An investigation of the killing of Mycobacterium tuberculosis by macrophages and the acid stress response of Mycobacterium smegmatis.

Attempts were made to activate human monocytes with immunomodulators and human macrophages with T-cell supernatants for in vitro antimycobacterial activity. Both these approaches failed. Alveolar macrophages from Mycobacterium bovis BCG-vaccinated guinea pigs have previously been shown to kill Mycobacterium tuberculosis in vitro. The guinea pig was therefore used to investigate macrophage antimycobacterial mechanisms. Tuberculocidal factors were found within lysosomes. Lysosomal fractions of macrophages from BCG-vaccinated guinea pigs significantly (P 0.001) killed M. tuberculosis. Tuberculocidal activity was not observed with macrophage lysosomal fractions from non-vaccinated guinea pigs. The specific activities of enzymes in macrophage homogenates were tested. None of the lysosomal enzymes had significantly different activities in macrophages from vaccinated and non-vaccinated guinea pigs. Strains of M. tuberculosis were sensitive to reactive nitrogen intermediates (RNI). However, guinea pig alveolar macrophages did not generate RNI on infection with mycobacteria. Macrophages from BCG-vaccinated guinea pigs killed M. tuberculosis in the presence of an inhibitor of RNI synthesis. Thus, no evidence was gained to suggest that RNI are responsible for the tuberculocidal activity of guinea pig macrophages. Aminoaldehydes have been shown to be toxic to M. tuberculosis. Ornithine decarboxylase (ODC) is the first enzyme in the pathway that synthesises aminoaldehydes. ODC activity was not elevated in macrophages twenty-four hours and six days after BCG vaccination. Increased ODC activity was not observed in macrophages infected with M. bovis BCG for twenty-four hours. ODC is therefore not responsible for any prolonged increase in aminoaldehyde production that may take place when macrophages are infected with mycobacteria. Mycobacterium tuberculosis was passaged once through the mouse. After passaging, the mycobacteria exhibited increased resistance to hydrogen peroxide but not RNI. This indicates that hydrogen peroxide is generated during the murine immune response to mycobacteria. Culturing Mycobacterium smegmatis at pH5.0 allowed the bacteria to survive subsequent incubation at pH3.5 better than bacteria grown solely at pH7.6. Mycobacterium smegmatis could therefore be adapted to lethal pH values by pre-exposing the bacteria to mildly acidic conditions.