured for 4 days and 14 days, respectively, before being harvested, washed and resuspended in Ca2+-free Hanks balanced salt solution (HBSS) (Highveld Biological (Pty) Ltd, Kelvin, South Africa) to a concentration of 1 x 107 for M. aurum A+ and 2 x 107 cfu/mL for the M. tuberculosis H37R isolates. The bacteria were then exposed to 45Ca2+ 4 mCi/L (calcium-45 chloride, 185 MBq), containing 20 µM cold carrier CaCl2, for 15 min at 37°C. After the addition of B663 or B669 (0.15–2.5 mg/L), the mycobacteria were incubated for a further 15 min at 37°C. They were then washed with ice-cold Ca2+-supplemented HBSS and, after disruption of the pellets by the addition of 0.4 ml of warm 5% TCA, the incorporated 45Ca2+ was determined in a liquid scintillation spectrometer. To eliminate the complicating effects of non-specific binding of the radiolabelled cation to the bacteria, net uptake of 45Ca2+ was taken as the difference in uptake of 45Ca2+ in the tubes at 37°C and the controls kept on ice. In kinetic studies, the time-course of Ca2+ uptake by control and B669 (0.6 and 2.5 mg/L)-treated bacteria was measured at 30 s and 1, 3, 5 and 15 min after addition of the riminophenazines.

Phospholipase A2 activity

PLA2 (phosphatidylcholine 2-acyl-hydrolase) activity in mycobacteria suspended (1–2 x 107 cfu/mL) in HBSS containing 1.25 mM CaCl2 was measured according to the release of [ 14C]arachidonate from the sn-2 position of added phosphatidylcholine (L-{alpha}-1-palmitoyl-2-arachidonyl, (arachidonyl-1-14C), 1.48–2.22 GBq, 0.5 mCi/L). The mycobacteria were treated with the riminophenazines (0.15–2.5 mg/L) for 30 min at 37°C, after which [14C]arachidonate was extracted and measured using high-performance thin layer chromatography. 3,15

In kinetic experiments, the effects of B669 (2.5 mg/L) on the PLA2 activity in M. aurumA+ at early time intervals (0, 10 and 30 s, and 1, 3 and 5 min) were investigated.

ATP concentrations

Microbial ATP concentrations were determined using a sensitive luciferin–luciferase chemiluminescence method. 16,17 The bacterial cells (0.01 mg protein per 10 mL K0N0 buffer) were coincubated for varying times (0 and 30 s, and 1, 3, 5, 10, 15 and 30 min) at 37°C with or without the riminophenazines (0.15 and 2.5 mg/L), after which the cells were concentrated by centrifugation, lysed with a nucleotide releasing agent (Lumac, Landgraaf, The Netherlands) and assayed for ATP, over a 10 s period, using a chemiluminometer (Biocounter M2010 Multijet, Lumac Systems Inc., Titusville, FL, USA).

{alpha}-tocopherol

The effects of a fixed concentration (25 mg/L) of {alpha}-tocopherol (DL-{alpha}-tocopherol, F. Hoffmann–La Roche, Basel, Switzerland), a membrane-stabilizing agent that neutralizes the antimicrobial activity of the riminophenazines,3 on mycobacterial growth, cation (K+ and Ca2+) transport and PLA2 activity in the presence and absence of clofazimine and B669 (0.6 and 2.5 mg/L) were also investigated. The {alpha}-tocopherol was added to the mycobacteria either 5 min before or 15 min after the riminophenazines.

Ca2+-chelating agents

The effects of chelation of Ca2+ on B669-mediated (0.6 mg/L) alterations in PLA2 activity and K+ transport were investigated in M. aurum A+ by preincubating the bacteria with extracellular (N,N,N',N'-tetraacetic acid, EGTA) and intracellular (1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, BAPTA, or 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'-amino-5'-methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid pentaacetoxy methylester oil, FURA-2–AM (Calbiochem, La Jolla, CA, USA)) Ca2+-chelating agents for 15 min at 37°C before addition of the riminophenazine. EGTA and BAPTA or FURA-2 were used at final concentration ranges of 0.2–7.2 g/L and 0.2–9.5 mg/L, respectively.

The uptake of FURA-2 and BAPTA by mycobacteria was ascertained in a series of preliminary experiments using a spectrophotometric procedure in which we demonstrated that exposure of FURA-2 and BAPTA-loaded mycobacteria to the calcium ionophore, 4-bromo A23187, was accompanied by an abrupt, transient increase in fluorescence intensity indicative of interaction of Ca2+ with the intracellular probes.

Statistical analysis

The results of each series of experiments are expressed as the mean values ± the standard error of the mean (SEM). Levels of statistical significance were calculated by the paired Student's t-test. Significance levels were taken at a P value of <=0.05.


    Results
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 Materials and methods
 Results
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 References
 
Effects of clofazimine and B669 on growth of, cation uptake by, and phospholipase A2 activity in mycobacteria

The effects of clofazimine and B669 on mycobacterial growth, cation uptake and phospholipase A2 activity are shown inFigures 1 and2 for M. tuberculosis H37Ra. The corresponding results for M. aurumA+ and M. tuberculosisH37Rv were almost identical (not shown).



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Figure 1. Effects of clofazimine (B663) on the growth of, cation uptake by and PLA2 activity of M. tuberculosis H37Ra. The bacteria were exposed to B663 0.15 mg/L ({square}), 0.3 mg/L ({blacksquare}), 0.6 mg/L (), 1.25 mg/L () and 2.5 mg/L (//). The results of between two and four experiments, each with triplicate determinations, are presented as the mean percentage of the untreated control system ± SEM.

 


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Figure 2. Effects of B669 on the growth of, cation uptake by and PLA2 activity of M. tuberculosis H37Ra. The bacteria were exposed to B669 0.15 mg/L ({square}), 0.3 mg/L ({blacksquare}), 0.6 mg/L (), 1.25 mg/L () and 2.5 mg/L (//). The results of between two and four experiments, each with triplicate determinations, are presented as the mean percentage of the untreated control system ± SEM.

 
At concentrations of 0.15–2.5 mg/L, both B663 and B669 caused statistically significant inhibition of the growth of M. tuberculosis H37Ra (P <= 0.0007 to P <= 0.0001). The growth of M. aurum A+ and M. tuberculosisH37Rv was also inhibited in a dose-dependent manner (P <= 0.0001 and P <= 0.0006, respectively).

Exposure of M. tuberculosis H37Ra, as well as M. aurum A+ and M. tuberculosis H37Rv to either clofazimine or B669 (0.15–2.5 mg/L) resulted in statistically significant, dose-related inhibition of the uptake of 86Rb+ and 42K+ (P <= 0.05 to P <= 0.0001), which was associated with enhancement of PLA2 activity over the same concentration range (P <= 0.02 to P <= 0.0001), according to increased release of [14C]arachidonate from riminophenazine-treated bacteria. Treatment of the mycobacteria with both riminophenazines at 1.25 and 2.5 mg/L, but not at lower concentrations, was accompanied by a significant increase (P <= 0.005 to P <= 0.002) in the influx of 45Ca2+.

Effects of ouabain and standard antimycobacterial agents on growth and K+ transport

Ouabain at concentrations of up to 0.15 g/L potentiated the growth of the mycobacteria without affecting uptake of 86Rb+. The mean values for the growth (percentage of control) of M. aurum A+ and M. tuberculosisH37Ra exposed to 0.15 g/L ouabain were 188 ± 31% and 163 ± 40%, respectively. As was the case with ouabain, a 60 min exposure of M. tuberculosis H37Ra to the various standard antituberculosis agents at two to five times their MIC did not affect the uptake of 86Rb+ (not shown).

Effect of clofazimine and B669 on the influx and efflux of K+

The effects of B663 and B669 (0.6 mg/L) on the influx of 42K+ using M. tuberculosis H37Ra are shown inFigure 3. Both riminophenazines inhibited the net influx of 42K+ into M. tuberculosis H37Ra throughout the 90 min duration of the experiment, whereas no accelerated efflux of the cation was noted over the same period of time in the drug-treated bacteria (data not shown).



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Figure 3. Kinetics of net influx of K+ from control ({blacksquare}), B663 (•) and B669 ({blacktriangleup}) (0.6 mg/L)-treated M. tuberculosis H37Ra. The results are expressed as the mean actual counts (cpm) ± SEM of three experiments, each with triplicate determinations.

 
Time-course of altered uptake of 86Rb+ and 45Ca2+ by, and PLA2 activity in B669-treated M. aurum A+

The effects of B669 (0.6 mg/L and 2.5 mg/L) on the uptake of 86Rb+ and 45Ca2+ by, and PLA2 activity in M. aurum A+ are shown inFigure 4. Exposure to B669 caused an immediate dose-related increase in PLA2 activity and inhibition of the uptake of 86Rb+, whereas increased uptake of 45Ca2+ was noted only 1 min after exposure of the mycobacteria to 2.5 mg/L of the antimicrobial agent.



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Figure 4. The time-course of altered 86Rb+ uptake ({blacksquare}) and 45Ca2+ uptake (•) by, and PLA2 activity ({blacktriangleup}) in B669-treated (a) 0.6 mg/L and (b) 2.5 mg/L M. aurum A+. The results are expressed as the mean percentage of the untreated control system ± SEM of two experiments, each with triplicate determinations.

 
Effects of {alpha}-tocopherol

{alpha}-tocopherol per se was found to have no effect on bacterial growth or cation transport. However, this agent was found to neutralize the inhibitory effects of the more potent riminophenazine, B669 (0.6 mg/L) on the growth of, and uptake of K+ by all the mycobacteria tested. The mean percentages of growth, relative to the control, were 28 ± 4 and 86 ± 3 for M. aurum A+ exposed to B669 only or to B669 preceded by treatment with {alpha}-tocopherol, respectively. The mean uptakes (percentages of control) of 86Rb+ by M. aurumA+ exposed to B669 in the presence and absence of {alpha}-tocopherol were 19 ± 3 and 83 ± 6, respectively. The addition of {alpha}-tocopherol 15 min after exposure of the mycobacteria to the antimicrobial agent also neutralized the inhibitory effects of B669 on the uptake of 86Rb+ to the same extent as with pretreatment. Similar results were obtained when 42K+ was used as tracer.

Treatment of M. aurum A+ with {alpha}-tocopherol added either 1 min before or 15 min after B669 (2.5 mg/L) caused antagonism and reversal, respectively, of the influx of 45Ca2+ into the mycobacteria. The mean uptakes (percentages of control) of 45Ca2+ by M. aurum A+ exposed to B669 for 15 min in the absence of {alpha}-tocopherol, or in the presence of this agent added before or 15 min after the riminophenazine were 209 ± 10, 107 ± 4 and 136 ± 6, respectively.

Ca2+-chelating agents

When added to M. aurumA+ before B669, either alone or in combination, the Ca2+-chelating agents EGTA and BAPTA at concentrations of up to 7.6 g/L and 9.5 mg/L, respectively, did not antagonize the riminophenazine-mediated inhibition of 86Rb+ uptake or the potentiation of PLA2 activity (results not shown).

Effects of clofazimine and B669 on ATP concentrations

Exposure of the mycobacteria to clofazimine or B669 at a fixed concentration of 0.6 mg/L did not significantly affect bacterial ATP concentrations. The respective concentrations of ATP for control, B663- and B669-treated M. aurum A+ were 6.7 ± 1.0, 6.0 ± 0.8 and 4.2 ± 0.8 nmol/mg protein. The corresponding values for M. tuberculosisH37Ra were 4.5 ± 0.6, 4.9 ± 0.5 and 5.6 ± 0.7 nmol/mg protein, whereas those for M. tuberculosisH37Rv were 2.0 ± 0.1, 2.3 ± 0.4 and 2.1 ± 0.3 nmol/mg protein, respectively. However, at concentrations of 2.5 mg/L and higher, exposure to B669 was accompanied by a significant reduction in mycobacterial ATP concentrations. In the case of M. tuberculosisH37Ra, the mean ATP concentration for the B669-treated M. tuberculosis H37Ra was 2.5 ± 0.2 nmol/mg protein compared with that of the control system, which was 4.5 ± 0.6 nmol/mg protein (P <= 0.0001).


    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
The primary objectives of the present study were to investigate the effects of the riminophenazine antimicrobial agents, clofazimine and B669, on mycobacterial PLA2 activity, K+ transport and growth, and to identify possible relationships between these. Exposure of M. aurum A+, a rapidly proliferating, non-pathogenic mycobacterium, as well as M. tuberculosis H37R, to the riminophenazines was accompanied by enhancement of PLA2 activity and inhibition of uptake of K+. These effects of clofazimine and B669 were dose related and observed at concentrations of these agents that inhibited the growth of the mycobacteria. As K+ is essential for bacterial proliferation, 18,19 these findings suggest that riminophenazine-induced dysregulation of uptake of this cation and inhibition of bacterial growth may be related events. Short-term (60 min) exposure of the mycobacteria to supra-MICs of streptomycin, isoniazid, rifampicin, ethambutol and pyrazinamide was not associated with alterations in microbial K+ transport.

In short duration time-course experiments, addition of B669 (0.6 and 2.5 mg/L) to the mycobacteria resulted in immediate enhancement of PLA2 activity and inhibition of K+ transport, with no clear chronological separation between these events, suggesting a mechanistic interrelationship. The absence of detectable efflux of K+, or influx of Ca2+, a divalent cation that is normally excluded from both prokaryotic and eukaryotic cells, 20 in the setting of sustained ATP concentrations in mycobacteria treated with B669 0.6 mg/L, suggests that the effects of the riminophenazines on microbial PLA2 activity and K+ transport precede and are not secondary to microbial damage or interference with energy metabolism. Although exposure of the mycobacteria to higher concentrations of B669 (2.5 mg/L) did result in a significant drop in microbial ATP concentrations and influx of Ca2+, these also appeared to be secondary events, as they were preceded by enhancement of PLA2 activity and inhibition of uptake of K+.

A negative association between activation of PLA2 and inhibition of K+ transport is well established in eukaryotic cells. Lysophosphatidylcholine and arachidonic acid, the primary hydrolysis products generated during cleavage of phosphatidylcholine by PLA++2, are inhibitors of Na+K+-ATPase. 5,6 Although the exact molecular mechanism by which these effects are achieved has not been established, it has been proposed that lysophospholipids, the most potent inhibitors, may compromise the activity of Na+K+-ATPase by interfering with essential interactions of this cation transporter with boundary phospholipids in the inner membrane.21 It is noteworthy that clofazimine and B669 have been reported to inhibit Na+K+-ATPase activity in human lymphocytes and cancer cell lines by a PLA2-dependent, lysophospholipid-mediated mechanism.22,23

Additional evidence linking riminophenazine-mediated enhancement of microbial PLA2 activity to inhibition of uptake of K+ and growth was derived from experiments using {alpha}-tocopherol. This agent, at the fixed concentration (25 mg/L) and incubation conditions used in the present study, does not appear to inhibit the activity of PLA2, 24 but rather interacts with lysophospholipids and unsaturated fatty acids to neutralize their membrane-destabilizing activity, a property not shared by {alpha}-tocopherol acetate or other lipid-soluble antioxidants.4 Pretreatment of mycobacteria with {alpha}-tocopherol antagonized the inhibitory effects of the riminophenazines on mycobacterial K+ transport and growth, as well as the influx of Ca2+ into mycobacteria treated with B669 at 2.5 mg/L. These effects of {alpha}-tocopherol were observed in the setting of sustained riminophenazine-mediated enhancement of PLA2 activity, suggesting that neutralization of PLA2+-derived membrane-destabilizing phospholipid hydrolysis products, rather than inhibition of this enzyme, is the primary mechanism of antagonism.

Delayed addition (15 min after exposure to the antimicrobial agents) of {alpha}-tocopherol to riminophenazine-treated mycobacteria was found to reverse B669-mediated (0.6 mg/L) inhibition of K+ transport and growth, and to restore Ca2+ homeostasis in bacteria treated with this agent (2.5 mg/L). This latter observation suggests that intra-membrane accumulation of lysophospholipids eventually causes an {alpha}-tocopherol-reversible increase in the permeability of the outer membrane to Ca2+. This may occur indirectly as a consequence of inactivation of microbial K+ transporters, and/or directly as a result of lysophospholipid-mediated damage to the cell membrane. Irrespective of the mechanism, this is clearly a source of additional stress to the bacteria, resulting in consumption of ATP as a result of activation of Ca2+ efflux systems.

Extracellular and intracellular Ca2+-chelating agents were used to investigate the involvement of this cation in riminophenazine-mediated enhancement of mycobacterial PLA2 activity. Treatment of the bacteria with EGTA and BAPTA/FURA-2–AM individually or in combination did not prevent, but rather potentiated, B669-mediated augmentation of PLA2 activity and inhibition of uptake of K+, suggesting that the riminophenazines may cause activation of a Ca2+-independent PLA2.25 However, our recent unpublished data from experiments using the Ca2+ ionophore, A23187, indicate that this is unlikely, as we have observed that exposure of M. aurum A+ and M. tuberculosis to low micromolar concentrations (0.1–5 µM) of A23187 is accompanied by immediate influx of Ca2+ into the mycobacteria. This in turn is associated with activation of PLA2 and {alpha}-tocopherol-reversible inhibition of K+ transport and growth. Although it cannot be completely discounted, it seems unlikely that the PLA2-related antimycobacterial activity of the riminophenazines and A23187 would be achieved by alterations in the activity of distinct phospholipases with different requirements for Ca2+.

The contention that riminophenazine-mediated alterations in the hydrolysis of mycobacterial membrane phospholipids are not achieved by effects of these agents on the activation of PLA2 is supported by the Ca2+ independence of these effects in intact bacteria, and by previous findings which demonstrated that neither clofazimine nor B669 affect the activity of purified PLA2 in vitro.3 Interestingly, it has previously been reported that bilayer packing stresses of the cell membrane during phase changes increase the sensitivity of the integral phospholipids to PLA2. 26,27 Riminophenazines, which are extremely lipophilic, may cause alterations in lipid packing in the outer membrane, resulting in increased susceptibility of phospholipids to PLA2. Such effects have previously been reported for membrane-interactive antimicrobial peptides, including gramicidins.26 These peptides were found to induce non-bilayer, or HII phases in membranes, apparently increasing and decreasing the accessibility of the acyl chains and head groups, respectively, with the net effect of enhancing PLA2 activity.26

Although relatively little is known about the K+-transporting systems of mycobacteria, the susceptibility of these to riminophenazine-mediated inactivation suggests structural similarities to those operative in Gram-positive bacteria 7 and eukaryotic cells.22,23 In all three cases inhibition of K+ transport is achieved by indirect, PLA2-dependent mechanisms. These indiscriminate inhibitory effects of the riminophenazines on K+ transport do not, however, eliminate microbial K+ transporters as possible novel and selective targets for antimicrobial chemotherapy. This is based on the observed absence of effects of ouabain, a selective and potent inhibitor of Na+K+-ATPase in eukaryotic cells, on mycobacterial growth and uptake of K+, indicating the existence of structural differences between microbial and eukaryotic K+-transporting systems. An ideal inhibitor of microbial K+ transport should, however, interact directly and selectively with the cation transporter, rather than by the non-selective, indirect, PLA2-dependent mechanisms described here for the riminophenazines.


    Acknowledgments
 
This study forms part of the continuing Glaxo–Wellcome Action TB Program, an international research initiative of Glaxo–Wellcome plc in collaboration with the South African Medical Research Council.


    Notes
 
* Correspondence address: Department of Immunology, PO Box 2034, Pretoria 0001, South Africa. Tel: +27-12-3192425; Fax: +27-12-3230732. Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Barry, V. C., Belton, J. G., Conalty, M. L., Denneny, J. M., Edward, D. W., O'Sullivan, J. F. et al. (1957). A new series of phenazines (rimino-compounds) with high anti-tuberculosis activity. Nature 179, 1013–15.[ISI][Medline]

2 . Atkinson, A. J., Sheagren, J. N., Rubio, J. B. & Knight, V. (1967). Evaluation of B663 in human leprosy. International Journal of Leprosy 35, 119–27.

3 . Van Rensburg, C. E. J., Jooné, G. K., O'Sullivan, J. F. & Anderson, R. (1992). Antimicrobial activities of clofazimine and B669 are mediated by lysophospholipids. Antimicrobial Agents and Chemotherapy 36, 2729–35.[Abstract]

4 . Kagan, V. E. (1989). Tocopherol stabilizes membrane against phospholipase A, free fatty acids and lysophospholipids. Annals of the New York Academy of Sciences 570, 121–35.[ISI][Medline]

5 . Oishi, K., Zheng, B. & Kuo, J. F. (1990). Inhibition of Na, K-ATPase and sodium pump by protein kinase C regulators sphingosine, lysophosphatidylcholine and oleic acid. Journal of Biological Chemistry 265, 70–5.[Abstract/Free Full Text]

6 . Okafor, M. C., Schiebinger, R. J. & Yingst, D. R. (1997). Evidence for a calmodulin-dependent phospholipase A2 that inhibits Na–K-ATPase. American Journal of Physiology 272, C1365–72.[Abstract/Free Full Text]

7.De Bruyn, E. E., Steel, H. C., Van Rensburg, C. E. J. & Anderson, R. (1996). The riminophenazines, clofazimine and B669, inhibit potassium transport in Gram-positive bacteria by a lysophospholipid-dependent mechanism. Journal of Antimicrobial Chemotherapy 38, 349–62.[Abstract]

8 . Rhoads, D. B., Waters, F. B. & Epstein, W. (1976). Cation transport in Escherichia coli. VIII. Potassium transport mutants. Journal of General Physiology 67, 325–41.[Abstract]

9 . Epstein, W. & Laimins, L. (1980). Potassium transport in Escherichia coli: diverse systems with common control by osmotic forces. Trends in Biochemical Science 5, 21–3.[ISI]

10 . Kakinuma, Y. (1993). K+ transport in Enterococcus hirae. In Alkali Cation Transport Systems in Prokaryotes (Bakker, E. P., Ed.), pp. 277–90. CRC Press, London.

11 . Skou, J. C. (1988). Overview: the Na+,K+-pump. Methods in Enzymology 156 , 1–25.[ISI]

12 . Abrams, A. & Smith, J. B. (1971). Increased membrane ATPase and K+ transport rates in Streptococcus faecalis induced by K+ restriction during growth. Biochemical and Biophysical Research Communications 44, 1488–95.[ISI][Medline]

13 . Rhoads, D. B., Woo, A. & Epstein, W. (1977) Discrimination between Rb+ and K+ by Escherichia coli. Biochimica et Biophysica Acta 469, 45–51.[ISI][Medline]

14 . Bakker, E. P. & Harold, F. M. (1980). Energy coupling to potassium transport in Streptococcus faecalis. Interplay of ATP and the proton motive force. Journal of Biological Chemistry 255, 433–40.[Abstract/Free Full Text]

15 . Bradova, V., Smid, F., Ledinova, J. & Michalec, C. (1990). Improved one-dimensional thin layer chromatography for the separation of phospholipids in biological material. Journal of Chromatography 533, 297–9.[Medline]

16 . Holmsen, H., Holmsen, I. & Bernhardsen, A. (1966). Microdetermination of adenosine diphosphate and adenosine triphosphate in plasma with the firefly luciferase system. Analytical Biochemistry 17, 456–73.[ISI][Medline]

17 . Holmsen, H., Storm, E. & Day, H. J. (1972). Determination of ATP and ADP in blood platelets: a modification of the firefly luciferase assay for plasma. Analytical Biochemistry 46, 481–501.

18 . Epstein, W. & Davies, M. (1970). Potassium-dependent mutants of Escherichia coli K-12. Journal of Bacteriology 101, 836–43.[Medline]

19 . Bakker, E. P. (1993). Cell K+ and K+ transport systems in prokaryotes. In Alkali Cation Transport Systems in Prokaryotes (Bakker, E. P., Ed.), pp. 205–25. CRC Press, London.

20 . Norris, V., Grant, S., Freestone, P., Canvin, J., Sheikh, F. N., Toth, I. et al. (1996). Calcium signalling in bacteria. Journal of Bacteriology 178, 3677–82.[Free Full Text]

21 . Oishi, K., Raynor, R. L., Charp, P. A. & Kuo, J. F. (1988). Regulation of protein kinase C by lysophospholipids. Journal of Biological Chemistry 263, 6865–71.[Abstract/Free Full Text]

22 . Anderson, R. & Smit, M. J. (1993). Clofazimine and B669 inhibit the proliferative responses and Na+, K+-adenosine triphosphatase activity of human lymphocytes by a lysophospholipid-dependent mechanism. Biochemical Pharmacology 46, 2029–38.[ISI][Medline]

23 . Van Rensburg, C. E. J., Van Staden, A. M. & Anderson, R. (1993). The riminophenazine agents clofazimine and B669 inhibit the proliferation of cancer cell lines in vitro by phospholipase A2-mediated oxidative and nonoxidative mechanisms. Cancer Research 53, 318–23.[Abstract]

24 . Pentland, A. P., Morrison, A. R., Jacobs, S. C., Hurza, L. L., Hebert, J. S. & Packer, L. (1992). Tocopherol analogs suppress arachidonic acid metabolism via phospholipase inhibition. Journal of Biological Chemistry 267, 15578–84.[Abstract/Free Full Text]

25 . Dennis, E. A. (1983). Phospholipases. In The Enzymes, 3rd edn (Boyer, P. D., Ed.), pp. 307–53. Academic Press, New York.

26 . Rao, N. M. (1992). Differential susceptibility of phosphatidylcholine small unilamellar vesicles to phospholipase A2, C and D in the presence of membrane active peptides. Biochemical and Biophysical Research Communications 182, 682–8.[ISI][Medline]

27 . Rao, N. M. & Nagaraj, R. (1993). Interaction of wild-type signal sequences and their charged variants with model and natural membranes. Biochemical Journal 293,43 –9.[ISI][Medline]

Received 3 November 1998; returned 11 February 1999; revised 16 March 1999; accepted 22 March 1999