Journal of Antimicrobial Chemotherapy (1999) 44, 209-216
© 1999 The British Society for Antimicrobial Chemotherapy
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
Medical Research Council Unit for Inflammation and Immunity, Department of Immunology, Institute for Pathology, University of Pretoria, Pretoria, South Africa
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 
-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.
* Correspondence address: Department of Immunology, PO Box 2034, Pretoria 0001, South Africa. Tel: +27-12-3192425; Fax: +27-12-3230732.
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