Inhibition of potassium transport and growth of mycobacteria exposed to clofazimine and B669 is associated with a calcium-independent increase in microbial phospholipase A(2) activity
Hc. Steel et al., Inhibition of potassium transport and growth of mycobacteria exposed to clofazimine and B669 is associated with a calcium-independent increase in microbial phospholipase A(2) activity, J ANTIMICRO, 44(2), 1999, pp. 209-216
Altered phospholipase A(2) (PLA(2)) 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 PLA, activity was measured using a radiometric thin-layer ch
romatography procedure, whereas K+ and Ca2+ transport were measured using R
b-86(+) or K-42(+) and Ca-45(2+), 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-re
lated enhancement of microbial PLA, activity, which was associated with inh
ibition of K+ influx and growth. Uptake of Ca2+ by mycobacteria was unaffec
ted, or minimally affected, by the riminophenazines at concentrations of le
ss than or equal to 0.6 mg/L, whereas higher concentrations resulted in inc
reased uptake of the cation in the setting of decreased microbial ATP conce
ntrations. The results of kinetic studies using a fixed concentration (2.5
mg/L) of B669 demonstrated that riminophenazine-mediated enhancement of PLA
, activity and inhibition of Kf uptake in mycobacteria are rapid and probab
ly related events that precede, by several minutes, any detectable effects
on microbial ATP concentrations and uptake of Ca2+. Inclusion of the extrac
ellular and intracellular Ca2+-chelating agents EGTA (0.2-7.2 g/L) and BAPT
A/FURA-2 (0.2-9.5 mg/L), individually or in combination, did not prevent th
e effects of B669 on mycobacterial PLA, activity or K+ transport, whereas a
-tocopherol, which neutralizes PLA(2) primary hydrolysis products, antagoni
zed the inhibitory effects of the riminophenazines on microbial K+ uptake a
nd growth. These results demonstrate that the antimycobacterial activities
of clofazimine and B669 are related to a Ca2+-independent increase in mycob
acterial PLA(2), leading to interference with microbial K+ transport.