Effects of isoniazid on ultrastructure of Mycobacterium aurum and Mycobacterium tuberculosis and on production of secreted proteins

Abstract

Isoniazid (INH), one of the most effective antimycobacterial drugs, specifically inhibits, at an early stage of its action, the biosynthesis of mycolic acids, specific mycobacterial lipids which play a central role in the cell envelope architecture of mycobacteria. In the present study, the consequences of the action of INH on the cell morphology of Mycobacterium tuberculosis and Mycobacterium aurum were examined. Electron microscopy was used to observe bacilli which were previously treated with either subinhibitory concentrations of INH or the MIC of the drug, leading to a decrease of 20 to 35% (by weight) of their mycolic acid contents. The earlier effect of INH on the ultrastructure of mycobacteria, as revealed by negative staining of bacilli, was the alteration of the bacterial poles; this event was observed prior to the bacteriostatic action of the drug and was accompanied by a release of material from the poles into the extracellular medium. In a later stage of the drug's action, cell deformation occurred and more extracellular material was seen. The material released following the action of the drug on susceptible mycobacterial cells was identified as being almost exclusively composed of proteins. Labeling of amino acids with 35S prior to and during the action of INH on M. aurum and subsequent analysis of the labeled proteins led to the conclusion that they consisted of secreted proteins which were up to 20-fold oversecreted in the presence of the drug. Competitive enzyme-linked immunosorbent assay with the secreted 45/47-kDa antigen complex of M. tuberculosis demonstrated up to 20-fold oversecretion of these proteins. Taken together, the production of oversecreted proteins following the decrease of the cell envelope mycolate content by INH strongly suggests that mycolic acids may act as a barrier in the export of proteins secreted by mycobacteria.

The draft genome of Mycobacterium aurum, a potential model organism for investigating drugs against Mycobacterium tuberculosis and Mycobacterium leprae

“…The analysis of loci involved in drug resistance demonstrated homology with M. tuberculosis and M. leprae. This insight corroborates earlier investigations of inhA gene mutants of M. aurum that showed similarity in drug resistance mechanisms against isoniazid and ethionamide between M. aurum and M. tuberculosis [6,40]. The draft M. aurum genome is larger than that of M. tuberculosis with an additional 2090 genes not observed in M. tuberculosis; it is also lacking 1002 of the genes found in M. tuberculosis.…”

Mycobacterium tuberculosis Proteins Involved in Mycolic Acid Synthesis and Transport Localize Dynamically to the Old Growing Pole and Septum

“…Mtb polar lysis has been observed in very early EM images of isoniazid-treated wild-type bacilli [67]. Isoniazid targets InhA and if we assume that synthesis and export of mycolic acids are primarily polar, InhA inhibition will leave “holes” in the polar mycomembrane and ultimatly induce specific unipolar lysis as previously reported [67]. More recently, polar lysis was also observed when arabinan synthesis was inhibited with benzothiazinone [31], [52].…”

“…Some reports can indeed be interpreted in this manner when compared with our results. Mtb polar lysis has been observed in very early EM images of isoniazid-treated wild-type bacilli [67]. Isoniazid targets InhA and if we assume that synthesis and export of mycolic acids are primarily polar, InhA inhibition will leave “holes” in the polar mycomembrane and ultimatly induce specific unipolar lysis as previously reported [67].…”

Dielectrophoretic characterization of antibiotic-treated Mycobacterium tuberculosis complex cells

“…The activated form of the drug is a potent inhibitor of synthesis of mycolic acid for cell wall [20,21], resulting in cell death. It has been reported that INH induces morphological changes in mycobacteria, such as loss of structure or appearance of surface wrinkles and bulging [22,23]. To date, the effects of RIF and INH on DEP behavior of MTB complex cells have not been investigated.…”