INTRODUCTION
Tuberculosis is one of the top ten causes of death worldwide (WHO,
2019). While a vaccine against the etiological agent, the intracellular
bacterium Mycobacterium tuberculosis (Mtb), has been available
for almost one hundred years, it is ineffective for the population most
at risk for pulmonary infection (Pai et al. , 2016, Philips &
Ernst, 2012). Furthermore, microbial resistance against frontline
antibiotics continues to rise (Gygli 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, 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 et al. , 1997). This process is dependent on bacterial
factors secreted through the SecA2 pathway of Mtb (Zulauf 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. M. tuberculosis disrupts mitochondrial membrane
potential post-infection resulting in the aberrant release of cytochrome
c (Abarca-Rojano et al. , 2003, Chen et al. , 2006, Duanet al. , 2002) and disruption of cellular bioenergetics
parameters, including ATP and ROS concentrations (reviewed in (Mohareeret 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 et al. ,
2017) and A549 airway epithelial cells (AECs) (Fine-Coulson 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 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 et al. , 2017) and can interact with other organelles
through membrane contact sites, expanding or contracting depending on
cellular metabolism (Pol et al. , 2014). Several studies have
shown that M. tuberculosis modifies the host environment to
induce the formation of lipid droplets and increase bacterial
intracellular survival, most notably within foamy macrophages (D’Avilaet al. , 2006, Peyron et al. , 2008, Singh et al. ,
2012, Singh 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 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 et al. , 1995). Several studies have shown that ESAT-6
plays an integral role in M. tuberculosis escape into the host
cytosol by rupturing phagosomal membranes (Houben et al. , 2012,
Smith 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 (Stammet al. , 2019).
Alternative methodologies designed to identify secreted Mtb proteins and
their interactors have been developed using genetic, proteomic and
transposon screens (Barczak et al. , 2017, Cao et al. ,
2019, McCann et al. , 2007). A unique approach to characterizing
some of these secreted proteins was developed by Stamm et al (Stammet 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: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 theM. tuberculosis genome (Cole et al. , 1998, Fishbeinet al. , 2015). PE17 is evolutionarily grouped with the PE_PGRS
proteins, although it lacks the characteristic PGRS domain (Talaricoet 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 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 M. tuberculosisentry into the macrophage, induce TNF-α expression, and cause cell death
(Basu et al. , 2007, Cadieux et al. , 2011, Palucci et
al. , 2016). Several studies have reported the colocalization of
PE_PGRS33 with mitochondria and effects on mitochondrial morphology
(Aguilar-Lopez et al. , 2019, Cadieux et al. , 2011,
Moreno-Altamirano et al. , 2012). Additionally, there is evidence
that PE/PPE family proteins co-evolved with the ESX gene regions (Gey
van Pittius et al. , 2006) and that PE_PGRS and PPE_MTPR
subfamilies are secreted in an ESX-5 dependent manner, including PE17
(Abdallah et al. , 2009). Despite these intriguing data, many
members of the PE/PPE families remain uncharacterized.
Recently, Li et al. published the first article directly
characterizing PE17 (Li et al. , 2019). The authors expressed PE17
in non-pathogenic 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-α were induced during
infection. This finding, coupled with the fact that PE17 was identified
as being expressed during M. tuberculosis infection in
macrophages (Vipond 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 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 duringM. 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.