The uptake and fate of Isoniazid in Mycobacterium tuberculosis var. bovis BCG

Abstract

S U M M A R YThe incorporation of 14C INH into sensitive and resistant bacteria has been investigated. Only organisms susceptible to INH took up the drug. Uptake was irreversible and became saturated at higher INH concentrations. Uptake was heat-labile, KCN-sensitive, partially inhibited by dinitrophenol and stimulated by SH group reagents. The activation energy of binding was of the same order as the activation energy of an enzyme catalysed reaction. Uptake at 37" was rapidly inhibited although at 4" it was not. The INH binding mechanism had many of the properties of catalase and peroxidase. It seems likely that all these functions were part of one enzyme which was missing from INH-resistant BCG.Chromatographic studies on hot-water extracts of INH-treated BCG indicated that INH was converted into several unidentified products within the organism. I N T R O D U C T I O NThe effect of isoniazid (INH) on the biosynthesis of a number of components of BCG has been examined in an earlier paper (Wimpenny, 1967). However, in attempting to elucidate both the mode of action and the specificity of INH as an anti-tubercular agent, the fate of the drug itselfin the sensitive organism must be traced. Several papers have been published which throw some light on this aspect of the problem. Barclay, Ebert & Koch-Weser (1953) showed that Mycobacterium tuberculosis var. hominis strain H 37 RV sensitive to INH took up the drug, whilst a resistant variant did not. In later experiments (Barclay, Koch-Weser & Ebert, 1954) using the same organism, they investigated the uptake of 14C isonicotinic acid, 14C nicotinamide and 14C nicotinic acid. Of these compounds, 14C INH was bound and then only by the drug-sensitive strain. Organisms killed by heat or with formalin did not fix INH. These workers indicated that uptake of INH was greater at 5" than at 37" in living bacteria and concluded that INH was bound by physical adsorption. Youatt ( 1 9 5 8~) confirmed that 14C INH was bound only by sensitive mycobacteria. She showed that the drug was fixed only under actively metabolizing conditions and that uptake in sensitive mycobacteria was sensitive to cyanide, azide and heat. She found that Staphylococcus aureus, Escherichia coli, Mycobacterium phlei, Proteus mirabilis, Bacillus megatherium and Candida albicans took up very little INH. In addition Youatt showed (19583) that the rate of breakdown of INH was greater in living BCG than in heat killed organisms.

Mechanism of isoniazid uptake in Mycobacterium tuberculosis

“…It has been shown that 4 × 10 −4 M dinitrophenol induces a 50% inhibition of labelling by [ 14 C]INH in Mycobacterium bovis BCG (Wimpenny, 1967), suggesting that uptake could result from active secondary transport. However, this experiment again used a long uptake time (40 h).…”

Isoniazid Uptake in Relation to Growth Inhibition ofMycobacterium tuberculosis

“…1). In fact, the level of cell-bound drug seemed to fall off gradually, which might reflect INH metabolism by the tubercle bacillus (11,13). In early preliminary work, we observed that NH protects tubercle bacilli from INH growth inhibition and is itself not inhibitory to the organism at a concentration of 10 Ag/ml.…”

“…It is also possible that, after incubation of "4C-INH-containing cells for several hours in a medium devoid of INH, a significant proportion of the cell-bound 14C activity is no longer INH, but rather some product of its metabolism (11,13). If this were true, it would not necessarily weaken the idea that a critical level of active INH is maintained under conditions where further INH uptake is blocked by NH.…”

“…A close direct relationship exists between a cell's capacity to bind or take up INH and its susceptibility to the drug. Thus, sensitive tubercle bacilli readily bind INH, whereas resistant strains and other microbes do not (3, 4, [10][11][12]. Therefore, regardless of the primary target site(s) of INH action, the effectiveness of the drug is closely governed by the uptake or binding process.…”

Antigenic Differences Between Mycobacterium bovis Strain BCG and an Isoniazid-Resistant Mutant

“…Some of the reported differences involve growth rate, virulence, and degrees of drug resistance (21). Most strains which develop INH-resistance also undergo enzymatic changes, such as losses in catalase and peroxidase activity (7,14), and lose the capacity to accumulate the drug intracellularly (19). The experiments reported here were carried out to investigate the possibility that these changes are associated with alterations in antigenicity of these organisms, and significant antigenic differences were found between an INH-resistant mutant and its parent strain.…”