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.
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 < or = 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.
Steel
H C
HC
Department of Immunology, Institute for Pathology, University of Pretoria, South Africa.
Matlola
N M
NM
Anderson
R
R
D000255:Adenosine Triphosphate / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology*; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002991:Clofazimine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D009161:Mycobacterium / Q000187:drug effects* / Q000254:growth & development; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000254:growth & development; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D011188:Potassium / Q000378:metabolism*
D000900:Anti-Bacterial Agents; C030428:B 669; D000255:Adenosine Triphosphate; D002991:Clofazimine; D010741:Phospholipases A; D054467:Phospholipases A2; D011188:Potassium; D002118:Calcium