Macrophages infected with MAC undergo apoptosis, in contrast to macrophages infected with M. tuberculosis
]. Apoptosis, therefore, is an effective host innate immunity mechanism to control the progression of the infection [8
]. MAC, however, has evolved strategies to overcome host killing. We report that MAC can survive in apoptotic macrophages, and many of the organisms escape the dying cells and infect other, adjacent, macrophages.
Fratazzi and colleagues found that 90% of MAC from macrophages that are undergoing apoptosis lose viability [8
]. In contrast, Pais and colleagues determined that MAC viability was unchanged upon treatment of macrophages with staurosporine, an inducer of apoptosis [27
]. The data shown in the present study indicate that some MAC can survive the apoptotic process and then escape apoptotic bodies.
Escape from the phagocyte requires that the bacterium lyse the vacuole and the cytoplasm membrane. Listeria monocytogenes
is the classical example of a bacterium that actively escapes the phagosome [28
]. We had hypothesized that MAC would rupture the vacuole membrane by an active mechanism; however, our results suggest that, upon induction of apoptosis, the vacuole membrane loses integrity and MAC is delivered to the cytoplasm. Microscopic observation indicates that the encounter between the bacterium and the macrophage’s cytoplasmic membrane was randomized and became more frequent when apoptotic bodies were formed. As far as we can tell from video observation, MAC depends on Brownian movement to encounter the macrophage cytoplasmic membrane. How MAC lyses the macrophage membrane is unknown. The genomic RD1 region appears to have a role in cell-to-cell spread of M. tuberculosis
]. However, MAC lacks genes of close sequence similarity to genes in the M. tuberculosis
region. Mycobacterium marinum
can also escape the phagosome and likely uses actin tails to spread from host cells [30
], suggesting that MAC uses a different mechanism of dissemination from both M. marinum
and M. tuberculosis
. A recent report by Hagedorn and colleagues confirmed our observations [31
] that MAC does not exit phagocytic cells in a similar fashion to M. marinum
and M. tuberculosis
. In fact, MAC has been shown to disrupt actin fibers [32
] and, therefore, tail formation in cytosol would be difficult to happen. Recent work in the laboratory has identified MAC mutants that cannot escape apoptotic macrophages and disseminate (data shown). Further study of these mutants may shed some light on the mechanism of escape.
It would not be unusual for MAC to contain genetic baggage that allows it to survive under diverse conditions for short periods of time. Alternatively, MAC within apoptotic bodies is also able to cause infection to secondary macrophages ingesting the apoptotic body without exposure to the extra-cellular space. The relative occurrence of these three modes of spreading is not currently known, and further research would shed light onto the most predominant mechanism of dissemination used by MAC. Our results showed that once MAC associated with apoptotic macrophages is taken up by fresh macrophages, a percentage of MAC organisms are killed, while the remaining viable bacteria replicate intracellularly.
The virulent M. tuberculosis
strain H37Rv induces less macrophage apoptosis than the attenuated H37Ra strain, suggesting an apoptosis block by virulent M. tuberculosis
], nonetheless, apoptosis still occurs upon infection by either strain. Hayashi and colleagues [34
] found that MAC sonicate induced apoptosis in both human monocyte derived and THP-1 macrophages. Similarly, we found murine Raw 264.7 and human THP-1 macrophages to have similar amounts of apoptosis triggered by live MAC. However, without side by side comparison of intact and lysed MAC or virulent and avirulent MAC strains, we are unable to say if apoptosis is actively induced or inhibited by live and virulent MAC compared to heat-killed or avirulent MAC.
In macrophages infected by M. tuberculosis
, autophagy results in double-membrane phagosomes that mature into phagolysosomes and inhibit bacterial survival [23
]. Similarly, we found a reduction in the ability of MAC to survive once macrophages become autophagic, although a fraction of the intracellular bacteria is still capable of surviving. The observation by de Chastellier and Thilo [5
] indicates that MAC in cholesterol-deprived macrophages is located in double-membrane bound vacuoles, but upon the addition of cholesterol, MAC was again located in typical vacuoles, suggesting that some MAC strains survive autophagy in macrophages. Since MAC is known to live within environmental amoeba [35
], and amoeba has been shown to undergo autophagy [36
], it would be of value to determine if MAC survives amoebic autophagy, and if the MAC survival mechanism in macrophages evolved due to the bacterial environmental lifestyle.
Based on the evidence gathered in this study, we propose a model for how MAC spreads from macrophage to macrophage. In this model, bacteria are phagocytosed into the macrophage, and apoptosis is induced a few days later. Upon apoptosis, some bacteria are killed, while other bacteria escape the apoptotic bodies to the extra-cellular space, where they can be efficiently phagocytosed by another macrophage and the cycle likely repeats. Other bacteria, however, stay in the apoptotic bodies, and once those are taken up by new macrophages, some bacteria effectively infect the new macrophage, while others are killed. One of the main questions in the interaction between MAC and macrophages regards if MAC tries to kill the host macrophages, inducing apoptosis, or is it the host cell that tries to eliminate the pathogen. So far, no definitions exist to this question.
Future studies will dissect the molecular mechanisms of apoptosis and autophagy in macrophages and how MAC deals with them.