Medical Research Council Unit for Inflammation and Immunity, Department of Immunology, Institute for Pathology, University of Pretoria, Pretoria, South Africa
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Abstract |
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Introduction |
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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 -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.
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Materials and methods |
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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 PontNEN Research Products (Boston, MA, USA).
Bacteria
The mycobacterial reference isolates used in this study were Mycobacterium aurum A+, provided by the GlaxoWellcome 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 (0.152.5 mg/L).
The effects of ouabain (0.070.15 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 (0.110 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 (0.152.5 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 (0.070.15 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 (0.152.5 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
(12 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--1-palmitoyl-2-arachidonyl, (arachidonyl-1-14C),
1.482.22 GBq, 0.5 mCi/L). The mycobacteria were treated with the riminophenazines
(0.152.5 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 luciferinluciferase 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).
-tocopherol
The effects of a fixed concentration (25 mg/L) of -tocopherol (DL-
-tocopherol, F. HoffmannLa 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
-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-2AM (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 0.27.2 g/L and 0.29.5 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.
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Results |
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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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Exposure of M. tuberculosis H37Ra, as well as M. aurum A+
and M. tuberculosis H37Rv to either clofazimine or B669 (0.152.5 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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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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-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
-tocopherol, respectively. The mean uptakes (percentages of
control) of
86Rb+ by M. aurumA+ exposed to B669 in the
presence and absence of
-tocopherol were 19 ± 3 and 83 ± 6, respectively.
The addition of
-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 -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
-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).
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Discussion |
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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 -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
-tocopherol acetate or other lipid-soluble antioxidants.4 Pretreatment of mycobacteria with
-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
-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 -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
-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-2AM
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 (0.15 µM) of A23187 is
accompanied by immediate influx of Ca2+ into the mycobacteria. This in turn is
associated with activation of PLA2 and -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.
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Acknowledgments |
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Notes |
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References |
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Received 3 November 1998; returned 11 February 1999; revised 16 March 1999; accepted 22 March 1999