Intracellular expression of ESAT-6 in A549 AECs does not result
in increased mitochondrial fragmentation
We previously reported that ESAT-6 is required for Mtb to elicit both
morphological changes in mitochondria and a reduction in mass
(Fine-Coulson et al. , 2015). In order to explore the possibility
that ESAT-6 was directly responsible for these mitochondrial changes, we
expressed myc-tagged ESAT-6 in A549 AECs and assessed mitochondrial
morphology by immunofluorescence microscopy. ESAT-6 was present
throughout the cytoplasm and at the cell surface but did not colocalize
with mitochondria and did not induce the fragmented mitochondrial
profile seen 48 h after Mtb infection (Figure 1A; compare mitochondria
detected by COXIV labelling after ESAT-6 transfection in top panel
(green) to mitochondria post-Mtb infection detected using mitotracker
red in lower left panel). To account for potential effects on
mitochondrial morphology caused by protein over-expression alone, we
also transfected cells with pcDNA3.1-mEmerald and quantified
mitochondrial morphology in the mEmerald-positive cells. In keeping with
the dynamic nature of mitochondrial membranes, there was a mixture of
both fragmented and tubular morphologies in both ESAT-6-expressing and
mEmerald-expressing cells (Figure 1B). Surprisingly, more extensive
mitochondrial fragmentation was observed in the mEmerald-expressing
cells versus the ESAT-6-expressing cells, indicating that ESAT-6 alone
does not increase mitochondrial fragmentation (Figure 1B). Taken
together, these findings suggest that ectopically-expressed ESAT-6 does
not affect mitochondrial morphology directly by binding to mitochondrial
membranes, nor does it directly induce the morphological changes
observed after Mtb infection.