PE17 localizes in close proximity to mitochondrial membranes and results in extensive changes to mitochondrial morphology and mass
To test the hypothesis that mitochondrial fragmentation is induced by other secreted Mtb proteins which require release to the host cytoplasm via ESAT-6 activity, we scanned the literature for reports citing localization or effects of secreted Mtb proteins on mitochondria. In a screen of Mtb secreted proteins, Stamm et al. reported that PE17 localizes to the mitochondria in HeLa cells (Stamm et al. , 2019). To confirm this observation, we transiently transfected A549 AECs with a constitutively-expressing myc-tagged PE17 construct and performed immunofluoresence microscopy using anti-myc and anti-COXIV antibodies to determine whether PE17 localized to mitochondria. Interestingly, in our hands, PE17 did not completely co-localize with mitochondria and was located perinuclearly, adjacent to the few remaining mitochondrial structures (Figure 1C). In addition, the PE17-positive structures correlated with large occlusions in the cytoplasm in transmitted light images (Figure 1C, arrows) and appeared as clusters of circular structures (Figure 1D). As there appeared to be a visible decrease in mitochondrial mass in PE17-expressing cells, we analyzed the percent area of mitochondria within transfected cells that did and did not express PE17. As predicted, expression of PE17 significantly decreased mitochondrial mass (Figure 1E, p-value <0.0001).