Killing of mycobacteria by macrophage cathepsin D.
Tuberculosis (TB) is the fifth largest cause of death in South Africa, with one in ten cases being resistant to treatment due to the development of multidrug-resistance and extensively drug-resistance in the agent responsible for this disease, Mycobacterium tuberculosis. This pathogen has developed mechanisms to evade killing by immune cells such as macrophages. Mycobacterium smegmatis, a non-pathogen, that does not evade killing by the macrophage, is often used to gain a better insight into the bacteriocidal pathways used to kill mycobacteria, and those potentially blocked by M.tuberculosis. In such studies nitric oxide and “lysosomal” proteases have emerged as major bacteriocidal pathways. Studies on the role of aspartic protease, cathepsin D, in killing green fluorescent protein- (GFP-) tagged-M.smegmatis in J774 macrophages required antibodies that would not cross-react with mycobacterial antigens. These were raised in chickens, using alum and saponin as adjuvants, and porcine and human cathepsin D. Using such antibodies, quantitative colocalization analysis using ImageJ and the JACoP colocalization plugins showed a greater degree of colocalization between cathepsin D and LysoTracker Red DND-99 in M.smegmatis-infected J774 macrophages than in uninfected cells. This indicates the possible presence of active, bacteriocidal cathepsin D in acidic, and hence matured phagosomes. A higher colocalization between cathepsin D and LAMP-1 and cathepsin D and LAMP-2 in uninfected cells possibly indicates the recycling of these two markers from vesicles not containing killed bacteria. Propidium iodide (PI) labelling and loss of GFP fluorescence appeared reliable indicators of M.smegmatis death or viability, respectively, as myobacteria that took up PI also lost green fluorescence, while M.smegmatis that exhibited green fluorescence (viable) were not observed to take up propidium iodide (dead). Faint colocalization between cathepsin D, LAMP-1 and -2 with dead, and to a lesser extent with live M.smegmatis occurred. Besides intensity correlation values other colocalization programs indicate the absence of colocalization between these markers and dead M.smegmatis, but, together with in vitro killing experiments (cathepsin D, 0.0098 units/ml resulting in 59% killing in 4 h) these appear to indicate a possible role of cathepsin D in killing of M.smegmatis.