DISCUSSION
Examples of bacterial pathogenicity factors being directly targeted to
– and imported into – mitochondria have been described for a number of
pathogens (Kozjak-Pavlovic et al. , 2008, Spier et al. ,
2019) and numerous studies have explored the effects of mycobacterial
infection on the function of mitochondria; however, there have been
relatively few studies investigating the direct association of
mycobacterial proteins with mitochondrial membranes at an intracellular
level. Cpn60.2 is an example of a secreted Mtb protein (Joseph et
al. , 2017) which was expressed intracellularly in RAW macrophages and
was partially localized to mitochondrial membranes by both immuno-EM and
immunofluorescence localization. However, there was no indication of the
disruption of mitochondrial membranes that we observed at 48 hrs
post-infection of Mtb H37Rv in A549 AECs (Fine-Coulson et al. ,
2015) and mitochondrial morphology was not quantitatively assessed. Due
to its membrane lytic activity, ESAT-6 was a likely candidate to cause
disruption of the mitochondrial membranes directly; however,
intracellular expression within A549 AECs in this study indicated a lack
of colocalization with the mitochondria. Indeed, comparison of the
degree of fragmentation induced by ESAT-6 versus a control GFP protein
suggested that either high-level expression of GFP was sufficient to
fragment mitochondria, or ESAT-6 induces increased tubulation, thus
working in an opposite manner to GFP. These results support our
conclusion that other secreted Mtb proteins may be directly responsible
for the mitochondrial fragmentation phenotypes observed during Mtb
pathogenesis, and not an activity of ESAT-6.
We initially became interested in PE17 following a report that PE17
localized to the mitochondria and decreased host cell secretion upon
expression in epithelial cells (Stamm et al. , 2019). We found
that PE17 did not completely co-localize with mitochondria when
expressed in A549 cells but was found adjacent to fragmented
mitochondria. Although PE17 did not induce exactly the same
mitochondrial fragmentation pattern as was seen after M.
tuberculosis infection of A549 cells, we did observe that PE17
expression resulted in an extensive decrease in mitochondrial mass
(Figure 1), which corresponds to our previous finding (Fine-Coulsonet al. , 2015). This result indicates mitochondrial fragmentation
can be dissociated from decreases in mitochondrial mass, suggesting that
multiple bacterial factors are likely involved in the mitochondrial
changes observed during M. tuberculosis infection. In 2019,
Aguilar-Lopez et al. investigated two mycobacterial virulence factors
which had been previously demonstrated to have N-terminal mitochondrial
targeting sequences, LprG and PE_PGRS33 (Aguilar-Lopez et al. ,
2019). Recombinant his-tagged proteins were added extracellularly to
human monocyte derived macrophages (MDMs) and size, interconnectivity
and elongation of mitochondria assessed. Surprisingly, these two
proteins had opposite effects; in LprG stimulated cells there was a
decrease in size, interconnectivity and elongation suggesting an
increase in mitochondrial fission whereas the opposite was seen in
PE_PGRS33 stimulated cells indicating increased fusion. Further
evidence for a role of other members of the PE/PPE family in
mitochondrial dynamics was reported in an elegant study by Cadieuxet al . A stably-transfected cell line expressing an inducible
PE_PGRS33 was generated and the mitochondrial localisation confirmed by
immunofluorescence microscopy (Cadieux et al. , 2011). They also
detected a swelling of mitochondria in these cells compared to controls.
Localisation of three additional PGRS-containing proteins, PE_PGRS1,
PE_PGRS18 and PE_ PGRS24, was also determined and although none
colocalized completely with mitochondria, both PE_PGRS18 and PE_PGRS24
localized in a compact structure close to the nucleus. Intriguingly,
PE_PGRS24 cells also appeared to be closely adjacent to, possibly
surrounding, spherical compact mitochondrial structures reminiscent of
the pattern seen after PE17 expression. Thus, it is possible that PE17
and other mitochondrially targeted Mtb proteins, including other members
of the PE/PPE family, can work cooperatively to alter the host
mitochondria during M. tuberculosis infection and should be a
focus of future studies.
An unexpected finding of this study was the extensive disruption in the
mass and morphology of the Golgi stack and TGN (Figure 2) in response to
the expression of PE17. Previous work had detected a secretion defect in
PE17-expressing HeLa cells (Stamm et al. , 2019), and it is
therefore possible that the structural changes identified in the present
study contribute to the observed changes in host secretion during PE17
expression. Indeed, chemical disruption of the Golgi stack, commonly
achieved through the fungal metabolite brefeldin A, decreases secretion
(Misumi et al. , 1986, Oda et al. , 1987). Interestingly,
the mass and morphology of the ER was unaffected by PE17 expression.
We also found a significant decrease in the mass of late endosomes and
lysosomes – but not early endosomes or recycling endosomes – upon PE17
expression. In macrophages, M. tuberculosis is known to prevent
fusion of the bacterium-containing compartment with late endosomes,
increasing bacterial viability (Via et al. , 1997, Westman &
Grinstein, 2020, Zulauf et al. , 2018). In A549 cells, it has been
shown that the M. tuberculosis bacterium-containing compartment
does acquire late endosomal markers, but avoids lysosomal fusion through
some involvement of the autophagy pathway (Fine et al. , 2012). It
is possible that the PE17-mediated decrease in the mass of the late
endosomes and lysosomes is another mechanism by which M.
tuberculosis avoids lysosomal degradation. Alternatively, as PE17
causes massive disruption of the TGN, which is a major source of input
into late endosomes and lysosomes, a reduction of flux through this
post-Golgi trafficking pathway could result in the observed loss in mass
of endo-lysosomal organelles.
One of our most striking results is the association of PE17 with the
surface of lipid droplets. Expression of PE17 in M. smegmatisincreased the bacterial viability during macrophage infection while
increasing macrophage necrosis (Li et al. , 2019). WhileMycobacterium smegmatis is known to utilize lipids and fatty
acids as a carbon source for growth, this bacterium lacks the molecular
machinery to persist intracellularly within macrophages. It is possible
that by providing PE17 to M. smegmatis , it allowed this bacterium
to acquire enough host cell lipids to allow for some intracellular
persistence in this study, unlike the wild-type parent. Therefore, it is
tempting to speculate that PE17 is directly involved in either inducing
the synthesis of, or ‘packaging’ of, host organellar membranes into
lipid droplets to serve as a source of nutrients for the intracellularMycobacterium . Alternatively, perhaps PE17 coats host lipid
droplets to prevent the host cell from consuming its own lipid droplets,
thereby ensuring a pool of lipids to be acquired byMycobacterium .
Several infectious diseases caused by intracellular pathogens have been
associated with effects on LD-organelle interfaces (Kory et al. ,
2016), including the association of Mtb-containing phagosomes with host
LDs within foamy macrophages (Peyron et al. , 2008). This process
is mediated by Rab7 (Roque et al. , 2020) and is also
characterized by LD redistribution and/or clustering upon infection. In
this study, PE17 positive LDs appear to be largely localized
perinuclearly and in close proximity to other LDs suggesting the
potential of PE17 to cluster lipid droplets in vivo. Increasing lipid
flux towards lipid droplet production without concomitant lipid
replacement of affected membrane compartments could lead to the drastic
PE17-dependent organellar phenotypes we have observed in this study.
Alternatively, or in addition, the widespread effects seen during PE17
expression may result from direct interaction with a host protein that
has a regulatory role in organelle homeostasis.
PE17 was detected in large spherical structures apparent in transmitted
light images as well as surrounding BODIPY-positive structures in
fluorescence images, a pattern reminiscent of perilipin A labelled LDs
(Garcia et al. , 2003). Although there is no consensus regarding a
specific protein targeting signal associated with proteins that bind
lipid droplets from the cytosol (Class II proteins) (Ingelmo-Torreset al. , 2009, Nakamura & Fujimoto, 2003, Olzmann & Carvalho,
2019), truncation experiments indicate the central domain of perilipin,
containing a combination of hydrophobic domains in conjunction with an
acidic domain, is required for targeting and anchoring to LDs (Garciaet al. , 2003). In this report we show the unique C-terminal
domain of PE17 is required for association with LDs in A549 cells. This
domain does not contain any previously identified targeting motifs, nor
is it predicted to be helical in nature, suggesting that PE17 may be
binding to a LD associated protein rather than directly interacting with
the LD membrane. A recent study used a proteomic approach to identify
proteins associated with LDs after infection with live Mtb (Menonet al. , 2019). Unexpectedly, as vesicular transport from LDs does
not occur, proteins associated with vesicular transport and lysosomal
biogenesis were found to accumulate on LDs during Mtb infection. As we
have demonstrated an effect of PE17 expression on multiple organelles,
it is tempting to speculate that PE17 may interact with a protein or
proteins associated not only with LDs, but with mitochondria, Golgi, and
lysosomes as well. One candidate identified in the proteomic screen
(Menon et al. , 2019) was Rab7, which increased its association
with lipid droplets after Mtb infection and has been implicated in
regulating LD-phagosome interactions (Roque et al. , 2020) as well
as promoting lysosome-mitochondria contacts (Wong et al. , 2018)
in addition to its more traditional role in Golgi-endosome trafficking
(Guerra & Bucci, 2016). Another potential candidate would be Arf1. Arf1
is the substrate for the guanine exchange factor GBF1, both of which are
perhaps best known for their role in recruiting COPI to coat vesicle for
retrograde traffic from the Golgi stack to the ER (Kaczmarek et
al. , 2017). At the LD surface, Arf1/COPI facilitates the transfer of
proteins from the ER to LD, and regulates LD surface tension (Thiamet al. , 2013, Wilfling et al. , 2014). Arf1 is also
associated with mitochondria as well as Golgi stack so offers a
potential link between the organelles most affected by PE17 expression.
PE17 may interact with and/or sequester Arf1/GBF1 at the LD surface,
inhibiting its many cellular roles.
Although the accumulation and redistribution of LDs within the host cell
is a hallmark of Mtb infection, the mechanisms involved in this process
are still poorly understood and require further investigation. Our
current findings suggest a potential role for PE17 in manipulation of
host LDs, but further research is needed to determine how PE17
expression leads to changes in organelle mass and if these changes are
mediated through either PE17:host protein or direct PE17:lipid
interactions.