Phagosomal maturation and intracellular survival of Mycobacterium avium subspecies paratuberculosis in J774 cells

The mechanisms by which Mycobacterium avium subspecies paratuberculosis (M. a. ptb) survives within macrophages are not well characterized. One strategy for intracellular survival developed by Mycobacterium tuberculosis is inhibition of phagosomal maturation. In this study it was hypothesized that M. a. ptb is capable of survival within macrophages by residing within a phagosomal compartment that does not mature into a functional phagolysosome. To test this hypothesis the following objectives were determined. Phagosomal maturation was evaluated by comparison of stage specific markers on the membranes of phagosomes containing live M. a. ptb with those containing killed M. a. ptb, Mycobacterium smegmatis, and zymosan A using immunofluorescent labelling and confocal microscopy. Intracellular survival of live M. a. ptb within J774 macrophages was compared to that of M. smegmatis by direct determination of bacterial viability by differential live/dead staining. The results of this study show that the phagosomes containing live M. a. ptb had increased levels of an early marker (transferrin receptor [TFR]) and decreased levels of a late maturation marker (lysosome associated membrane protein one [Lamp-1]), relative to those containing killed M. a. ptb, M. smegmatis, and zymosan A. Additionally, compared to M. smegmatis, M. a. ptb has enhanced ability to survive within cultured macrophages. These data indicate that M. a. ptb resists intracellular killing by residing within a phagosomal compartment that retains the characteristics of early phagosomes and resists maturation into functional phagolysosome..