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\begin{document}

\title{Mycobacterium Tuberculosis Protein PE17 Induces Organelle Disruptions
Upon Heterologous Expression in A549 Human Airway Epithelial Cells}



\author[1]{Allyson T. Loy}%
\author[1]{Lucinda N. Shaffer}%
\author[1]{Sophia T. Gavalas}%
\author[1]{Anna A. Mermilliod}%
\author[2]{Frederick Quinn}%
\author[2]{Adrian J. Wolstenholme}%
\author[2]{Barbara J. Reaves}%
\author[1]{Vincent Starai}%
\affil[1]{University of Georgia Department of Microbiology}%
\affil[2]{University of Georgia Department of Infectious Diseases}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
\emph{Mycobacterium tuberculosis} (Mtb) is the causative agent of
tuberculosis. Its success as a pathogen is dependent on an arsenal of
secreted virulence factors which target host cell organelles. In this
report, we have characterized the effects of two proteins known to be
secreted by Mtb on eukaryotic mitochondria: ESAT-6 and a member of the
poorly-described PE/PPE protein family, PE17. Intracellular expression
of ESAT-6 in A549 cells did not induce mitochondrial fragmentation. In
contrast, expression of PE17 caused extensive mitochondrial
fragmentation and an overall reduction in mass. Further characterization
of the effects of PE17 within host cells revealed a dramatic decrease in
the mass of the trans-Golgi network and the Golgi stack with lesser but
significant decreases in late endosomes and lysosomes. The endoplasmic
reticulum and organelles of the early endocytic pathway were not
significantly affected. PE17 specifically colocalized with large,
cytoplasmic lipid droplets via a C-terminal domain, suggesting a
significant role for PE17 in the disruption of multiple host cell
organelles and a novel interaction with host lipid droplets.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

Tuberculosis is one of the top ten causes of death worldwide (WHO,
2019). While a vaccine against the etiological agent, the intracellular
bacterium \emph{Mycobacterium tuberculosis} (Mtb), has been available
for almost one hundred years, it is ineffective for the population most
at risk for pulmonary infection (Pai \emph{et al.} , 2016, Philips \&
Ernst, 2012). Furthermore, microbial resistance against frontline
antibiotics continues to rise (Gygli \emph{et al.} , 2017, WHO, 2019).
Therefore, increasing knowledge about how Mtb interacts with -- and
survives within -- host cells during infection may lead to new vaccine
targets and treatment options, which are desperately needed to control
the global tuberculosis incidence.

As an intracellular pathogen, \emph{M. tuberculosis} has been shown to
manipulate or hijack cellular processes within host cells to promote its
own survival upon phagocytic uptake. One of the best studied strategies
employed by the bacteria to evade destruction is to prevent fusion of
the bacteria-containing compartment with lysosomes (Armstrong \& Hart,
1971, Via \emph{et al.} , 1997). This process is dependent on bacterial
factors secreted through the SecA2 pathway of Mtb (Zulauf \emph{et al.}
, 2018) as well as perturbation of phagosomal pH (reviewed in (Westman
\& Grinstein, 2020)). Due to their essential roles in host cellular
processes such as energy production, calcium storage, and induction of
cell death pathways, mitochondria are also key organelles targeted by
pathogens. \emph{M. tuberculosis} disrupts mitochondrial membrane
potential post-infection resulting in the aberrant release of cytochrome
c (Abarca-Rojano \emph{et al.} , 2003, Chen \emph{et al.} , 2006,
Duan\emph{et al.} , 2002) and disruption of cellular bioenergetics
parameters, including ATP and ROS concentrations (reviewed in
(Mohareer\emph{et al.} , 2020)). Strikingly, these Mtb-dependent
biochemical changes of mitochondrial metabolism are often accompanied by
gross alterations in mitochondrial morphology. In healthy cells,
mitochondria are dynamic organelles concurrently exhibiting both tubular
and spherical morphologies as a result of continuous homotypic fusion
and fissioning to maintain proper metabolic homeostasis. During Mtb
infection of the human monocytic cell line, THP-1 (Asalla \emph{et al.}
, 2017) and A549 airway epithelial cells (AECs) (Fine-Coulson \emph{et
al.} , 2015), mitochondria appeared fragmented with small spherical
structures clustered around the nucleus. In contrast, TEM micrographs of
THP-1 macrophages infected with virulent Mtb caused mitochondria to
become elongated and increased in electron density (Jamwal \emph{et al.}
, 2013).

Although less studied, lipid droplet (LD) homeostasis is also disrupted
during Mtb infection, but the role that LDs play in the infection has
not been well characterized. Once believed to be solely involved in
lipid storage, it is now recognized that lipid droplets are dynamic
organelles that respond to the conditions of the cellular environment.
LDs are composed of a phospholipid monolayer and a neutral lipid core,
which contains triacyglycerol (TAGs) and cholesterol esters (CE)
(Walther \emph{et al.} , 2017) and can interact with other organelles
through membrane contact sites, expanding or contracting depending on
cellular metabolism (Pol \emph{et al.} , 2014). Several studies have
shown that \emph{M. tuberculosis} modifies the host environment to
induce the formation of lipid droplets and increase bacterial
intracellular survival, most notably within foamy macrophages
(D'Avila\emph{et al.} , 2006, Peyron \emph{et al.} , 2008, Singh
\emph{et al.} , 2012, Singh \emph{et al.} , 2015). In conditions that
mimic the hypoxic environment within granulomas, PBMC-derived
macrophages infected with Mtb accumulate lipid bodies and utilize host
lipids for accumulating triacylglycerol inside the bacteria (Daniel
\emph{et al.} , 2011). The mechanism underlying the manipulation of host
cell lipids and induction of lipid body synthesis by Mtb requires
further investigation.

Mtb proteins that are secreted into the host cell cytoplasm are
perfectly localized to interact with host organelles and induce the
changes seen in the endo-lysosomal system, mitochondria and lipid
droplets (reviewed in (Augenstreich \& Briken, 2020)). Early efforts to
identify these proteins relied mainly on biochemical isolation and
characterization (Wiker \& Harboe, 1992). One of the earliest
characterized and most intensively studied of the cohort of secreted Mtb
proteins is the 6 kDa early secretory antigenic target (ESAT-6)
(Sorensen \emph{et al.} , 1995). Several studies have shown that ESAT-6
plays an integral role in \emph{M. tuberculosis} escape into the host
cytosol by rupturing phagosomal membranes (Houben \emph{et al.} , 2012,
Smith \emph{et al.} , 2008). In addition, the actions of ESAT-6 may also
be important in allowing cytosolic access to other secreted proteins
whose targets lie outside of the bacterium-containing compartment
(Stamm\emph{et al.} , 2019).

Alternative methodologies designed to identify secreted Mtb proteins and
their interactors have been developed using genetic, proteomic and
transposon screens (Barczak \emph{et al.} , 2017, Cao \emph{et al.} ,
2019, McCann \emph{et al.} , 2007). A unique approach to characterizing
some of these secreted proteins was developed by Stamm et al
(Stamm\emph{et al.} , 2019) and specifically identified mycobacterial
secreted proteins that interact with yeast membranes and disrupt
eucaryotic secretory pathways. Using GFP fusions of predicted Mtb
secreted effectors found to bind membranes in yeast, they then localized
each of these proteins to organelles within human HeLa cells. Although
the largest proportion of the fusion proteins colocalized with the ER
resident protein, calreticulin, several others were observed to localize
to the Golgi apparatus, mitochondria, and peroxisomes. When considering
secreted Mtb proteins that could both bind membranes and disrupt host
protein secretion pathways, only five such proteins remained. Four
localized to the ER, but one was found to localize to
mitochondria:\emph{Rv1646} (PE17).

PE17 belongs to the family of PE (Pro-Glu) proteins, which along with
the PPE (Pro-Pro-Glu) family, account for approximately 7\% of
the\emph{M. tuberculosis} genome (Cole \emph{et al.} , 1998,
Fishbein\emph{et al.} , 2015). PE17 is evolutionarily grouped with the
PE\_PGRS proteins, although it lacks the characteristic PGRS domain
(Talarico\emph{et al.} , 2008). PE\_PGRS proteins have been found to be
secreted through ESX-5, and contain a YXXXE/D ESX general signal
sequence C-terminal to the PE domain (Daleke \emph{et al.} , 2012).
While many of the PE/PPE proteins remain uncharacterized, there are some
indications that they may be involved in virulence. The best studied
member of the PE family, PE\_PGRS33, has been shown to mediate \emph{M.
tuberculosis}entry into the macrophage, induce TNF-\selectlanguage{greek}α \selectlanguage{english}expression, and
cause cell death (Basu \emph{et al.} , 2007, Cadieux \emph{et al.} ,
2011, Palucci \emph{et al.} , 2016). Several studies have reported the
colocalization of PE\_PGRS33 with mitochondria and effects on
mitochondrial morphology (Aguilar-Lopez \emph{et al.} , 2019, Cadieux
\emph{et al.} , 2011, Moreno-Altamirano \emph{et al.} , 2012).
Additionally, there is evidence that PE/PPE family proteins co-evolved
with the ESX gene regions (Gey van Pittius \emph{et al.} , 2006) and
that PE\_PGRS and PPE\_MTPR subfamilies are secreted in an ESX-5
dependent manner, including PE17 (Abdallah \emph{et al.} , 2009).
Despite these intriguing data, many members of the PE/PPE families
remain uncharacterized.

Recently, \emph{Li} et al. published the first article directly
characterizing PE17 (Li \emph{et al.} , 2019). The authors expressed
PE17 in non-pathogenic \emph{Mycobacterium smegmatis} , which does not
natively encode the protein and does not persist intracellularly. It was
found that expression of PE17 significantly increased bacterial survival
and caused higher rates of necrosis in mouse peritoneal macrophages.
Expression of PE17 also significantly decreased the degree to which the
proinflammatory cytokines IL-6, IL-2, and TNF-\selectlanguage{greek}α \selectlanguage{english}were induced during
infection. This finding, coupled with the fact that PE17 was identified
as being expressed during \emph{M. tuberculosis} infection in
macrophages (Vipond \emph{et al.} , 2006), strengthens the hypothesis
that PE17 plays an important role in Mtb pathogenesis.

As we have previously observed that an Mtb ESAT-6 deletion strain does
not disrupt mitochondrial dynamics or mass upon infection of A549 AECs
when compared to the wild type H37Rv strain (Fine-Coulson \emph{et al.}
, 2015), we addressed the following two hypotheses in the present study:
1) ESAT-6 directly disrupts mitochondrial membranes, particularly in
light of its pore-forming ability; 2) ESAT-6 pore formation in the
bacteria-laden compartment is required for the release of another
protein that directly impacts mitochondrial dynamic, such as PE17.
Accordingly, we sought to learn more about mitochondrial changes
during\emph{M. tuberculosis} infection by heterologous expression of
both ESAT-6 and PE17 in A549 AECs. Surprisingly, we report that
expression of ESAT-6 alone does not result in mitochondrial changes.
However, PE17 expression leads to alterations in mass and distribution
not only of mitochondria, but of a variety of different organelles.
Furthermore, PE17 specifically localizes to intracytoplasmic lipid
droplets, suggesting that PE17 has the capacity to directly alter host
membrane dynamics.

\textbf{RESULTS}

\textbf{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 \emph{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.

\textbf{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 \emph{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 \textless{}0.0001).

\textbf{PE17 alters Golgi stack and trans-Golgi network (TGN) morphology
and mass, and inhibits protein secretion}

Our findings indicated that PE17 does not localize completely with
mitochondria, but rather shows a distinct distribution that does not
appear to overlap with major intracellular compartments. As PE17 has
been reported to decrease protein secretion in transfected cells
(Stamm\emph{et al.} , 2019), however, we hypothesized that PE17 might
localize to, or disrupt, an organelle involved in the secretory pathway.
To determine if PE17 localized to (or perturbed) the morphology of the
endoplasmic reticulum (ER) we performed colocalization experiments with
a marker for the ER, calnexin. We found that PE17 positive structures
did not strongly colocalize with the ER marker calnexin (Figure 2A). We
found that the overall morphology of the ER network was unaltered by
expression of PE17 (Figure 2A). To assess any possible changes of ER
mass, we analyzed the percent area of the ER in cells expressing PE17
and cells that were not expressing PE17 and found no significant
difference (Figure 2B, p\textgreater{}0.05).

As secretory cargo is transported from the ER to the Golgi stack, we
also investigated whether PE17 may affect this part of the secretory
pathway. Immunolocalisation of the Golgi stack-associated protein,
Gpp130, in PE17-transfected cells indicated that, while PE17 did not
localize to the Golgi stack, expression of PE17 dramatically altered
Golgi stack morphology (Figure 2C). In cells expressing PE17, compact
Golgi cisternae were no longer observed, but rather Gpp130 labelling
identified a series of short, disconnected tubules. While the Golgi
stack is typically located in a perinuclear domain, these short tubules
were found throughout the cytoplasm. Additionally, we noticed an overall
decrease in the amount of Gpp130 staining in cells expressing PE17 as
compared to cells not expressing PE17. Upon evaluating the percent area
of Gpp130 staining in both conditions, we found that cells expressing
PE17 had significantly less Golgi stack mass (Figure 2D,
p\textless{}0.0001).

As the TGN receives modified proteins from the Golgi stack, we
postulated that the TGN might also be impacted by PE17 expression. As
predicted, when PE17 was expressed, we observed similar morphological
changes to those observed with the Golgi stack (Figure 2E). While
typically compact, the TGN in PE17-expressing cells is fragmented and
dispersed throughout the cell with some cells appearing to have lost
most of this structure. Notably, the mass of the TGN was significantly
decreased in cells expressing PE17 (Figure 2F, p\textless{}0.0001).

Although loss of both Golgi stack and TGN would be sufficient to account
for a loss of secretion, we also took advantage of a novel
characteristic of the type II A549 alveolar epithelial cell line to
further analyze this finding. Surfactant protein C is expressed in the
lung and is secreted predominantly from type II alveolar cells after
undergoing a proteolytic maturation in multivesicular bodies and
subsequent delivery to the plasma membrane via exocytosis
(Conkright\emph{et al.} , 2001). Therefore, we transiently transfected
A549 AECs with PE17 and probed for total prosurfactant-C. Analysis of
fluorescence intensity in PE17 expressing versus non-expressing cells
demonstrated that total intracellular prosurfactant-C staining was
significantly brighter in cells expressing PE17 than in cells that were
not expressing PE17 (Figure 2G,H)(p\textless{}0.0001), suggesting a
disruption in secretory traffic from the ER to later compartments along
the secretory pathway.

One mechanism of organelle fragmentation is through the disruption of
cytoskeletal elements, such as microtubules and actin filaments. Since
PE17 expression causes severe changes in mitochondria, Golgi stack, and
TGN morphology and distribution, we next sought to determine if these
changes were due to disruption in F-actin or microtubule dynamics. Upon
PE17-myc expression and labelling with phalloidin to image F-actin and
anti-tubulin antibodies to visualize microtubules, we found that there
was no difference in the morphology of F-actin or microtubules in cells
expressing PE17 and cells that are not expressing PE17 (Figure 3A,B),
indicating that the changes in organelle morphology previously observed
in PE17-expressing strains are not due to large-scale cytoskeletal
disruptions.

\textbf{Differential effects of PE17 expression on early, late and
recycling endosomes and lysosomes}

As PE17 expression affected the Golgi stack, TGN, and mitochondria, but
not the ER, we investigated whether PE17 has a broad effect on most
organelles or these were specifically targeted by PE17 activity.
Inhibition of trafficking through early endosomal compartments by
bafilomycin A - or late endosomal compartments by wortmannin -- results
in the accumulation of enlarged spherical endosomes (Reaves \& Banting,
1994, Reaves \emph{et al.} , 1996). As the PE17 positive structures were
often distinctly spherical and perinuclear, we labelled cells
transfected with PE17-myc with antibodies to markers of early (EEA1) and
recycling endosomes (Tfr, transferrin receptor), respectively. There was
no colocalization between PE17 and EEA1 or Tfr (Figure 4A,C). No changes
in the morphology of the recycling or early endosomes were apparent.
Upon quantifying the mass, we found no difference between cells
expressing PE17 and those that were not (p = 0.4033, p = 0.0576,
respectively) (Figure 4B,D).

We next investigated effects on late endosomes and lysosomes following
transfection with PE17 using antibodies against CD63 and LAMP1,
respectively. In both instances, we again saw no colocalization of these
markers with PE17 and saw no obvious changes in organelle morphology
(Figure 4E,G). However, we measured a significant reduction in late
endosomes (p \textless{} 0.0001) and a significant, but milder, effect
on lysosomal mass (p \textless{} 0.0001) (Figure 4F,H). These results
support a specific effect on a subset of organelles and preclude a
general ablation of organelle integrity.

\textbf{PE17 Associates with Lipid Droplets via its C-terminal domain}

Lipid droplets (LDs) are monolayered organelles involved in nutrient
storage, trafficking, and toxicity prevention. Recent investigations
into the interaction between host lipid droplets and infection with Mtb
highlight the importance of these for the intracellular survival of the
bacterium (Lovewell \emph{et al.} , 2016). Lipid droplets can
translocate to the bacteria-laden vacuole after infection, possibly
serving as an important nutrient source (Daniel \emph{et al.} , 2011).
Due to the circular shape of lipid droplets, we wondered if the
PE17-positive clusters we observed might be localizing to the LD
surface. Post-transfection with PE17-myc, A549 cells were labelled with
anti-myc antibodies to visualize PE17 and BODIPY 493/503, which stains
neutral lipids (lipid droplets). The large spherical structures observed
in the light micrographs were positive for both BODIPY and PE17 (Figure
5A), suggesting these large accumulations contain neutral lipids and are
therefore likely lipid droplets. On closer inspection, the staining for
PE17 forms a ring surrounding the LDs, with the central portion of the
structure staining for a neutral lipid core (Figure 5B).

Lipid droplets have a unique membrane structure in comparison to other
organelles, consisting of a phospholipid monolayer surrounding a core of
neutral lipids, comprised primarily of steryl esters and
triacylglycerol. Proteins can associate with the cytosolic face of the
lipid droplet but are not found in the inner core (Olzmann \& Carvalho,
2019). To determine which portion of PE17 is important for LD
association, a truncated form of PE17 was generated lacking the
C-terminal 77 amino acids, PE17\textsubscript{1-233}-Myc (Figure 5C).
The PE17\textsubscript{1-233} truncation did not localize to or cause
redistribution of LDs (Figure 5D), rather it was distributed throughout
the cell. In addition, it did not elicit the changes in Golgi stack
morphology compared to the full-length protein (Figure 5E) but did
result in alterations of mitochondrial morphology (Figure 5F). These
findings indicate that the C-terminus of PE17 is not only important for
localization to LDs, but also for its effects on organelle morphology
and distribution.

In addition to perturbing organelle integrity, PE17-myc-his expression
appeared to diminish cell size in most of our micrographs. To confirm
this finding, we found that A549 cells expressing PE17-myc-his were
indeed smaller, overall, than a control strain (Figure 6A). Therefore,
we investigated whether PE17 was also inducing cell death upon
expression. As our protocol is based on transient transfections making
analysis of cell death in the population more difficult, we utilized a
yeast-based system to address this question. This approach is relevant
due to the general conservation of lipid droplet synthesis pathways
across eukaryotes. Upon high-level expression of PE17 driven by a
galactose-inducible promoter (using \selectlanguage{greek}β-\selectlanguage{english}estradiol for induction), we noted
strong inhibition of growth when compared to the empty vector control
(Figure 6B). Therefore, ectopic expression of PE17 in eukaryotic cells
appears to be inhibitory to cellular viability.

In order to localize PE17 in yeast cells and compare this localization
to what we previously observed in A549 cells, we fused the fluorescent
protein, mRuby2, to the C-terminus of PE17 in the same
galactose-inducible vector; high level expression of this construct
inhibited yeast cell growth to similar levels as the untagged protein
(Figure 6C) and full-length expression of PE17-mRuby2 in yeast was
observed via immunoblot (Figure 6D). We then moved this construct into
yeast strains harboring a plasmid constitutively expressing GFP-Erg6,
the yeast [?]24-sterol methyltransferase known to strongly associate with
the surface of lipid droplets (Leber \emph{et al.} , 1994). GFP-Erg6 was
observed to localize to punctate structures defining lipid droplets, as
expected (Figure 6E). PE17-mRuby also localized to punctate structures
which strongly overlapped with GFP-Erg6 localization (Figure 6E),
suggesting that PE17-mRuby also bound lipid droplets in yeast.

Yeast strains lacking the two sterol acetyltransferases, Are1p and
Are2p, and two enzymes with diacylglycerol O-acyltransferase activity,
Lro1p and Dga1p, are unable to accumulate lipid droplets (Joshi \emph{et
al.} , 2018); expression of only a single one of these genes allows the
formation of lipid droplets in vivo (Jacquier \emph{et al.} , 2011). As
lipid droplets are synthesized at the ER, lipid droplet-binding proteins
are known to redistribute to the ER in the absence of free lipid
droplets. Therefore, to confirm that PE17 is specifically recruited to
lipid droplets, we expressed GFP-Erg6 and the galactose-inducible
PE17-mRuby in either the \emph{are1[?] are2[?] lro1[?] dga1[?]} (3[?]\emph{dga1[?]}
) background, or an isogenic genetic background that contains a
galactose-inducible \emph{DGA1} gene (3[?] \emph{GAL-DGA1} ) (Jacquier
\emph{et al.} , 2011)\emph{.} Under glucose growth conditions for both
yeast strains, the constitutively-expressed GFP-Erg6 protein accumulates
on both the perinuclear and cortical ER instead of punctate lipid
droplets (Figure 6F), as expected. After induction of PE17-mRuby under
galactose conditions, PE17-mRuby localizes to cortical membrane regions
in the 3[?] \emph{dga1[?]} strain, similar to the localization of GFP-Erg6
(Figure 6F). In contrast, however, the 3[?] \emph{GAL-DGA1}strain under
galactose conditions now begins to accumulate cytosolic lipid droplets
marked by GFP-Erg6, which also recruit PE17-mRuby (Figure 6F). Finally,
we isolated membrane fractions highly enriched for lipid droplets from
strains expressing GFP-Erg6 and either a vector control or PE17-mRuby.
Direct observation of these lipid droplets shows distinct labeling with
GFP-Erg6, as anticipated (Figure 6G). Lipid droplets isolated from the
PE17-mRuby strains show near-perfect colocalization of GFP-Erg6 and
PE17-mRuby on the lipid droplet surface membrane (Figure 6G). Taken
together, the localization patterns of PE17-mRuby strongly support the
hypothesis that PE17 specifically binds free lipid droplets in both A549
and yeast cells, and likely the lipid droplets contained within host
cells during an infection.

\textbf{DISCUSSION}

Examples of bacterial pathogenicity factors being directly targeted to
-- and imported into -- mitochondria have been described for a number of
pathogens (Kozjak-Pavlovic \emph{et al.} , 2008, Spier \emph{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 \emph{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 \emph{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 \emph{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 \emph{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-Coulson\emph{et 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 \emph{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
\emph{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
Cadieux\emph{et al} . A stably-transfected cell line expressing an
inducible PE\_PGRS33 was generated and the mitochondrial localisation
confirmed by immunofluorescence microscopy (Cadieux \emph{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 \emph{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 \emph{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 \emph{et al.} , 1986, Oda \emph{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, \emph{M. tuberculosis} is known to prevent
fusion of the bacterium-containing compartment with late endosomes,
increasing bacterial viability (Via \emph{et al.} , 1997, Westman \&
Grinstein, 2020, Zulauf \emph{et al.} , 2018). In A549 cells, it has
been shown that the \emph{M. tuberculosis} bacterium-containing
compartment does acquire late endosomal markers, but avoids lysosomal
fusion through some involvement of the autophagy pathway (Fine \emph{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
\emph{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 \emph{M.
smegmatis}increased the bacterial viability during macrophage infection
while increasing macrophage necrosis (Li \emph{et al.} , 2019).
While\emph{Mycobacterium 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 \emph{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
intracellular\emph{Mycobacterium} . 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
by\emph{Mycobacterium} .

Several infectious diseases caused by intracellular pathogens have been
associated with effects on LD-organelle interfaces (Kory \emph{et al.} ,
2016), including the association of Mtb-containing phagosomes with host
LDs within foamy macrophages (Peyron \emph{et al.} , 2008). This process
is mediated by Rab7 (Roque \emph{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 \emph{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-Torres\emph{et 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 (Garcia\emph{et 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 (Menon\emph{et 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 \emph{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 \emph{et al.} , 2020) as well as promoting
lysosome-mitochondria contacts (Wong \emph{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 \emph{et
al.} , 2017). At the LD surface, Arf1/COPI facilitates the transfer of
proteins from the ER to LD, and regulates LD surface tension
(Thiam\emph{et al.} , 2013, Wilfling \emph{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.

\textbf{MATERIALS AND METHODS}

\textbf{Cell Lines}

A549 cells were grown in Dulbecco's modified Eagle medium (DMEM) (Gibco)
supplemented with 10\% heat inactivated fetal bovine serum (FBS)
(Sigma), penicillin-streptomycin (100 U/mL, Gibco), and L-glutamine
(2mM, Gibco), at 37\selectlanguage{ngerman}°C in 5\% CO\textsubscript{2}. All\emph{Saccharomyces
cerevisiae} strains used were derivatives of BY4742 (MAT
\(\alpha\) \emph{his3} \emph{1 leu2} \emph{0 lys2} \emph{0 ura3}
\emph{0} ), some of which were modified to induce all\emph{GAL1}
promoters via addition of \(\beta\)-estradiol due to the
incorporation of the gene for Gal4-estrogen receptor-VP16 protein within
the \emph{leu2} \emph{0} locus (Louvion \emph{et al.} , 1993). Yeast
strains either lacking lipid droplets (VCY2; BY4742 \emph{are1::KanMX
are2::KanMX trp1::URA3 lro1::TRP1 dga1::lox-HIS-lox} ) or containing the
galactose-inducible \emph{DGA1} gene (VCY3; BY4742 \emph{are1::KanMX
are2::KanMX trp1::URA3 lro1::TRP1 GAL-DGA1::HIS3} ) were kind gifts from
Dr. William Prinz (NIDDK).

\textbf{Plasmids and Transfections}

Plasmid pMRLB.7 containing gene Rv3875 (Protein ESAT-6) from
Mycobacterium tuberculosis, NR-50170 and genomic DNA from Mycobacterium
tuberculosis, Strain H37Rv, NR-48669 were obtained through BEI Resources
(National Institutes of Health, NIAID). The expression vector
mEmerald-N1 used as a control in the ESAT-6 studies, was a gift from
Michael Davidson (Addgene plasmid \# 53976;
http://n2t.net/addgene:53976; RRID:Addgene\_53976).

Gene Rv3875 containing the coding region for Protein ESAT-6 was
amplified from plasmid pMRLB.7 using primers Esat6XbaF
(5'-CGAGTCTAGATGCCACCATGACAGAGCAG-3') and Esat6ApaR
(5'-GTTCGAAGGGCCCGAGTGCGAACATCCCAGTGAC-3'). The fragment was then cloned
in-frame with the myc tag in pcDNA3.1(+)Myc-His A (Thermofisher) using
the XbaI and ApaI restriction sites to create the plasmid
pcDNA3.1myc-hisA-ESAT6.

The Mtb genomic region containing Rv1646 (PE17) was amplified from

The Mtb genomic region containing Rv1646 (PE17) was amplified from
Mycobacterium tuberculosis H37Rv genomic DNA and inserted into
pcDNA3.1(+) using the following primers: PE17HindIIF
(5'-GGATTGCTAAGCTTTAGACTTTATTTC-3') and PE17XbaR
(5'-GTCTCTAGACGGTTCGAAACCG-3'). PE17-Myc-His and
pcDNA3.1-PE17\textsubscript{1-233}-Myc-His were generated by cloning
into the \emph{Hind} III and \emph{Xba} I (New England BioLabs)
restriction sites of pcDNA3.1(+)Myc-His A (Thermofisher). Identical
forward primers were used in generation of both constructs
(5'-GGATTGCTAAGCTTTAGACTTTATTTC). Reverse primers are as follows: PE17,
5'-GTCTCTAGACGGTTCGAAACCG; PE17\textsubscript{1-223},
5'-GCCGGTCACCGCTCTAGAGGGAGCTTGCAG. All constructs were confirmed by DNA
sequencing. The plasmids were amplified in RapidTrans TAM1
competent\emph{E.coli} (Active Motif) and purified using a genElute
plasmid miniprep kit or HiSpeed plasmid maxi kit prep kit (Sigma,
Qiagen, respectively). DNA concentrations were quantified using a
NanoDrop 2000 Spectrophotometer (Thermofisher).

pYES2-PE17 was constructed by amplifying PE17 with primers (forward: 5'-
CGGGATCTGTACGACGATGACGATAAGGTAATGTCGTTTCTCACCGTGGC; reverse:
5'-GAGACCGAGGAGAGGGTTAGGGATAGGCTTTTAGAAACCGTTGAGTA

GGGCGGGAAG) containing 30 bp of homology upstream and downstream of the
pYES2/NT A (Thermo Scientific) multiple cloning site, thereby fusing the
N-terminal Xpress epitope to the PE17 open reading frame. pYES2/NT A was
linearized via \emph{Bam} HI digestion, and linear plasmid was
co-transformed into yeast with the above PCR amplicon to form the
pYES2-PE17 construct via gap repair. Successful incorporation of PE17
was verified by sequencing.

pYES2-PE17-mRuby2 was constructed by amplifying the mRuby2 open reading
frame from pFA6a-link-yomRuby2-SpHis5 (Lee \emph{et al.} , 2013) using
the primers 5'- GGCCTTCTTCCCGCCCTACTCAAC\emph{GGTTTCGGTGGTGGTGCTTCT}
ATGGTGTCCAAAGGAGAGGAGTTAATC and 5'-
ATAACTAATTACATGATGCGGCCCTCTAGGGATCTATATTACCCTGTTATCCCTAGCG, which
contain 30-bp homology to both the C-terminus of PE17 and the pYES2-PE17
vector, as well as introducing a {[}G\textsubscript{4}A{]} linker
sequence (underlined) between the PE17 and mRuby2 open reading frames.
The resultant amplicon was co-transformed into yeast with the pYES2-PE17
plasmid that had been previously linearized with PmeI, and selected for
growth on CSM plates lacking uracil to construct pYES2-PE17-mRuby2 via
gap repair. When used in the 3\selectlanguage{english}[?] \selectlanguage{english}\emph{dga1[?]} or 3\selectlanguage{english}[?]\emph{GAL-DGA1}
backgrounds, pYES2-PE17-mRuby2 was converted from a\emph{URA3} selection
to \emph{LYS2} by transforming a BY4742 strain harboring
pYES2-PE17-mRuby2 with HindIII-digested pM2660 (ATCC plasmid \#87559
(Cross, 1997), and selected for growth on CSM plates lacking lysine.
Resultant colonies were then screened for no growth on CSM plates
lacking uracil, creating the pYES2-PE17-mRuby2 (\emph{ura3::LYS2} )
plasmid.

To construct a plasmid expressing GFP-Erg6, the \emph{ERG6} open reading
frame was amplified from the BY4742 yeast genome using the primer pair
5'- ATGGATGAACTA TACAAGTCCGGACTCAGATCTATGAGTGAAACAGAATTGAGAAAAAG and 5'-
GTC GACTGCAGAATTCGAAGCTTGAGCTCGAGATCTTTATTGAGTTGC TTCTTGGGAAG. The
resultant amplicon was co-transformed into yeast with plasmid pGO36
(Odorizzi \emph{et al.} , 1998) which had been previously linearized
with BglII. Resultant gap-repaired plasmids were selected on CSM plates
lacking uracil, thus constructing the pGO36-Erg6 (GFP-Erg6) plasmid. In
order to convert the pGO36-Erg6 plasmid from uracil to leucine
selection, BY4742 harboring pGO36-Erg6 was transformed with
SmaI-digested pUL9 (ATCC plasmid \#87552 (Cross, 1997)) and
transformants were selected on CSM plates lacking leucine. Resultant
colonies were then screened for no growth on plates lacking uracil,
creating the pGO36-Erg6 (\emph{ura3::LEU2} ) plasmid.

Mammalian cells were grown on sterilized coverlips for 24 hours prior to
transient transfection using jetPRIME (Polyplus) according to the
manufacturers protocol with the following exception: for each well of a
six well plate, 1 \selectlanguage{greek}µ\selectlanguage{english}g DNA was used per transfection. Transient
transfections were allowed to incubate for 48 hours before processing.

Infection of A549 cells with Mtb and post-fixation staining with
Mitotracker was performed as described previously (Fine-Coulson \emph{et
al.} , 2015).

\textbf{Immunofluorescence and Antibodies}

At the appropriate time-point, coverslips were washed with PBS prior to
fixation with 4\% formaldehyde for 20 minutes. Slides were again washed
with PBS prior to permeabilization (0.1\% Triton-X 100) for 5 minutes
and blocking (3\% BSA, 1 \% serum) for 20 minutes. Slides were incubated
with primary antibodies suspended in blocking solution for one hour.
Slides were washed with 10\% blocking solution in PBS, followed by PBS.
All following steps were completed in the dark. Cells were incubated in
secondary antibodies, DAPI, and/or Alexa Fluor 488 Phalloidin
(Invitrogen) suspended in blocking solution for 20 minutes. Slides were
washed as above, and appropriate slides were incubated in a 1\selectlanguage{greek}µ\selectlanguage{english}g/ml
preparation of BODIPY 493/503 (Thermofisher) for 30 minutes. These
slides were washed with PBS, and all slides were mounted with Mowiol
4-88 (Sigma), and allowed to dry overnight before imaging.

Primary antibodies used in immunofluorescence and immunoblot experiments
are as follows: Myc (9E10, Invitrogen), Myc (4A6, Millipore) COXIV
(3E11, Cell Signaling Technology), Calnexin (ab22595, Abcam), Gpp130
(Poly19238, BioLegend), TGN46 (AHP500, Bio-Rad), Alpha tubulin (DM1A,
Cell Signaling Technology), Beta tubulin (TBN06, Thermofisher),
Transferrin Receptor (CBL47, Millipore), EEA1(clone 14, BD Biosciences),
CD63 (ab59479, Abcam), Lamp1 (D2D11 XP, Cell Signaling),
Anti-Xpress(46-0528, Invitrogen), and polyclonal Sec17p serum (Haas and
Wickner, 1996).

\textbf{Yeast lipid droplet isolation}

Yeast membrane fractions highly enriched for lipid droplets were
prepared essentially as described in (Ding \emph{et al.} , 2013), with
minor modifications. Briefly, 25 ml yeast strains harboring pGO36-Erg6
(\emph{ura3:LEU2} ) and either the pYES2 vector control or the
galactose-inducible pYES2-PE17-mRuby2 were grown to saturation at 30\selectlanguage{ngerman}°C
in selective media. These cultures were then used to inoculate 800 ml
fresh selective medium containing 1 \selectlanguage{greek}µ\selectlanguage{english}M \selectlanguage{greek}β-\selectlanguage{english}estradiol to induce the
expression of PE17 and grown for 16h at 30\selectlanguage{ngerman}°C. Cells were harvested via
centrifugation and washed twice with 30 ml PBS buffer. Cell pellets were
suspended in 7 ml Buffer A (20 mM tricine, 250 mM sucrose, pH = 7.8)
containing EDTA-free protease inhibitor cocktail, and left on ice 20
min. Suspended cells were lysed in a OneShot cell disruptor (Constant
Systems) with three passes at 22000psi (\textasciitilde{}1500 bar), and
cell debris was cleared via centrifugation (3000 x \emph{g} , 10 min).
10 ml resultant supernatants were transferred to a SW41 ultracentrifuge
tubes and overlayed with Buffer B (20 mM HEPES, 100 mM KCl, 2 mM
MgCl\textsubscript{2}, pH = 7.4). Tubes were centrifuged (10500
x\emph{g} , 4°C, 1 h), then immediately recentrifuged (185000 x \emph{g}
, 4°C, 1h). Lipid droplets were removed from the top layer, then
centrifuged (20000 x \emph{g} , 4°C, 5 min) and washed 3 times in Buffer
B.

\textbf{Confocal Microscopy and Image Analysis}

All slides were imaged on a Nikon A1R confocal microscope using either a
40X or 60X objective. To analyze the percent area occupied by a specific
organelle, 10 fields per slide were imaged using the 40X objective.
These frames were processed using an in-house plugin for FIJI (National
Institutes of Health), which contained components inspired by the ImageJ
Mitochondrial Morphology Macro (Ruben K. Dagda Ph.D.; University of
Nevada School of Medicine, Stefan Strack Ph.D.; University of Iowa, and
Charleen Chu; University of Pittsburgh). Briefly, cell perimeters were
manually outlined, the channels were split, thresholded, and analyzed
for percent area of the appropriate channel.

\textbf{Statistical Analysis}

Cells analyzed from three slides for each condition were pooled.
Gaussian distribution was visually assessed through histograms.
Mann-Whitney U test was used for

analyzing statistical significance. In all instances, a p-values \selectlanguage{english}[?] 0.05
were considered significant. Statistical analyses were performed using
Prism version 6 (GraphPad).

\textbf{Acknowledgements}

The authors would like to thank Drs. Greg Odorizzi and William Prinz for
providing essential reagents. The expression plasmid, mEmerald-N1, was a
gift from Michael Davidson (Addgene plasmid \# 53976). This work was
supported by a grant from the National Institute of Allergy and
Infectious Diseases (R21-AI140448) to A.J.W. V.J.S. was supported in
part by the University of Georgia Department of Microbiology. The
content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Institutes of
Health. The Authors have no conflicts of interest to declare.

\textbf{References}

Abarca-Rojano, E., Rosas-Medina, P., Zamudio-Cortez, P.,
Mondragon-Flores, R., and Sanchez-Garcia, F.J. (2003)\emph{Mycobacterium
tuberculosis} virulence correlates with mitochondrial cytochrome c
release in infected macrophages. \emph{Scand J Immunol} \textbf{58} :
419-427.

Abdallah, A.M., Verboom, T., Weerdenburg, E.M., Gey van Pittius, N.C.,
Mahasha, P.W., Jimenez, C., Parra, M., Cadieux, N., Brennan, M.J.,
Appelmelk, B.J., and Bitter, W. (2009) PPE and PE\_PGRS proteins
of\emph{Mycobacterium marinum} are transported via the type VII
secretion system ESX-5. \emph{Mol Microbiol} \textbf{73} : 329-340.

Aguilar-Lopez, B.A., Correa, F., Moreno-Altamirano, M.M.B., Espitia, C.,
Hernandez-Longoria, R., Oliva-Ramirez, J., Padierna-Olivos, J., and
Sanchez-Garcia, F.J. (2019) LprG and PE\_PGRS33 \emph{Mycobacterium
tuberculosis} virulence factors induce differential mitochondrial
dynamics in macrophages. \emph{Scand J Immunol} \textbf{89} : e12728.

Armstrong, J.A., and Hart, P.D. (1971) Response of cultured macrophages
to \emph{Mycobacterium tuberculosis} , with observations on fusion of
lysosomes with phagosomes. \emph{J Exp Med} \textbf{134} : 713-740.

Asalla, S., Mohareer, K., and Banerjee, S. (2017) Small Molecule
Mediated Restoration of Mitochondrial Function Augments
Anti-Mycobacterial Activity of Human Macrophages Subjected to
Cholesterol Induced Asymptomatic Dyslipidemia. \emph{Front Cell Infect
Microbiol} \textbf{7} : 439.

Augenstreich, J., and Briken, V. (2020) Host Cell Targets of Released
Lipid and Secreted Protein Effectors of \emph{Mycobacterium
tuberculosis} . \emph{Front Cell Infect Microbiol} \textbf{10} : 595029.

Barczak, A.K., Avraham, R., Singh, S., Luo, S.S., Zhang, W.R., Bray,
M.A., Hinman, A.E., Thompson, M., Nietupski, R.M., Golas, A.,
Montgomery, P., Fitzgerald, M., Smith, R.S., White, D.W., Tischler,
A.D., Carpenter, A.E., and Hung, D.T. (2017) Systematic, multiparametric
analysis of \emph{Mycobacterium tuberculosis} intracellular infection
offers insight into coordinated virulence. \emph{PLoS Pathog}\textbf{13}
: e1006363.

Basu, S., Pathak, S.K., Banerjee, A., Pathak, S., Bhattacharyya, A.,
Yang, Z., Talarico, S., Kundu, M., and Basu, J. (2007) Execution of
macrophage apoptosis by PE\_PGRS33 of \emph{Mycobacterium
tuberculosis}is mediated by Toll-like receptor 2-dependent release of
tumor necrosis factor-alpha. \emph{J Biol Chem} \textbf{282} :
1039-1050.

Cadieux, N., Parra, M., Cohen, H., Maric, D., Morris, S.L., and Brennan,
M.J. (2011) Induction of cell death after localization to the host cell
mitochondria by the \emph{Mycobacterium tuberculosis} PE\_PGRS33
protein. \emph{Microbiology (Reading)} \textbf{157} : 793-804.

Cao, T., Lyu, L., Jia, H., Wang, J., Du, F., Pan, L., Li, Z., Xing, A.,
Xiao, J., Ma, Y., and Zhang, Z. (2019) A Two-Way Proteome Microarray
Strategy to Identify Novel \emph{Mycobacterium tuberculosis} -Human
Interactors. \emph{Front Cell Infect Microbiol} \textbf{9} : 65.

Chen, M., Gan, H., and Remold, H.G. (2006) A mechanism of virulence:
virulent \emph{Mycobacterium tuberculosis strain} H37Rv, but not
attenuated H37Ra, causes significant mitochondrial inner membrane
disruption in macrophages leading to necrosis. \emph{Journal of
immunology} \textbf{176} : 3707-3716.

Cole, S.T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris,
D., Gordon, S.V., Eiglmeier, K., Gas, S., Barry, C.E., 3rd, Tekaia, F.,
Badcock, K., Basham, D., Brown, D., Chillingworth, T., Connor, R.,
Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd,
S., Hornsby, T., Jagels, K., Krogh, A., McLean, J., Moule, S., Murphy,
L., Oliver, K., Osborne, J., Quail, M.A., Rajandream, M.A., Rogers, J.,
Rutter, S., Seeger, K., Skelton, J., Squares, R., Squares, S., Sulston,
J.E., Taylor, K., Whitehead, S., and Barrell, B.G. (1998) Deciphering
the biology of \emph{Mycobacterium tuberculosis} from the complete
genome sequence. \emph{Nature} \textbf{393} : 537-544.

Conkright, J.J., Bridges, J.P., Na, C.L., Voorhout, W.F., Trapnell, B.,
Glasser, S.W., and Weaver, T.E. (2001) Secretion of surfactant protein
C, an integral membrane protein, requires the N-terminal
propeptide.\emph{J Biol Chem} \textbf{276} : 14658-14664.

Cross, F.R. (1997) 'Marker swap' plasmids: convenient tools for budding
yeast molecular genetics. \emph{Yeast} \textbf{13} : 647-653.

D'Avila, H., Melo, R.C., Parreira, G.G., Werneck-Barroso, E.,
Castro-Faria-Neto, H.C., and Bozza, P.T. (2006) \emph{Mycobacterium
bovis} bacillus Calmette-Guerin induces TLR2-mediated formation of lipid
bodies: intracellular domains for eicosanoid synthesis in
vivo.\emph{Journal of immunology} \textbf{176} : 3087-3097.

Daleke, M.H., Ummels, R., Bawono, P., Heringa, J., Vandenbroucke-Grauls,
C.M., Luirink, J., and Bitter, W. (2012) General secretion signal for
the mycobacterial type VII secretion pathway. \emph{Proc Natl Acad Sci U
S A} \textbf{109} : 11342-11347.

Daniel, J., Maamar, H., Deb, C., Sirakova, T.D., and Kolattukudy, P.E.
(2011) \emph{Mycobacterium tuberculosis} uses host triacylglycerol to
accumulate lipid droplets and acquires a dormancy-like phenotype in
lipid-loaded macrophages. \emph{PLoS Pathog} \textbf{7} : e1002093.

Ding, Y., Zhang, S., Yang, L., Na, H., Zhang, P., Zhang, H., Wang, Y.,
Chen, Y., Yu, J., Huo, C., Xu, S., Garaiova, M., Cong, Y., and Liu, P.
(2013) Isolating lipid droplets from multiple species. \emph{Nat
Protoc}\textbf{8} : 43-51.

Duan, L., Gan, H., Golan, D.E., and Remold, H.G. (2002) Critical role of
mitochondrial damage in determining outcome of macrophage infection
with\emph{Mycobacterium tuberculosis} . \emph{Journal of
immunology}\textbf{169} : 5181-5187.

Fine, K.L., Metcalfe, M.G., White, E., Virji, M., Karls, R.K., and
Quinn, F.D. (2012) Involvement of the autophagy pathway in trafficking
of \emph{Mycobacterium tuberculosis} bacilli through cultured human type
II epithelial cells. \emph{Cellular microbiology} \textbf{14} :
1402-1414.

Fine-Coulson, K., Giguere, S., Quinn, F.D., and Reaves, B.J. (2015)
Infection of A549 human type II epithelial cells with\emph{Mycobacterium
tuberculosis} induces changes in mitochondrial morphology, distribution
and mass that are dependent on the early secreted antigen, ESAT-6.
\emph{Microbes Infect} \textbf{17} : 689-697.

Fishbein, S., van Wyk, N., Warren, R.M., and Sampson, S.L. (2015)
Phylogeny to function: PE/PPE protein evolution and impact
on\emph{Mycobacterium tuberculosis} pathogenicity. \emph{Mol
Microbiol}\textbf{96} : 901-916.

Garcia, A., Sekowski, A., Subramanian, V., and Brasaemle, D.L. (2003)
The central domain is required to target and anchor perilipin A to lipid
droplets. \emph{J Biol Chem} \textbf{278} : 625-635.

Gey van Pittius, N.C., Sampson, S.L., Lee, H., Kim, Y., van Helden,
P.D., and Warren, R.M. (2006) Evolution and expansion of
the\emph{Mycobacterium tuberculosis} PE and PPE multigene families and
their association with the duplication of the ESAT-6 (esx) gene cluster
regions. \emph{BMC Evol Biol} \textbf{6} : 95.

Guerra, F., and Bucci, C. (2016) Multiple Roles of the Small GTPase
Rab7. \emph{Cells} \textbf{5} .

Gygli, S.M., Borrell, S., Trauner, A., and Gagneux, S. (2017)
Antimicrobial resistance in \emph{Mycobacterium tuberculosis} :
mechanistic and evolutionary perspectives. \emph{FEMS Microbiol
Rev}\textbf{41} : 354-373.

Houben, D., Demangel, C., van Ingen, J., Perez, J., Baldeon, L.,
Abdallah, A.M., Caleechurn, L., Bottai, D., van Zon, M., de Punder, K.,
van der Laan, T., Kant, A., Bossers-de Vries, R., Willemsen, P., Bitter,
W., van Soolingen, D., Brosch, R., van der Wel, N., and Peters, P.J.
(2012) ESX-1-mediated translocation to the cytosol controls virulence of
mycobacteria. \emph{Cellular microbiology} \textbf{14} : 1287-1298.

Ingelmo-Torres, M., Gonzalez-Moreno, E., Kassan, A., Hanzal-Bayer, M.,
Tebar, F., Herms, A., Grewal, T., Hancock, J.F., Enrich, C., Bosch, M.,
Gross, S.P., Parton, R.G., and Pol, A. (2009) Hydrophobic and basic
domains target proteins to lipid droplets. \emph{Traffic} \textbf{10} :
1785-1801.

Jacquier, N., Choudhary, V., Mari, M., Toulmay, A., Reggiori, F., and
Schneiter, R. (2011) Lipid droplets are functionally connected to the
endoplasmic reticulum in \emph{Saccharomyces cerevisiae} . \emph{J Cell
Sci} \textbf{124} : 2424-2437.

Jamwal, S., Midha, M.K., Verma, H.N., Basu, A., Rao, K.V., and Manivel,
V. (2013) Characterizing virulence-specific perturbations in the
mitochondrial function of macrophages infected with \emph{Mycobacterium
tuberculosis} . \emph{Sci Rep} \textbf{3} : 1328.

Joseph, S., Yuen, A., Singh, V., and Hmama, Z. (2017)\emph{Mycobacterium
tuberculosis} Cpn60.2 (GroEL2) blocks macrophage apoptosis via
interaction with mitochondrial mortalin. \emph{Biol Open}\textbf{6} :
481-488.

Joshi, A.S., Nebenfuehr, B., Choudhary, V., Satpute-Krishnan, P.,
Levine, T.P., Golden, A., and Prinz, W.A. (2018) Lipid droplet and
peroxisome biogenesis occur at the same ER subdomains. \emph{Nat
Commun}\textbf{9} : 2940.

Kaczmarek, B., Verbavatz, J.M., and Jackson, C.L. (2017) GBF1 and Arf1
function in vesicular trafficking, lipid homoeostasis and organelle
dynamics. \emph{Biol Cell} \textbf{109} : 391-399.

Kory, N., Farese, R.V., Jr., and Walther, T.C. (2016) Targeting Fat:
Mechanisms of Protein Localization to Lipid Droplets. \emph{Trends Cell
Biol} \textbf{26} : 535-546.

Kozjak-Pavlovic, V., Ross, K., and Rudel, T. (2008) Import of bacterial
pathogenicity factors into mitochondria. \emph{Current opinion in
microbiology} \textbf{11} : 9-14.

Leber, R., Zinser, E., Zellnig, G., Paltauf, F., and Daum, G. (1994)
Characterization of lipid particles of the yeast, \emph{Saccharomyces
cerevisiae} . \emph{Yeast} \textbf{10} : 1421-1428.

Lee, S., Lim, W.A., and Thorn, K.S. (2013) Improved blue, green, and red
fluorescent protein tagging vectors for \emph{S. cerevisiae} .
\emph{PLoS One} \textbf{8} : e67902.

Li, Z., Liu, H., Li, H., Dang, G., Cui, Z., Song, N., Wang, Q., Liu, S.,
and Chen, L. (2019) PE17 protein from \emph{Mycobacterium
tuberculosis}enhances \emph{Mycobacterium smegmatis} survival in
macrophages and pathogenicity in mice. \emph{Microb Pathog} \textbf{126}
: 63-73.

Louvion, J.F., Havaux-Copf, B., and Picard, D. (1993) Fusion of
GAL4-VP16 to a steroid-binding domain provides a tool for gratuitous
induction of galactose-responsive genes in yeast.
\emph{Gene}\textbf{131} : 129-134.

Lovewell, R.R., Sassetti, C.M., and VanderVen, B.C. (2016) Chewing the
fat: lipid metabolism and homeostasis during \emph{M.
tuberculosis}infection. \emph{Current opinion in microbiology}
\textbf{29} : 30-36.

McCann, J.R., McDonough, J.A., Pavelka, M.S., and Braunstein, M. (2007)
Beta-lactamase can function as a reporter of bacterial protein export
during \emph{Mycobacterium tuberculosis} infection of host
cells.\emph{Microbiology (Reading)} \textbf{153} : 3350-3359.

Menon, D., Singh, K., Pinto, S.M., Nandy, A., Jaisinghani, N., Kutum,
R., Dash, D., Prasad, T.S.K., and Gandotra, S. (2019) Quantitative Lipid
Droplet Proteomics Reveals \emph{Mycobacterium tuberculosis} Induced
Alterations in Macrophage Response to Infection. \emph{ACS Infect
Dis}\textbf{5} : 559-569.

Misumi, Y., Misumi, Y., Miki, K., Takatsuki, A., Tamura, G., and
Ikehara, Y. (1986) Novel blockade by brefeldin A of intracellular
transport of secretory proteins in cultured rat hepatocytes. \emph{J
Biol Chem} \textbf{261} : 11398-11403.

Mohareer, K., Medikonda, J., Vadankula, G.R., and Banerjee, S. (2020)
Mycobacterial Control of Host Mitochondria: Bioenergetic and Metabolic
Changes Shaping Cell Fate and Infection Outcome. \emph{Front Cell Infect
Microbiol} \textbf{10} : 457.

Moreno-Altamirano, M.M., Paredes-Gonzalez, I.S., Espitia, C.,
Santiago-Maldonado, M., Hernandez-Pando, R., and Sanchez-Garcia, F.J.
(2012) Bioinformatic identification of \emph{Mycobacterium
tuberculosis}proteins likely to target host cell mitochondria: virulence
factors?\emph{Microb Inform Exp} \textbf{2} : 9.

Nakamura, N., and Fujimoto, T. (2003) Adipose differentiation-related
protein has two independent domains for targeting to lipid
droplets.\emph{Biochem Biophys Res Commun} \textbf{306} : 333-338.

Oda, K., Hirose, S., Takami, N., Misumi, Y., Takatsuki, A., and Ikehara,
Y. (1987) Brefeldin A arrests the intracellular transport of a precursor
of complement C3 before its conversion site in rat
hepatocytes.\emph{FEBS Lett} \textbf{214} : 135-138.

Odorizzi, G., Babst, M., and Emr, S.D. (1998) Fab1p PtdIns(3)P 5-kinase
function essential for protein sorting in the multivesicular
body.\emph{Cell} \textbf{95} : 847-858.

Olzmann, J.A., and Carvalho, P. (2019) Dynamics and functions of lipid
droplets. \emph{Nat Rev Mol Cell Biol} \textbf{20} : 137-155.

Pai, M., Behr, M.A., Dowdy, D., Dheda, K., Divangahi, M., Boehme, C.C.,
Ginsberg, A., Swaminathan, S., Spigelman, M., Getahun, H., Menzies, D.,
and Raviglione, M. (2016) Tuberculosis. \emph{Nat Rev Dis
Primers}\textbf{2} : 16076.

Palucci, I., Camassa, S., Cascioferro, A., Sali, M., Anoosheh, S.,
Zumbo, A., Minerva, M., Iantomasi, R., De Maio, F., Di Sante, G., Ria,
F., Sanguinetti, M., Palu, G., Brennan, M.J., Manganelli, R., and
Delogu, G. (2016) PE\_PGRS33 Contributes to \emph{Mycobacterium
tuberculosis} Entry in Macrophages through Interaction with
TLR2.\emph{PLoS One} \textbf{11} : e0150800.

Peyron, P., Vaubourgeix, J., Poquet, Y., Levillain, F., Botanch, C.,
Bardou, F., Daffe, M., Emile, J.F., Marchou, B., Cardona, P.J., de
Chastellier, C., and Altare, F. (2008) Foamy macrophages from
tuberculous patients' granulomas constitute a nutrient-rich reservoir
for \emph{M. tuberculosis} persistence. \emph{PLoS Pathog} \textbf{4} :
e1000204.

Philips, J.A., and Ernst, J.D. (2012) Tuberculosis pathogenesis and
immunity. \emph{Annu Rev Pathol} \textbf{7} : 353-384.

Pol, A., Gross, S.P., and Parton, R.G. (2014) Review: biogenesis of the
multifunctional lipid droplet: lipids, proteins, and sites. \emph{J Cell
Biol} \textbf{204} : 635-646.

Reaves, B., and Banting, G. (1994) Vacuolar ATPase inactivation blocks
recycling to the trans-Golgi network from the plasma membrane.\emph{FEBS
Lett} \textbf{345} : 61-66.

Reaves, B.J., Bright, N.A., Mullock, B.M., and Luzio, J.P. (1996) The
effect of wortmannin on the localisation of lysosomal type I integral
membrane glycoproteins suggests a role for phosphoinositide 3-kinase
activity in regulating membrane traffic late in the endocytic
pathway.\emph{J Cell Sci} \textbf{109 ( Pt 4)} : 749-762.

Roque, N.R., Lage, S.L., Navarro, R., Fazolini, N., Maya-Monteiro, C.M.,
Rietdorf, J., Melo, R.C.N., D'Avila, H., and Bozza, P.T. (2020) Rab7
controls lipid droplet-phagosome association during mycobacterial
infection. \emph{Biochim Biophys Acta Mol Cell Biol Lipids}\textbf{1865}
: 158703.

Singh, V., Jamwal, S., Jain, R., Verma, P., Gokhale, R., and Rao, K.V.
(2012) Mycobacterium tuberculosis-driven targeted recalibration of
macrophage lipid homeostasis promotes the foamy phenotype. \emph{Cell
Host Microbe} \textbf{12} : 669-681.

Singh, V., Kaur, C., Chaudhary, V.K., Rao, K.V., and Chatterjee, S.
(2015) \emph{M. tuberculosis} Secretory Protein ESAT-6 Induces Metabolic
Flux Perturbations to Drive Foamy Macrophage Differentiation. \emph{Sci
Rep} \textbf{5} : 12906.

Smith, J., Manoranjan, J., Pan, M., Bohsali, A., Xu, J., Liu, J.,
McDonald, K.L., Szyk, A., LaRonde-LeBlanc, N., and Gao, L.Y. (2008)
Evidence for pore formation in host cell membranes by ESX-1-secreted
ESAT-6 and its role in \emph{Mycobacterium marinum} escape from the
vacuole. \emph{Infect Immun} \textbf{76} : 5478-5487.

Sorensen, A.L., Nagai, S., Houen, G., Andersen, P., and Andersen, A.B.
(1995) Purification and characterization of a low-molecular-mass T-cell
antigen secreted by \emph{Mycobacterium tuberculosis} . \emph{Infect
Immun} \textbf{63} : 1710-1717.

Spier, A., Stavru, F., and Cossart, P. (2019) Interaction between
Intracellular Bacterial Pathogens and Host Cell
Mitochondria.\emph{Microbiol Spectr} \textbf{7} .

Stamm, C.E., Pasko, B.L., Chaisavaneeyakorn, S., Franco, L.H., Nair,
V.R., Weigele, B.A., Alto, N.M., and Shiloh, M.U. (2019)
Screening\emph{Mycobacterium tuberculosis} Secreted Proteins Identifies
Mpt64 as a Eukaryotic Membrane-Binding Bacterial Effector.
\emph{mSphere}\textbf{4} .

Talarico, S., Zhang, L., Marrs, C.F., Foxman, B., Cave, M.D., Brennan,
M.J., and Yang, Z. (2008) \emph{Mycobacterium tuberculosis} PE\_PGRS16
and PE\_PGRS26 genetic polymorphism among clinical
isolates.\emph{Tuberculosis (Edinb)} \textbf{88} : 283-294.

Thiam, A.R., Antonny, B., Wang, J., Delacotte, J., Wilfling, F.,
Walther, T.C., Beck, R., Rothman, J.E., and Pincet, F. (2013) COPI buds
60-nm lipid droplets from reconstituted
water-phospholipid-triacylglyceride interfaces, suggesting a tension
clamp function. \emph{Proc Natl Acad Sci U S A} \textbf{110} :
13244-13249.

Via, L.E., Deretic, D., Ulmer, R.J., Hibler, N.S., Huber, L.A., and
Deretic, V. (1997) Arrest of mycobacterial phagosome maturation is
caused by a block in vesicle fusion between stages controlled by rab5
and rab7. \emph{J Biol Chem} \textbf{272} : 13326-13331.

Vipond, J., Vipond, R., Allen-Vercoe, E., Clark, S.O., Hatch, G.J.,
Gooch, K.E., Bacon, J., Hampshire, T., Shuttleworth, H., Minton, N.P.,
Blake, K., Williams, A., and Marsh, P.D. (2006) Selection of novel TB
vaccine candidates and their evaluation as DNA vaccines against aerosol
challenge. \emph{Vaccine} \textbf{24} : 6340-6350.

von der Haar, T. (2007) Optimized protein extraction for quantitative
proteomics of yeasts. \emph{PLoS One} \textbf{2} : e1078.

Walther, T.C., Chung, J., and Farese, R.V., Jr. (2017) Lipid Droplet
Biogenesis. \emph{Annu Rev Cell Dev Biol} \textbf{33} : 491-510.

Westman, J., and Grinstein, S. (2020) Determinants of Phagosomal pH
During Host-Pathogen Interactions. \emph{Front Cell Dev Biol}\textbf{8}
: 624958.

Wiker, H.G., and Harboe, M. (1992) The antigen 85 complex: a major
secretion product of \emph{Mycobacterium tuberculosis} . \emph{Microbiol
Rev} \textbf{56} : 648-661.

Wilfling, F., Thiam, A.R., Olarte, M.J., Wang, J., Beck, R., Gould,
T.J., Allgeyer, E.S., Pincet, F., Bewersdorf, J., Farese, R.V., Jr., and
Walther, T.C. (2014) Arf1/COPI machinery acts directly on lipid droplets
and enables their connection to the ER for protein
targeting.\emph{Elife} \textbf{3} : e01607.

Wong, Y.C., Ysselstein, D., and Krainc, D. (2018) Mitochondria-lysosome
contacts regulate mitochondrial fission via RAB7 GTP
hydrolysis.\emph{Nature} \textbf{554} : 382-386.

World Health Organization. (2019) Global tuberculosis report 2019.
In\emph{.} , pp.

Zulauf, K.E., Sullivan, J.T., and Braunstein, M. (2018) The SecA2
pathway of M\emph{ycobacterium tuberculosis} exports effectors that work
in concert to arrest phagosome and autophagosome maturation. \emph{PLoS
Pathog} \textbf{14} : e1007011.

\textbf{FIGURE LEGENDS}

\textbf{FIGURE 1. Mitochondria morphology remains tubular after ESAT-6
expression in A549 AECs. (} A) A549 cells were transfected with
pcDNA3.1-ESAT-6-myc for 48 hours prior to fixation and processing with
anti-myc antibodies (ESAT-6, red) and anti-COXIV (mitochondria,
green)(top panel). Mitochondrial morphology in cells expressing ESAT-6
appears similar to untransfected cells. For comparison, A549 cells were
infected with Mtb and cells fixed and stained for mitochondria using
Mitotracker Red(r) (lower panel in A), as in (Fine-Coulson \emph{et al.} ,
2015). After infection with Mtb, mitochondrial morphology appears
concentrated and fragmented. Scale bar = 10 \selectlanguage{greek}µ\selectlanguage{english}m. (B) The percentage of
cells with tubular, intermediate or fragmented mitochondria was
determined in cells transfected with ESAT-6-myc or with pcDNA3-mEmerald
as a transfection control. N=3, \textgreater{}100 cells counted per
transfection. \textbf{PE17 does not localize to the mitochondria of
A549~cells but~does decrease mitochondrial mass.~(} C) AECs were
transfected with pcDNA3.1-PE17-myc-his6 48 hours prior to fixation and
processing. Cells were probed with anti-COXIV (mitochondria,
green),~anti-myc~(PE17, red), and~stained with~DAPI (nucleus, blue).
Images were~captured by a~confocal microscope~and~are representative of
three independent experiments. Arrows highlight inclusions apparent in
the transmitted light images that correspond to PE17 positive
structures. Scale bar = 20 \selectlanguage{greek}µ\selectlanguage{english}m. (D) PE17 (red) appears as clusters of
empty spheres adjacent to the nucleus (blue). (E) Images captured from
the slides prepared in (C) were analyzed for organelle mass using an
in-house~FIJI~macro. Over 130 cells between three slides were measured
for the percent area of the cell containing mitochondria for each
condition. N = 3. Error bars indicate SEM. Significance was measured
using~a~Mann-Whitney~U~test. ***Indicates p-values \textless{}
0.0001.~\emph{~}

\textbf{FIGURE 2. Expression of PE17 in A549 AECs alters~Golgi~stack and
trans-Golgi~network, but not ER, morphology and mass.} ~AECs were
transfected with pcDNA3.1-PE17-Myc-His6 48 hours prior to fixation and
processing.~A,C,E) Cells were~probed~with
anti-myc~(PE17),~anti-calnexin~(ER; A),~anti-Gpp130 (golgi~stack; C),
anti-TGN46 (trans Golgi~network; E) and stained with DAPI
(nucleus).~Images were~captured on~a confocal microscope~and~are
representative of three independent experiments. Scale bar = 20 \selectlanguage{greek}µ\selectlanguage{english}m. B,
D, F) Images captured from the slides prepared in~A,C,E,
respectively,~~were analyzed using in-house FIJI macros. Over~90~cells
between three slides were measured for the percent area of the cell
containing the respective organelle for each condition. Error bars
indicate SEM. Significance was measured using~a~Mann-Whitney test.
***Indicate p-values \textless{} 0.0001.~Values \textgreater{}0.05 were
considered not significant.

\textbf{FIGURE 3. PE17 expression does not cause disruptions in the
cytoskeleton.} ~AECs were transfected with
pcDNA3.1-PE17-myc-His\textsubscript{6} 48 hours prior to fixation and
processing.~Cells were~stained with DAPI (nucleus) and phalloidin
(F-actin; A) and~probed~with anti-myc~(PE17)~and~anti-alpha
tubulin~(microtubules; B).~Images were~captured by~a confocal
microscope~and~are representative of two~independent experiments. Scale
bar = 25 \selectlanguage{greek}µ\selectlanguage{english}m.

\textbf{FIGURE 4. Expression of PE17 in A549 AECS does not alter the
mass of recycling or early endosomes but alters the mass of late
endosomes and lysosomes.} AECs were transfected with pcDNA3.1-PE17 48
hours prior to fixation and processing. (A,C,E,G) Cells were probed with
anti-myc (PE17), anti-transferrin receptor (recycling endosomes; A),
anti-EEA1 (early endosomes; C), anti-CD63 (late endosomes; E),
anti-lamp1 (lysosomes; G) and stained with DAPI (nucleus). Images were
captured on a confocal microscope and are representative of three
independent experiments. Scale bar \emph{=} 20 \selectlanguage{greek}µ\selectlanguage{english}m. (B, D, F, H) Images
captured from the slides prepared in A,C,E,G, respectively, were
analyzed using in-house macros in FIJI. Over 90 cells in B, D, and H and
over 60 cells in E were measured between experiments for each condition.
Error bars indication SEM. Significance was measured using a
Mann-Whitney U test. ***Indicate a p-values \textless{} 0.001. Values
\textgreater{}0.05 were considered nonsignificant.

\textbf{FIGURE 5. PE17 localizes to lipid droplets via the C-terminal
domain.} AECs were transfected with pcDNA3.1-PE17-Myc-His6 (A) or
pcDNA3.1-PE17\textsubscript{1-233}-Myc-His6 (D-F) 48 hours prior to
fixation and processing.~(A) Cells transfected with
pcDNA3.1-PE17-Myc-His6 were~stained with DAPI (nucleus, blue), BODIPY
(lipid droplets, green), and~probed~with
anti-myc~(PE17\textsubscript{;}red). Images were~captured by~a confocal
microscope~and~are representative of two~independent experiments. Scale
bar = 5 \selectlanguage{greek}µ\selectlanguage{english}m. B) Magnified view of the region of interest shown by the
boxed in area in A. Scale bar = 20 \selectlanguage{greek}µ\selectlanguage{english}m. C) Schematic cartoon showing the
domain structure of full-length PE17 and the truncated form,
PE17\textsubscript{1-233.}D-F) AECs were transfected with
pcDNA3.1-PE17\textsubscript{1-233}-Myc-His6 48 hours prior to fixation
and processing.~Cells were~stained with DAPI (nucleus, blue)
and~probed~with anti-myc~(PE17\textsubscript{1-233,} red) and (D) BODIPY
(lipid droplets, green), (E) anti-Gpp130 (Golgi~stack, green) or (D)
anti-COXIV (mitochondria, green). Insets show magnified views of areas
indicated by boxes. Scale bar = 5 \selectlanguage{greek}µ\selectlanguage{english}m.

\textbf{FIGURE 6. PE17 inhibits eukaryotic cell growth and binds yeast
lipid droplets.} (A) Yeast strains modified to induce
\emph{GAL}promoters with \selectlanguage{greek}β-\selectlanguage{english}estradiol (Methods) and harboring either
vector controls or (B) pYES-PE17 or (C) pYES-PE17-mRuby2 were grown to
saturation in selective medium, diluted to OD\textsubscript{600} = 1.0
in sterile water, then serially diluted 10-fold. 5 \selectlanguage{greek}µ\selectlanguage{english}l of each dilution
were spotted to CSM plates lacking uracil either lacking or containing 1
\selectlanguage{greek}µ\selectlanguage{english}M \selectlanguage{greek}β-\selectlanguage{english}estradiol. Plates were incubated for 48h at 30\selectlanguage{ngerman}°C and imaged. (D)
Saturated cultures from (C) were subcultured to 5 ml fresh medium either
lacking or containing 1 \selectlanguage{greek}µ\selectlanguage{english}M \selectlanguage{greek}β-\selectlanguage{english}estradiol and outgrown for 6h at 30\selectlanguage{ngerman}°C.
Total cellular proteins were extracted from equivalent cell densities as
in (von der Haar, 2007), separated via SDS-PAGE, and visualized via
immunoblot. Membrane was cut between the 25 and 37 kDa molecular weight
markers to blot for both PE17-mRuby2 (anti-Xpress, top) and Sec17p
(bottom). (E) Yeast strain harboring both GFP-Erg6 and the
galactose-inducible pYES-PE17-mRuby2 was grown to saturation in
selective medium at 30°C, subcultured to fresh medium containing 1 \selectlanguage{greek}µ\selectlanguage{english}M
\selectlanguage{greek}β-\selectlanguage{english}estradiol to induce PE17-mRuby2 expression, outgrown for 6h at 30\selectlanguage{ngerman}°C,
then visualized. Scale bar = 5 \selectlanguage{greek}µ\selectlanguage{english}m. (F) Yeast strains either lacking
(3[?]\emph{dga1[?]} ) or producing lipid droplets in the presence of
galactose (3[?] \emph{GAL-DGA1} ) and harboring GFP-Erg6 and the
galactose-inducible pYES-PE17-mRuby2 plasmids were grown to saturation
in selective medium containing 2\% glucose. Strains were subcultured to
fresh selective medium containing either 2\% glucose or 2\% galactose as
a carbon source. Cultures were grown for 18h at 30\selectlanguage{ngerman}°C and cells were
visualized via epifluorescent microscopy. Scale bar = 5 \selectlanguage{greek}µ\selectlanguage{english}m. (G) Lipid
droplets were purified (Materials and Methods) from yeast strains
harboring GFP-Erg6 and either vector control or PE17-mRuby2 plasmids. 15
\selectlanguage{greek}µ\selectlanguage{english}l from each lipid droplet fraction were quickly mixed into 15 \selectlanguage{greek}µ\selectlanguage{english}l molten
0.6\% agarose in 20 mM HEPES, 100 mM KCl, 2 mM MgCl\textsubscript{2}, pH
= 7.4. 15 \selectlanguage{greek}µ\selectlanguage{english}l of this mixture was mounted to a slide, allowed to solidify
for 1 min, and visualized via epifluorescent microscopy. Scale bar = 5
\selectlanguage{greek}µ\selectlanguage{english}m.

\selectlanguage{english}
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