Antimicrobial activity of clofazimine is not dependent on mycobacterial C-type phospholipases

M. C. Bopape1, H. C. Steel2, R. Cockeran2, N. M. Matlola2, P. B. Fourie1 and R. Anderson2,*

1 Tuberculosis Research Lead Programme, Medical Research Council, Pretoria; 2 Medical Research Council Unit for Inflammation and Immunity, Department of Immunology, Faculty of Health Sciences, University of Pretoria, PO Box 2034, Pretoria 0001, South Africa

Received 21 November 2003; accepted 27 February 2004


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have used a phospholipase C (PLC)-deletion mutant (plcABC) of the H37Rv strain of Mycobacterium tuberculosis (MTB), as well as a plcA-insertion mutant of Mycobacterium smegmatis, to investigate the possible involvement of PLCs in clofazimine-mediated inhibition of mycobacterial K+ transport and growth. Inactivation of the PLCs of MTB and insertion of the plcA gene into M. smegmatis resulted in a substantial reduction and increase in hydrolysis of phosphatidylcholine (PC), respectively. However, both the mutant and wild-type strains of MTB and M. smegmatis were equally sensitive to the inhibitory effects of clofazimine on K+ uptake and growth. These observations demonstrate that the PLCs of MTB are not involved in the antimicrobial activity of clofazimine.

Keywords: mycobacteria, phospholipase C, Mycobacterium tuberculosis, Mycobacterium smegmatis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have previously reported that exposure of mycobacteria, as well as Gram-positive and Gram-negative bacteria, to the riminophenazine antimicrobial agent clofazimine and its 2-alkylimino derivatives is accompanied by increased microbial phospholipase (PL) activity, which is coincident with decreased uptake of K+ and precedes inhibition of bacterial growth.13 The observed increased release of arachidonate (AA) following the addition of clofazimine to phosphatidylcholine (PC)-pulsed mycobacteria suggested the involvement of a microbial phospholipase A (PLA), as well as a mechanistic link between increased hydrolysis of membrane phospholipids, decreased uptake of K+, and inhibition of bacterial growth.13 Others have suggested that phospholipases C or D (PLC, PLD) are primarily involved in the hydrolysis of PC by Mycobacterium tuberculosis (MTB).4 Interestingly, no genes encoding conventional PLAs have been documented in the MTB genome, in contrast to the recognition of several genes encoding putative PLCs (plc A,B,C,D).5

In this study, we have used a triple PLC-deletion mutant (plcABC{Delta} knockout) of the H37Rv strain of MTB, which is also deficient in the plcD gene,6,7 as well as a plcA-insertion mutant of Mycobacterium smegmatis, which does not possess PLCs,8 to characterize the mechanism(s) of PC-degradation operative in MTB, as well as the involvement, if any, of mycobacterial PLCs in the antimicrobial activity of clofazimine.


    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], also known as B663, was synthesized by the late Dr J. F. O’Sullivan (Department of Chemistry, University College, Dublin, Republic of Ireland), and dissolved in dimethyl sulphoxide (DMSO) and used at a final concentration range of 0.015–2.5 mg/L. Unless otherwise stated, all the other chemicals used were obtained from Sigma Chemical Co. (St Louis, MO, USA). The radioisotopes, phosphatidylcholine (PC, L-{alpha}-1-palmitoyl-2-arachidonyl, [arachidonyl-1-14C], 1.48–2.22 GBq) and 86Rb (rubidium-86 chloride, 37 MBq) were purchased from Perkin Elmer Life Sciences (Boston, MA, USA).

Bacterial strains and mutants

The plcABC{Delta} M. tuberculosis mutant and its control strain, H37Rv, which were used in these studies, were provided by Dr T. Parish (St Bartholomews and The Royal London School of Medicine and Dentistry, London, UK). The plcA-insertion mutant of M. smegmatis, together with the corresponding control strains, namely the wild-type of M. smegmatis Mc2155 and Mc2155 transformed with the pMV262 vector, were provided by Dr S. C. Leão (Department of Microbiology, Immunology and Parasitology, Universidade Federal de São Paolo, Brazil). The procedures used to generate these mutants of MTB and M. smegmatis have been described in detail elsewhere.6,8

The MTB strains were cultured in 7H9 liquid medium (Difco) supplemented with 10% OADC (oleic acid, albumin, dextrose, catalase) and 0.05% Tween 80, whereas the M. smegmatis strains were cultured on OADC-supplemented 7H10 agar with (plcA insertion mutant and plasmid control strains) and without (wild-type strain) kanamycin (15 mg/L). One week- and 3-day-old cultures of the mutant and wild-type strains of MTB and M. smegmatis, respectively, were used for the assays described below.

Phospholipase activity

This procedure was used (i) to compare the magnitudes of PC hydrolysis by the mutant and wild-type strains of MTB and M. smegmatis, and (ii) to investigate the effects of the diacylglycerol (DAG) lipase inhibitor, RHC-80267 (Calbiochem),9 at final concentrations of 0.1, 1 and 10 mg/L on the generation of AA from added PC by the wild-type strain of MTB. The inhibitor was added to the bacteria 10 min before addition of [14C]PC. PC breakdown was measured according to the release of [14C]arachidonate from the sn-2 position of added radiolabelled phosphatidylcholine (0.5 mCi/L) as described previously.2 After incubation at 37°C, [14C]arachidonate was extracted with chloroform/methanol (2:1, v/v) from the wild-type and mutant strains of MTB or M. smegmatis, which had been pre-loaded with [14C]PC, and assayed by high performance thin layer chromatography.2

Uptake of rubidium-86 chloride

Rubidium-86 chloride (86Rb) was used as tracer for measuring the effects of clofazimine (0.15–2.5 mg/L) on K+ uptake by the control and mutant strains of MTB and M. smegmatis. Briefly, the bacteria were harvested and resuspended to 106 cfu/mL in K+-free buffer (KONO) containing 2 mCi/L 86Rb, with and without clofazimine, and uptake of 86Rb was determined after 90 min at 37°C as previously described.2

Measurement of bacterial growth

Growth curves for the wild-type and mutant strains of MTB and M. smegmatis were determined radiometrically using the BACTEC TB system (Becton Dickinson Diagnostic Instrument Systems, Towson, MD, USA). Seed culture (0.1 mL) of each strain was inoculated into a BACTEC 12B vial and cultured for 2–3 days, followed by sub-culture until growth index (GI) values of 200–300 were obtained. These cultures (0.1 mL) were used for inoculation and growth at 37°C and were monitored daily until GI values of 999 were reached.

A variation of this procedure was used to determine the sensitivity of the wild-type and mutant strains of MTB and M. smegmatis to clofazimine (0.015–2.5 mg/L). Briefly, synchronized cultures with GI values of 400–500 were inoculated in 0.1 mL volumes containing 104–105 cfu/mL into BACTEC vials with and without clofazimine, while a control system was inoculated with 0.1 mL of a 1:100 dilution of the inoculum. Vials were incubated at 37°C and GI values recorded daily until the value of the diluted control system reached 30, at which time the experiment was terminated. The {Delta}GI values were calculated by subtracting the values for each clofazimine-treated system measured on the day before the diluted control system attained a value of 30 from the corresponding values recorded on that day. The MIC value was taken as the lowest concentration of clofazimine with a {Delta}GI value less than that of the diluted control system.

Statistical analysis

The results are expressed as the mean values ± S.E.M. All statistical analyses were carried out with the INSTAT program using the paired Student’s t-test. The significance levels were taken at a P value less than 0.05.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Hydrolysis of radiolabelled PC

These results are shown in Figure 1. Hydrolysis of PC, as measured by the release of [14C]arachidonate, was significantly decreased and increased, respectively in the PLC-deficient mutant of MTB and the insertion mutant of M. smegmatis relative to the responses of the corresponding wild-type strains. The effects of the DAG lipase inhibitor, RHC-80267, on the release of AA from [14C]PC-loaded wild-type MTB are shown in Figure 2. Treatment of the bacteria with the inhibitor was associated with dose-related inhibition of the generation of AA from PC, which attained a value of about 90% with 10 mg/L RHC-80267.



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Figure 1. Generation of arachidonate from [14C]phosphatidylcholine by the wild-type, plasmid control and plcA-insertion mutant strains of M. smegmatis, as well as by the wild-type and plcABC-deletion mutant strains of M. tuberculosis (H37Rv). The results of three experiments are presented as the mean values in radioactive counts per minute (cpm) ± S.E.M. *P < 0.05.

 


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Figure 2. Effects of the diacylglycerol lipase inhibitor, RHC-80267, on the generation of arachidonate from [14C]phosphatidylcholine by the wild-type strain of M. tuberculosis (H37Rv). The results of a single representative experiment with five replicates for each system are presented as the mean values in radioactive counts per minute (cpm) ± S.E.M. *P < 0.05.

 
Uptake of rubidium-86 chloride

These results are shown in Figure 3. The extent of uptake of 86Rb by the mutant strain was similar to that of the wild-type strain of MTB following a 90 min incubation period at 37°C (23 373 ± 1921 versus 23 079 ± 2821 cpm for the mutant and wild-type strains, respectively; data from five experiments with five replicates in each). However, uptake of 86Rb by both the wild-type and mutant strains was sensitive to the inhibitory effects of clofazimine, with maximal inhibition of influx observed at 1.25–2.5 mg/L of the antimicrobial agent.



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Figure 3. Effects of clofazimine on K+ (86Rb) uptake by the wild-type, plasmid control and plcA-insertion mutant strains of M. smegmatis, as well as by the wild-type and plcABC-deletion mutant strains of M. tuberculosis. The results of three to five experiments are presented as the mean percentages ± S.E.M. of the corresponding clofazimine-free control systems.

 
In the case of M. smegmatis, the magnitudes of uptake of 86Rb by the wild-type and plasmid control strains were comparable, whereas that of the plcA-insertion mutant was significantly higher (P < 0.0001) (44 982 ± 3880, 41 880 ± 3559 and 142 386 ± 15 436 cpm, respectively, following 90 min of incubation at 37°C), with all three strains being more-or-less equally sensitive to the inhibitory effects of clofazimine (Figure 3).

Bacterial growth and MICs

The growth curves for both the wild-type and mutant strains of MTB (in the absence of clofazimine) were virtually superimposable, whereas the growth of the insertion mutant of M. smegmatis was somewhat slower than that of the wild-type and plasmid control strains (data not shown). In the presence of clofazimine, the MIC values for both the wild-type strain and deletion mutant of MTB were equivalent (0.06 mg/L for both), whereas the MIC values for the wild-type, plasmid control and insertion mutant of M. smegmatis were 0.3, 0.15 and 0.6 mg/L, respectively.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The results of this study confirm that the PLCs of MTB utilize PC as a substrate and demonstrate that this results in the formation of arachidonate, which based on the results with RHC-80267, appears to occur by a mechanism involving PLC-mediated formation of DAG from PC, which is in turn converted into arachidonate by a DAG lipase. This appears to be the primary mechanism of PC degradation by MTB, excluding significant involvement, if indeed any, of PLA and PLD. This contention is compatible with the MTB genome sequence, which has identified not only the plcABCD genes but also genes encoding acylglycerol lipase activity.6,10

PC is the predominant phospholipid in eukaryotic cell membranes and its transformation by PLC/DAG may provide a source of 3-carbon compounds to fuel the energy requirements and growth of intracellular MTB. Moreover, DAG and AA initiate intracellular signalling cascades which activate pro-inflammatory and antimicrobial activities of human phagocytes.1114 MTB-mediated conversion of PC into DAG and AA in infected macrophages may therefore result in over-exuberant, misdirected pro-inflammatory activity, which rather than eradicating this microbial pathogen, may favour persistence. This contention is supported by recent observations that the expression of plcA, plcB and plcC is strongly up-regulated during the first 24 h of macrophage infection by MTB, whereas deletion of these genes is associated with decreased virulence.15

We have previously reported that exposure of MTB to clofazimine, or to its 2-alkylimino derivatives, is accompanied by increased hydrolysis of PC and inhibition of the uptake of K+. Both events occur abruptly, have a similar concentration dependence and precede influx of Ca2+, reduced microbial ATP levels, and inhibition of growth.2,3 Although the exact identity of the mycobacterial PLs remained to be established, we suggested, based on this and other evidence, that the clofazimine-mediated augmentation of PL activity resulted in dysregulation of membrane cation transporters and inhibition of microbial growth.2,3

The results of this study appear to eliminate mycobacterial PLCs as being potential targets for clofazimine. This contention is based on observations that the absence of all four MTB PLCs, which is associated with marked attenuation of PC hydrolysis, does not influence the sensitivity of the deletion mutant to the inhibitory effects of clofazimine on either uptake of K+ or growth. Conversely, insertion of the MTB plcA gene into M. smegmatis, which results in impressive acquisition of the ability to hydrolyse PC, also has no detectable effects on the sensitivity of bacterial K+ transport and growth to clofazimine.

In conclusion, mycobacterial PLCs do not appear to be involved in the anti-mycobacterial activity of clofazimine, apparently excluding PL-mediated inactivation of bacterial K+ transporters as a mechanism of antimicrobial action. This raises the possibility that clofazimine may function as a direct inhibitor of mycobacterial K+-uptake systems, or alternatively, interference with uptake of the cation may be a consequence of the disruptive effects of this antimicrobial agent on membrane integrity and function. We are currently investigating these possibilities using K+-transport knockout mutants of MTB.


    Footnotes
 
* Corresponding author. Tel: +27-12-319-2425; Fax: +27-12-323-0732; E-mail: randerso{at}medic.up.ac.za Back


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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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