3. DISCUSSION
Mtb is one of the most successful
pathogens that has evolved since decades to be able to evade the host
mediated immune responses. Virulent Mtb possess the recently evolved
PE_PGRS subfamily proteins which have been reported to be crucial in
pathogenesis and immuno-modulation (Brennan, 2017). However, the
specific function of only some of these PE_PGRS proteins have been
investigated in Mtb pathogenesis.
One uncharacterized protein of the PE family is Rv2615c protein which
has been reported to be upregulated during dormant TB infection. This
study was conceived to investigate the possible role of PE_PGRS45
(Rv2615c) in host cell death and immune response modulation. I-TASSER
based homology mapping showed similarity between Rv2615c and
APAF1-Apoptosome complex. Additionally, functional and molecular Gene
Ontology predictions showed possible role of Rv2615c protein in host
cell death pathways particularly pinpointing its role in apoptosis and
cysteine type endo-peptidase activation. To experimentally evaluate the
role of this protein in host macrophage apoptosis, in-vitroexperiments were performed with recombinant Rv2615c protein. Our results
established that the Rv2615c protein reduces cell viability and induces
apoptotic cell death in THP-1 macrophages as analysed through MTT and
CellTiter Blue assay, Annexin V-FITC/PI staining and TUNEL assay. About
12 PE_PGRS proteins are known to be expressed at late stage (at 90 days
in guinea pig lungs upon Mtb infection) and some of them like PE_PGRS5
and PE_PGRS17 are reported to induce host cell death (Kruh et
al. , 2010), (Chen et al. , 2013)(Grover et al. , 2018)
(Medha, Sharma and Sharma, 2021). PE_PGRS33, one of the first
extensively studied protein of this subfamily is also a potent
Caspase-mediated apoptotic inducer of infected macrophages, although the
time of its expression is unknown (Basu et al. , 2007). Because
Rv2615c has been documented to be upregulated during dormant Mtb
infection, it may also be a late stage expressing protein of Mtb which
induces host macrophage apoptosis (Urán Landaburu et al. , 2020).In-silico analysis predicted Rv2615c to be involved in the
activation of cysteine type endo-peptidase and it was observed that
recombinant Rv2615c protein activated initiator Caspase9 and executioner
Caspases 3 and 7 in THP-1 macrophages. Inhibitor studies with Caspase
inhibitor led to decrease in apoptosis of THP-1 macrophages which
implicates that apoptosis induced by our protein is Caspase mediated.
Our results corroborates with the study of Augenstreich et al.,where the Mtb-induced apoptosis was blocked by Caspase inhibitors
(Augenstreich et al. , 2017). During chronic TB infection,
pathogen employs battery of proteins that can facilitate its survival
and spread in host. Based on our observations, we propose that Rv2615c
is one protein that is expressed to induce macrophage apoptosis within
the granulomatous lesion and help Mtb in expanding its niche.
The Pathogen Associated Molecular Patterns (PAMPs) within Mtb interact
with host TLRs activate macrophages and modulate various cell processes
such as apoptosis, induction of oxidative stress, antigen presentation
etc,.(Bansal et al. , 2010; Grover et al. , 2018; Liuet al. , 2020). Our in-silico results revealed preferential
binding of Rv2615c to TLR4 as compared to TLR2. It was observed that
stimulation with Rv2615c led to an increase in expression of TLR4,
adapter molecule MyD88 and NF-ƙB in THP-1 macrophages. Although PE_PGRS
proteins such as PE_PGRS17 and PE_PGRS33 are shown to induce host cell
apoptosis via TLR2 dependent pathway (Basu et al. , 2007; Chenet al. , 2013); our observation of Rv2615c’s interaction with TLR4
gets support from the recent studies where PE/PPE proteins such as
PE_PGRS5, PE6 and PE9-PE10 protein complex were found to induce
apoptosis via TLR4-mediated signalling (Tiwari, Ramakrishnan and
Raghunand, 2015; Grover et al. , 2018; Sharma et al. , 2021;
Medha, Priyanka, et al. , 2022). Both TLR2 and TLR4 are key
regulators in inducing apoptosis of Mtb infected macrophages. Signalling
via TLR4 is crucial for maintaining balance between apoptosis and
necrosis in Mtb infected macrophages (Sánchez et al. , 2010). A
PE/PPE family protein- PE13 functions by engagement of TLR4 mediated
up-regulation of NF-ƙB and implicated in modulation of the macrophage
activation and apoptosis (Li et al. , 2016). The
Toll/Interleukin-1 Receptor Homology (TIR) Domains mediate the
intracellular TLR associated signal transduction. TIR domain-activated
signalling induces the recruitment of other adaptor molecules such as
MyD88, which in turn triggers the activation of downstream effectors
NF-ƙB (Horng et al. , 2002; Yamamoto et al. , 2002).
Activation of TLR4-MyD88-NF-ƙB cascade is also responsible for
production of proinflammatory cytokines TNF-α and IL-1β (Yamamotoet al. , 2002; Amaral and Andrade, 2017). In our study, levels of
soluble TNF-α were observed to be up-regulated in Rv2615c-stimulated
macrophages whereas no significant release of IL-1β was observed. As a
key pro-inflammatory cytokine, TNF- also plays a significant role in the
development and maintenance of lung granulomas in advanced stages of
infection (Dorhoi and Kaufmann, 2014). Activation of TLR4-MyD88-NF-ƙB
signalling and increased levels of pro-inflammatory cytokines are
prerequisite for activation of macrophages (Saraav et al. , 2017).
To keep the macrophages active and conducive for Mtb proliferation,
bacilli modulates an endogenous host mechanism for the resolution of
exacerbated inflammation (Próchnicki and Latz, 2017). IL-1β is a
mediator of inflammasome activation and plethora of studies have
reported its heterogenous regulation (Mishra et al. , 2010;
Hackett et al. , 2020). The transcriptional regulation of IL-1β is
not only limited to TLR4-NFκB axis but is also controlled by macrophage
immune-metabolic responses (Gleeson et al. , 2016). Recent
findings established that persistent Mtb infection of macrophages was
associated with suppression of glycolysis and reduced production of
IL-1β, despite upregulated TNF-α levels (Próchnicki and Latz, 2017).
Therefore, our observation of selective pro-inflammatory cytokine
secretion of only TNF-α in response to Rv2615c indicates its role as
modulator of macrophage functions to ensure bacilli survival and
multiplication.
Although few PE/PPE proteins such as PE_PGRS47 are known to
down-regulate antigen presenting HLA-DR molecules (Saini et al. ,
2016); we observed stimulation of macrophages with Rv2615c led to an
increase in HLA-DR expression. Inhibitor studies with anti-TLR4 blocking
antibody showed down-regulation in HLA-DR and TNF-α expression which
suggests that
the macrophage
activation in response to Rv2615c could be TLR4 mediated. Observations
of selective release of pro-inflammatory cytokine TNF-α, macrophage
activation and induction of macrophage apoptosis in response to Rv2615c
indicate that this protein has role in maintenance of the granuloma,
dissemination and persistence of Mtb (Fig 8).
Since, we observed that Rv2615c shares homology with APAF1-Apoptosome
which is a multimeric protein adaptor complex responsible for activation
of initiator Caspase9, sequence scan of Rv2615c was done which
discovered the presence of CARD-like domain similar to CARD of
eukaryotic APAF1 and Caspase9. Interaction between APAF1-CARD and
Caspase9-CARD is indispensable for Caspase9 activation (Hu et
al. , 2014). This shows that Rv2615c could bind Caspase9 and APAF1
through its eukaryote-like CARD domain and may enhance Caspase9
activation. We found two adjacently placed critical residues (Leu116 and
Ile117) in the CARD-like domain of Rv2615c which are conserved or highly
similar and aligned with the conserved residues of APAF1-CARD and
Caspase9-CARD domain. Molecular docking and simulation studies confirmed
that the mutation and deletion of these two residues decreased the
stability of Rv2615c protein and its affinity for CARD domain of APAF1
and Caspase9. Pathogenic microorganisms have rapidly acquired mechanisms
to escape the host surveillance system through co-evolution with host
cells. They harbour proteins which can imitate the eukaryotic proteins
in terms of sequence, homology, function or possessing a short linear
motif (SLiMs) or domains- a mechanism termed as ‘molecular mimicry’.
Many intracellular bacterial pathogens demonstrate remarkable example of
molecular mimicry such as Legionella, Coxiella, Mycobacterium,
Helicobacter, Chlamydia, and Bacillus which have been reported to
contain eukaryote-like proteins with prominent role in infection
(Chmiela and Gonciarz, 2017) (Sonia et al. , 2021). Therefore, our
results suggest that Rv2615c protein could be a molecular mimic of CARD
domain of APAF-1 and Caspase9 orchestrating apoptotic cell death.
Furthermore, the significance of this CARD-like domain in Rv2615c needs
to be validated further through site directed mutagenesis studies.
In conclusion, activation of TLR4-MyD88-NF-ƙB-TNF-α signalling cascade,
up-regulation of HLA-DR molecules and apoptotic cell death of
macrophages induced by Rv2615c implicate that this protein has potential
to evoke moderate host immune response to recruit immune cells at the
site of granuloma and facilitates pathogen persistence by cell to cell
spread via apoptosis (Fig 8). The strategy of molecular mimicry adopted
by Rv2615c protein of Mtb enhances our understanding of Mtb pathogenesis
and needs to be explored experimentally. Further investigations focused
on Rv2615c may help in development of new TB therapeutics. Especially
the use of inhibitors of apoptosis in dormant stages of TB infection
could be a novel approach to prevent the spread of Mtb to new sites
within host.