Thermally adaptive changes of mycolic acids in Mycobacterium smegmatis.

The effect of growth temperature on mycolic acid composition in eight strains of Mycobacterium smegmatis was investigated by gas chromatography/mass spectrometry. A change in growth temperature from 45 to 20 degrees C caused a shift in the subclass and molecular species composition of mycolic acids. The relative amount of alpha'-mycolic acids to alpha-mycolic acids decreased, and that of hydroxy mycolic acids increased at lower temperatures. Moreover, the proportion of shorter-chain species of alpha-mycolic acids increased, and those of longer-chain species of alpha-mycolic and hydroxy mycolic acids decreased. This observation seems to be due to the changes of the chain length of meromycolates because the alpha-alkyl chain unit of mycolic acids was not affected. The ratio of odd to even carbon-numbered alpha-mycolates decreased as the growth temperature was lowered. In contrast, the molecular species composition of alpha'-mycolic acid was not influenced by the growth temperature.

Requirement of glucose for mycolic acid biosynthetic activity localized in the cell wall of Bacterionema matruchotii.

When the localization of mycolic acid biosynthetic activity was examined with Bacterionema matruchotii cells disrupted by the ultrasonic vibration method, activity was detected only in the cell wall fraction, not in the inner membrane nor in the 78,000g supernatant. Either the supernatant or sugar was absolutely required for the incorporation of [14C]palmitate into mycolic acids. Among sugars examined, glucose was most effective, with maltose being second. Unexpectedly, trehalose was inert. As to substrate, the present system utilized free palmitic acid rather than palmitoyl-CoA. The reaction products from palmitate and glucose were glucose mycolate and trehalose monomycolate, in which the label from [14C]palmitate or [14C]glucose was incorporated. Glucose palmitate was also formed. Addition of trehalose resulted in a shift from glucose mycolate to trehalose monomycolate. These data clearly indicate that sugars play an important role in the synthesis of mycolic acids from free fatty acids.

Mycobacterial lipids in infected tissue samples.

Mycobacteria synthesize characteristic lipids which can readily be distinguished from those synthesized by the host cells which they infect during pathogenesis. This fact can be exploited to provide information about mycobacteria growing in infected tissue samples: (a) qualitative analysis of lipid extracts from lepromatous leprosy skin biopsies reveals the presence of several mycobacterial lipids including an M. leprae-specific glycolipid, phthiocerol dimycocerosate and mycolic acids; (b) quantitative analysis shows that the amount of mycobacterial lipids present in lepromatous lesions is much greater than that expected on the basis of the number of acid-fast bacilli present; (c) incorporation of 14C-acetate into cell wall mycolic acids can be used to monitor the growth of intracellular mycobacteria.

Isoniazid inhibition of the synthesis of monounsaturated long-chain fatty acids in Mycobacterium tuberculosis H37Ra.

Isoniazid inhibited C(24) and C(26) monounsaturated fatty acid synthesis in Mycobacterium tuberculosis H37Ra. Time courses of this inhibition and that of mycolic acid synthesis were similar.

Effect of low temperature on growth, viability, and synthesis of mycolic acids of Mycobacterium tuberculosis strain H37Ra.

Cultures of Mycobacterium tuberculosis strain H37Ra were grownto early logarithmic phase at 37 degrees C and were incubated at 16 degrees, 20 degrees, and 25 degrees C. The decrease in this ability was more rapid at 20 degrees C than at 16 degrees C. Low-temperature incubation caused decreases in the ratios of mycolic acids and monounsaturated C16-19 fatty acids relative to the total of fatty acids synthesized. It also caused an increase in the ratio of saturated C24-26 fatty acids relative to the total of fatty acids synthesized. These ratios were based on the incorporation of radiolabel from 14C-acetate into fatty acids. These results showed that when M. tuberculosis H37Ra was incubated at low temperatures, it did not adapt by increasing the ratio of unsaturated to saturated fatty acids synthesized. The ability of the cells to synthesize mycolic acids was sharply decreased. These changes may lead to the loss of viability of M. tuberculosos H37Ra. Mycolic acid synthesis is similarly affected by exposure of cells to isoniazid, an antimycobacterial drug.

Relationship between the uptake of isoniazid and its action on in vivo mycolic acid synthesis in Mycobacterium tuberculosis.

A direct relationship was established between the rate of uptake of isoniazid and the action of this drug on in vivo mycolic acid synthesis in Mycobacterium tuberculosis H37Ra. The rate of uptake of isoniazid increased linearly with its external concentration and appeared to reach a maximal value of 52 pmoles per hr per 10(9) cells at an external concentration of about 13 mum. Correspondingly, the rate of inhibition of mycolic acid synthesis increased with the rise in the rate of uptake of the drug. A 50% inhibition of mycolic acid synthesis occurred when the uptake of isoniazid reached 5.2 pmoles per 10(9) cells. Calculations showed that this level of drug uptake represents an internal cellular concentration of 9 mum. These results show clearly that the action of isoniazid on the mycolate synthetase system of M. tuberculosis is rapid and that this enzyme system is highly sensitive to the drug.

Effect of isoniazid on the in vivo mycolic acid synthesis, cell growth, and viability of Mycobacterium tuberculosis.

When an actively growing culture of the H37Ra strain of Mycobacterium tuberculosis was exposed to isoniazid at a concentration of 0.5 mug/ml, the cells began to lose their ability to synthesize mycolic acids immediately. After 60 min, the cells had completely lost this ability. The synthesis of the three mycolate components-alpha-mycolate, methoxymycolate, and beta-mycolate-was inhibited. The viability of the isoniazid-treated cells was unaffected up to about 60 min of exposure, after which time there was a gradual decline in the viability to about 18% after 180 min. Correspondingly, growth of the drug-treated cells slowed down and stopped after 24 hr. The inhibition of the synthesis of mycolic acids was reversible if the drug was removed before the loss of viability set in. Incubation of the viable cells in the absence of the drug for 24 hr restored the mycolate synthesis. These results strongly suggest that the inhibition of the synthesis of the mycolic acids is closely associated with the primary mechanism of action of isoniazid on the tubercle bacilli. The sequence of events which leads to the loss of viability of cells exposed to isoniazid is described.