Selective autophagy gets more selective: Uncoupling of autophagy flux and xenophagy flux in Mycobacterium tuberculosis-infected macrophages

Abstract

Induction of autophagy has been reported as a potential means to eliminate intracellular pathogens. Corroborating that, many studies report inhibition of autophagy as a survival strategy of bacterial pathogens. Incidentally, autophagy at the basal level is critical for survival of host cells including macrophages. We asked how a bacterial pathogen could inhibit autophagy for its survival if the inhibition resulted in cell death. In a recent study we show distinct regulation of autophagy in Mycobacterium tuberculosis (Mtb)-infected macrophages where Mtb containing- and nonMtb-containing autophagosomes show different fates in terms of maturation. We show that upon Mtb infection, there is no dramatic change in the autophagy flux in macrophages. However, autophagosomes that contain the virulent strains of Mtb show selective resilience to the maturation phase of autophagy. Surprisingly, nonMtb-containing autophagosomes in the infected cells continue to mature into autolysosomes. The block in the xenophagy flux is missing in the case of avirulant infections. We show that this selectivity is achieved through selective exclusion of RAB7 from virulent Mtb-containing autophagosomes, thereby restricting the formation of amphisomes.

Autophagy is a cellular homeostatic mechanism involving degradation of cellular targets such as damaged organelle or misfolded proteins by selectively targeting them into a double-membrane structure called the phagophore, which subsequently matures into an autophagosome and eventually fuses with a lysosome. The degradation of autophagosome-associated LC3 molecules upon fusion with the lysosome can be used to indirectly monitor the rate of degradation of autophagic cargo, also known as monitoring autophagic flux. Autophagic flux at a basal level is essential for long-term cellular survival and, especially through mitophagy, it helps check the accumulation of damaged mitochondria in the cell, a byproduct of mitochondrial respiration. Inhibition of autophagy therefore results in increased mitochondrial depolarization, cellular reactive oxygen species production and subsequent cell death. To note, cell death is also an integral constituent of innate defense mechanisms.

While the role of autophagy in regulating intracellular Mtb survival has been studied in detail, we wanted to understand how autophagy flux was regulated in Mtb-infected macrophages. To address that, we selected one virulent (H37Rv) and one nonvirulent (H37Ra) strain of Mtb. The virulent strain survives much better in the macrophages upon infection with respect to the nonvirulent strain. We monitored autophagy flux using the vacuolar-type ATPase inhibitor bafilomycin A₁ (BafA1). In both H37Rv- and H37Ra-infected macrophages overall autophagy flux was similar as observed through LC3 western blot. In the confocal microscopy experiment, however, we could see increased LC3 localization of H37Ra upon BafA1 treatment. No such effect of BafA1 treatment could be observed in the case of H37Rv infections. It seems the maturation phase of autophagy is blocked in H37Rv-infected macrophages, however, that was not visible in the LC3 immunoblots. Surprisingly, in cells stained with LysoTracker, we could observe high colocalization between LC3 and lysosomes even in H37Rv-infected macrophages, suggesting maturation of autophagosomes. This was only possible if H37Rv inhibits maturation of only those autophagosomes that harbored them, i.e., xenophagosomes, while allowing the maturation of other autophagosomes. We then show that mycobacterial virulence factors PhoP and ESAT6 are required for this selective inhibition of xenophagy flux by the virulent infections.

To further understand how xenophagy flux is inhibited by the virulent infections, we critically compared the maturation of phagosomes and autophagosomes. Maturation of autophagosomes may require RAB7 to form amphisomes before fusing with lysosomes or the autophagosomes may directly fuse with lysosomes. Interestingly, in the case of virulent Mtb infections, phagosome maturation arrest has been extensively reported. Phagosomes containing the virulent Mtb avoid recruitment of RAB7 and thereby do not mature into phagolysosomes. We suspected this could also help the virulent Mtb to avoid getting targeted to the amphisomes, the penultimate step for autophagosome maturation. We indeed found higher targeting of the nonvirulent strain H37Ra to amphisomes as compared to the virulent strain H37Rv. Interestingly, when we knocked down RAB7 using specific siRNA the xenophagy flux of even H37Ra-containing xenophagosomes was abolished. Furthermore, in RAB7 knockdown cells, even basal autophagy flux was inhibited. Thus H37Rv selectively inhibits the recruitment of RAB7 to the autophagosomes harboring them. This selectivity helps them achieve 2 key objectives simultaneously—the H37Rv-containing autophagosomes do not fuse with the lysosomes, which helps with bacterial survival. Second, nonMtb-containing autophagosomes continue to fuse with the lysosomes unabated in both virulent and nonvirulent infections, helping the survival of host macrophages. This uncoupling of the microbicidal arm of autophagy from the homeostatic arm is the fascinating finding of this study.

Several studies in the past have reported on the regulation of autophagy in macrophages upon infection with Mtb and its influence on intracellular Mtb survival. However, the effect on the homeostatic arm of autophagy was largely ignored. Based on our study, we propose that an optimal balance between autophagy and xenophagy is required to eliminate intracellular bacterial pathogens. The selective xenophagy inhibition could serve as a deliberate ploy of the infecting Mtb. At a very low autophagy level cells may undergo apoptosis, which could lead to pathogen killing. When autophagy is induced, host-cell survival is maintained, however, pathogen killing will depend on whether xenophagosome maturation is also induced. At an optimal autophagy level, the virulent mycobacteria will reside in the xenophagosomes, which do not mature, and allow basal autophagy to continue, helping cellular survival while simultaneously maximizing bacterial survival (Fig. 1).

Figure 1.

Optimizing autophagy-mediated regulation of intracellular Mtb survival. At very low autophagy levels (shaded zone), cellular survival is compromised, killing Mtb collaterally. With an increase in autophagy flux (green line) cellular survival is maintained. When autophagy flux is coupled with xenophagy flux (red dotted line, as in the case of rapamycin treatment), bacterial survival again gets compromised. An interesting facet of this model is at the junction where an increase in autophagy flux rescues cell death. At this junction, an increase in autophagy could have probacterial effects rather than the established antibacterial function as long as the bacteria have a means of avoiding sequestration or (in the case of Mtb) maturation of bacteria-containing autophagosomes. The red inhibition mark highlights the point that Mtb selectively inhibits xenophagy flux.

Disclosure of potential conflicts of interest

The authors declare no conflicts of interest.