\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[T2A]{fontenc}
\usepackage[romanian,greek,ngerman,polish,english]{babel}



\usepackage{float}








\begin{document}

\title{Deciphering the role of PE\_PGRS45 (Rv2615c) protein of Mycobacterium
tuberculosis in host macrophage apoptosis: Possible effector for
persistence}



\author[1]{Monika Sharma}%
\author[1]{* Medha}%
\author[1]{* Priyanka}%
\author[1]{Sadhna Sharma}%
\affil[1]{University of Delhi Miranda House}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
\emph{Mycobacterium tuberculosis} has co-evolved with its host to evade
and modulate host cellular processes. Few PE/PPE proteins of
\emph{Mycobacterium} have been reported to modulate cell death pathways
and determine the infection outcome. This study investigates role of one
such uncharacterized protein PE\_PGRS45/Rv2615c in host cell death and
immuno-modulation. \emph{In-silico} analysis predicted similarity of
Rv2615c with APAF1-apoptosome and involvement in Cysteine-type
endo-peptidase activation during apoptosis. \emph{In-vitro} experiments
with THP-1 macrophages confirmed the Caspase-dependent apoptosis
inducing potential of Rv2615c. Several PE\_PGRS proteins have been
reported to be TLR-agonist. Docking showed preferential binding of
Rv2615c with TLR4 than TLR2. Up-regulation of
TLR4-HLA-DR-MyD88-NF-\selectlanguage{romanian}ƙB-TNF\selectlanguage{english}-\selectlanguage{greek}α \selectlanguage{english}in Rv2615c-stimulated THP-1 macrophages
was observed. To investigate the mechanism underpinning the apoptotic
function, a thorough sequence scan of Rv2615c revealed the presence of
eukaryotic CARD-like domain in it. \emph{In-silico} studies showed
binding affinity of Rv2615c's CARD-like domain with APAF1-CARD and
Caspase9-CARD, attributing Rv2615c's role in apoptosis. Since, Rv2615c
is reported to be upregulated in dormancy which suggest that it may be
one of the unexplored effectors employed by Mtb for its persistence via
silent cell-to-cell spread of infection by inducing macrophage
apoptosis. Additionally, Rv2615c's interaction with TLR4 implicates its
engagement in host-pathogen interaction.%
\end{abstract}%



\sloppy


\textbf{Deciphering the role of PE\_PGRS45 (Rv2615c) protein of
\emph{Mycobacterium tuberculosis} in host macrophage apoptosis: Possible
effector for persistence}

\emph{Medha} , Priyanka, Sadhna Sharma, Monika Sharma*

DSKC Bio Discovery Lab and

Department of Zoology, Miranda House, University of Delhi, New Delhi,
India- 110007

\textbf{*Corresponding author: Dr. Monika Sharma}

Address: Department of Zoology, Miranda House, University of Delhi,
Delhi, India -- 110007

Email- monika.sharma@mirandahouse.ac.in (*Correspondence and reprints'')

Other Contacts -- medha1.singh@mirandahouse.ac.in ,
priyanka.taank@mirandahouse.ac.in, sadhna.sharma@mirandahouse.ac.in

\textbf{Running Title:}

\textbf{PE\_PGRS45 protein induces host macrophage} \textbf{apoptosis}

\textbf{Summary}

\emph{Mycobacterium tuberculosis} has co-evolved with its host to evade
and modulate host cellular processes. Few PE/PPE proteins
of\emph{Mycobacterium} have been reported to modulate cell death
pathways and determine the infection outcome. This study investigates
role of one such uncharacterized protein PE\_PGRS45/Rv2615c in host cell
death and immuno-modulation. \emph{In-silico} analysis predicted
similarity of Rv2615c with APAF1-apoptosome and involvement in
Cysteine-type endo-peptidase activation during apoptosis.
\emph{In-vitro} experiments with THP-1 macrophages confirmed the
Caspase-dependent apoptosis inducing potential of Rv2615c. Several
PE\_PGRS proteins have been reported to be TLR-agonist. Docking showed
preferential binding of Rv2615c with TLR4 than TLR2. Up-regulation of
TLR4-HLA-DR-MyD88-NF-\selectlanguage{romanian}ƙB-TNF\selectlanguage{english}-\selectlanguage{greek}α \selectlanguage{english}in Rv2615c-stimulated THP-1 macrophages
was observed. To investigate the mechanism underpinning the apoptotic
function, a thorough sequence scan of Rv2615c revealed the presence of
eukaryotic CARD-like domain in it. \emph{In-silico} studies showed
binding affinity of Rv2615c's CARD-like domain with APAF1-CARD and
Caspase9-CARD, attributing Rv2615c's role in apoptosis. Since, Rv2615c
is reported to be upregulated in dormancy which suggest that it may be
one of the unexplored effectors employed by Mtb for its persistence via
silent cell-to-cell spread of infection by inducing macrophage
apoptosis. Additionally, Rv2615c's interaction with TLR4 implicates its
engagement in host-pathogen interaction.

\textbf{Keywords:}

\emph{Mycobacterium tuberculosis,} Rv2615c, Apoptosis, Toll-like
receptor 4, CARD-like domain

\textbf{Abbreviation:}

\emph{Mycobacterium tuberculosis} (Mtb), Apoptotic Peptidase Activating
Factor 1 (APAF1), Caspase Activation and Recruitment Domains (CARD),
Toll-like receptor (TLR), Tumor necrosis factor-\selectlanguage{greek}α \selectlanguage{english}(TNF-\selectlanguage{greek}α\selectlanguage{english})

\textbf{1. Introduction}

Tuberculosis (TB) caused by intracellular bacterium \emph{Mycobacterium
tuberculosis} (Mtb), remains a global health burden for decades now
(WHO, 2021). Many hypothetical proteins in different pathogenic bacteria
have been characterized and found to play role in pathogen persistence
and disease progression suggesting the importance of unravelling their
function (Desler \emph{et al.} , 2009; Kumar \emph{et al.} , 2014). The
Proline-Glutamate (PE)/ Proline-Proline-Glutamate (PPE) proteins encoded
within 7\% of Mtb genome are exclusive to the
pathogenic\emph{Mycobacterium} (Cole \emph{et al.} , 1998; Gey Van
Pittius \emph{et al.} , 2006; McGuire \emph{et al.} , 2012). The
PE\_PGRS proteins of this family have been studied for their immunogenic
potential and as modulators of host immune responses (Medha, Sharma and
Sharma, 2021). Many PE\_PGRS proteins are still uncharacterized and
therefore, investigating their role in Mtb pathogenesis and persistence
poses an interesting research question.

Various cell death modalities such as necrosis, pyroptosis, autophagy
and apoptosis have been reported in case of Mtb infection which are
immune defence mechanisms of host (Chai, Wang and Liu, 2020). It is
important to understand how these cell death modalities are exploited by
the pathogen for its benefit. There are approximately 12 PE\_PGRS
proteins observed to be expressed a late stage of infection (chronic
stage) i.e., at 90 days in infected guinea pig lungs (Kruh \emph{et al.}
, 2010). A recent study investigated the role of PE\_PGRS5 and revealed
the ability of this late stage expressing protein to induce apoptosis
(Grover \emph{et al.} , 2018). Reports on other PE proteins such as
PE\_PGRS33 and PE6 have also shown the apoptosis inducing potential of
these Mtb proteins; though the time of their expression is yet to be
investigated (Basu \emph{et al.} , 2007) (Sharma \emph{et al.} , 2021).
The apoptogenic function played by these PE family proteins has been
discussed to be beneficial for granuloma maintenance during later stages
and infection persistence in all the reported studies. On the basis of
these findings, we deduced that these PE/PPE proteins might be expressed
at later stages of infection within granuloma of infected host cell.
Apoptosis induction by these proteins may enable Mtb to expand its niche
without an exaggerated inflammatory response to promote its persistence
(Medha, Sharma and Sharma, 2021). It has been reported that Mtb escapes
the phagolysosome and translocate to cytosol where it triggers host cell
apoptosis. These apoptotic cells can be phagocytosed by na\selectlanguage{ngerman}ïve
infiltrated macrophages which accumulate to develop primary
granulomatous lesion. The bacilli infected macrophages then migrate to
different tissues and culminates in secondary granuloma development.
Findings of studies on Mtb infected mouse and zebrafish lung granuloma
suggest that apoptosis and bacilli survival could be positively
correlated; though needs further research (Davis \emph{et al.} , 2002),
(Volkman \emph{et al.} , 2004), (Saunders and Britton, 2007), (Seimon
\emph{et al.} , 2010), (Ramakrishnan, 2012). Mtb-infected lung
granulomas have reported to contain apoptotic macrophages. (Fayyazi
\emph{et al.} , 2000; Pan \emph{et al.} , 2005). While some studies
showed apoptosis as a host mediated defence mechanism against the
pathogens; recent studies also showed that several Mtb proteins could
either induce or inhibit apoptosis. Therefore, if Mtb has both
apoptogenic as well as apoptosis inhibiting proteins, it is important to
understand when these proteins express themselves during the course of
infection. When the apoptosis induction potential of these proteins was
correlated with their time of expression, it was observed that these
proteins might be expressed at late stages of infection at the site of
lung granuloma and aid in pathogen dissemination via apoptosis (Grover
\emph{et al.} , 2018) (Sharma \emph{et al.} , 2021). Additionally, one
study also revealed that to prevent excessive host inflammatory
responses and ensure long-term intracellular survival, Mtb regulates
intracellular bacterial burden by inducing apoptosis (Chai\emph{et al.}
, 2019). The PE PGRS proteins and the ESX-V secretory system have
co-evolved together and it has been reported that members of ESX-I and
ESX-V secretory system induce host cell apoptosis that aids in pathogen
persistence (Gey Van Pittius \emph{et al.} , 2006), (Abdallah\emph{et
al.} , 2007), (Abdallah \emph{et al.} , 2011), (Aguilo \emph{et al.} ,
2013). PE\_PGRS33 (Rv1818c) has been reported to trigger
mitochondria-mediated intrinsic apoptosis via TLR2-TNF-\selectlanguage{greek}α \selectlanguage{english}signalling and
facilitates survival of \emph{Mycobacterium smegmatis} expressing this
protein within host macrophages (Basu \emph{et al.} , 2007)
(Cadieux\emph{et al.} , 2011). Another protein Rv0297 {[}PE\_PGRS5{]}
expressed at 90 days of infection is reported to induce endoplasmic
reticulum stress mediated apoptosis via TLR4-TNF-\selectlanguage{greek}α \selectlanguage{english}signalling pathway
and favours Mtb dissemination (Grover \emph{et al.} , 2018).

In TDR Target database, Rv2615c is reported to be an essential protein
in Mtb growth and is up-regulated during latent/dormant TB infection
(Hasan \emph{et al.} , 2006; Murphy and Brown, 2007). Biochemical
characterization of Rv2615c revealed it to be an oxidoreductase which
could be targeted by drugs with anti-TB property using drug repurposing
approach (Medha, Joshi, \emph{et al.} , 2022). Given the essentiality of
this protein in Mtb pathogenesis, we designed this study to investigate
the molecular characterization of Rv2615c (PE\_PGRS45) protein in host
cell death and in modulation of host immune response.
Preliminary\emph{in-silico} profiling predicted Rv2615c to be an
antigenic protein (Vaxijen score of 1.73). Homology and function-based
characterization revealed high similarity of Rv2615c with
APAF1-apoptosome and involvement in Cysteine-type endo-peptidase
activation process during apoptotic pathway. To validate these
\emph{in-silico} results,\emph{in-vitro} experiments with recombinant
Rv2615c and THP-1 macrophages were conducted. Our results showed that
Rv2615c protein of Mtb induced host macrophage apoptosis and Caspase
activation indicating that this protein might be employed by the
pathogen during late stages of infection at the site of granuloma where
it may facilitate disease persistence through apoptosis induction.
During late stages of infection, the granuloma is maintained by
activated macrophages. To understand the role of Rv2615c in macrophage
activation and granuloma maintenance, we probed the expression of TLRs,
HLA-DR, NF-\selectlanguage{romanian}ƙB and pro-inflammatory cytokines. Experiments with
Rv2615c-stimulated THP-1 macrophages showed up-regulated levels of
TLR4-MyD88, antigen presenting HLA-DR molecules, NF-ƙB and inflammatory
TNF-\selectlanguage{greek}α \selectlanguage{english}cytokine. To get further insight into the apoptotic function of
Rv2615c, a thorough sequence scan was done which highlighted the
presence of eukaryotic CARD (Caspase Activation and Recruitment
Domains)-like domain in Rv2615c similar to CARD domains in Apoptotic
Peptidase Activating Factor 1 (APAF1) and Caspase9. Docking and
Molecular Dynamic (MD) simulation studies substantiated the high binding
stability of Rv2615c complex with CARD-APAF1 and CARD-Caspase9. Two
critical residues (Leu116 and Ile117) in the CARD-like domain of Rv2615c
was found to be either conserved or highly similar and aligned with the
conserved residues of APAF1-CARD and Caspase9-CARD domain. Docking and
MD simulation studies confirmed that the mutation and deletion of these
two residues decreased the stability of Rv2615c protein and its affinity
to bind CARD domain of APAF1 and Caspase9. Validation through
site-directed mutagenesis experiments will give further insight of
molecular mimicry adopted by Rv2615c protein of Mtb.

\textbf{2. RESULTS:}

\textbf{2.1 \emph{In-silico} characterization of Rv2615c}

\textbf{2.1.1 Rv2615c is an antigenic protein predicted to be involved
in host cell death pathways}

Vaxijen 2.0 server predicted Rv2615c to be a probable antigen with high
score of 1.73. 5 models were generated for secondary structure
prediction using I-TASSER server which were further subjected for energy
minimization in Chiron server. Model 1 was selected based on the C score
of -3.06 in I-TASSER. Ramachandran plot for Model 1 showed 97.5\%
residues were present in favoured and generously allowed region while
only 2.5\% residues were present in disallowed region. Errat score was
86.67 for Model 1 and Verify3D score was 81.13.

I-TASSER predictions related to functional role strongly pointed the
involvement of Rv2615c in host cell death pathways. Three top predicted
structural analogs and Gene ontology templates for Rv2615c were those of
Apoptosome complex (PDB ID: 5JUL, 3IZ8, 1VT4) and Apoptosome-ProCaspase9
CARD complex (PDB ID: 3IYT, 3IZA). Consensus GO terms for biological
process and molecular functions were -- GO: 0006917 (apoptotic process)
and GO: 006919, GO: 0043028, GO: 0016505 (apoptotic process involved in
activation of cysteine type endo-peptidase activity).

\textbf{2.2 In vitro assessment of Rv2615c for inducing apoptosis}

\textbf{3.2.1 Cloning, expression and purification of recombinant
Rv2615c protein}

His-tagged recombinant Rv2615c protein was successful with pGEMT easy
vector in DH5\selectlanguage{greek}α \selectlanguage{english}followed by pET28a{[}+{]} expression vector in
BL21\emph{DE3} and the clones were also confirmed by sequencing. Rv2615c
was successfully expressed and purified from insoluble fraction of
transformed BL21 \emph{DE3} culture by affinity chromatography. The
protein had a molecular weight of 39.3 kDa observed in SDS-PAGE and
anti-His antibody on western blotting (Fig S1). One liter of culture
yielded approximately 2 mg/mL of protein. The purified protein was
passed through polymyxin B-agarose beads to remove bacterial endotoxin
and Limulus Amoebocyte Lysate assay (Pierce, USA) was performed in
collected fractions which confirmed no endotoxin contamination.

\textbf{2.2.2 (a) Rv2615c protein induces cell death in THP-1
macrophages}

To determine the cell viability, MTT and CellTiter Blue assay were
performed to probe any change in viability of THP-1 cells in response to
Rv2615c stimulation. Three concentrations of Rv2615c (5\selectlanguage{greek}μ\selectlanguage{english}g/ml, 10\selectlanguage{greek}μ\selectlanguage{english}g/ml
and 15\selectlanguage{greek}μ/\selectlanguage{english}ml) were included for checking the dose dependent viability.
Cell viability was prominently affected in Rv2615c stimulated cells in a
dose dependent manner (Fig S2a). Based on these observations, we
selected 10\selectlanguage{greek}μ\selectlanguage{english}g/ml as stimulation concentration for Rv2615c protein in our
experiments. Unless otherwise stated, LPS and staurosporine served as
controls in each experiment. Viability of Rv2615c-stimulated and
control-stimulated THP-1 macrophages was significantly decreased at
all-time points when compared to unstimulated cells (Fig S2b and S2c).
In comparison to the unstimulated cells, Rv2615c-stimulation caused
approximately 50 to 60\% cell death till 48 hours that was comparable to
cell death induced by LPS stimulation (Fig S2b and S2c). These
observations indicate that Rv2615c protein affects viability and induce
cell death in THP-1 macrophages.

\textbf{2.2.2. (b) Rv2615c protein did not induce LDH, a marker for the
necrotic cell death}

LDH release usually determines the cellular cytotoxicity in response to
external stimulus; moreover, it also defines if the cells are undergoing
necrotic cell death. 1\% Triton-X, lysis buffer and a positive control
was included as controls in study. Rv2615c-stimulation led release of
LDH was observed to be at basal level, comparable to the unstimulated
cells (Fig S3). Levels of LDH were significantly high in each of the
positive controls included in the study at 16h, 24h and 48h. These
observations indicate that the cell death induced by Rv2615c protein is
not necrosis.

\textbf{2.2.2. (c) Rv2615c-stimulation leads to flipping of
phosphatidylserine} \textbf{on outer membrane and apoptosis of THP-1
macrophages}

Phosphatidylserine {[}PS{]} is flipped from the inner to the outer
leaflet of the plasma membrane as a result of biochemical alterations
associated with the initiation of apoptotic pathway. Rv2615c-stimulation
showed an increase in early apoptosis indicated by annexinV positive
cells (Fig 1b, bottom right quadrant) plus late apoptosis indicated by
annexinV/PI dual-positive cell population (Fig 1b, top right quadrant)
in a time-dependent manner. At 16h, there was no significant increase in
percentage of both the cell populations (early apoptotic annexinV
positive; late apoptotic annexinV/PI positive cells) on stimulation with
Rv2615c protein as compared to unstimulated control (Fig 1a). At 24h and
48h, Rv2615c-stimulation showed gradual and significant increase in
percentage of dual positive cell populations (Mean\selectlanguage{ngerman}±SEM in range of 37 to
49\%) as compared to unstimulated cells (Mean±SEM in range of 27 to
35\%) while the cell death was comparable to LPS stimulation (Mean±SEM
range of 33 to 47\%) at 48h. Since Staurosporine is a well-known cell
death inducer; we observed maximum cell death (Mean±SEM value of 62\%)
till 48h (Fig 1a).

\textbf{2.2.2 (d) Rv2615c stimulation leads to DNA fragmentation in
THP-1 macrophages}

DNA fragmentation is another distinct feature of cells undergoing
apoptosis which was studied by TUNEL assay. At 16h, 24h and 48h, the
percentage of TUNEL positive cells in response to Rv2615c-stimulation
(Mean±SEM range of 22 \% to 35\%) were significant than the unstimulated
cells (Mean±SEM range of 3 \% to 25\%) and comparable to positive
control (LPS/Staurosporine stimulated) cells (Fig 1c and 1d). These
results depicted that the putative Rv2615c protein induces apoptotic
cell death in THP-1 macrophages.

\textbf{2.2.2 (e) Rv2615c protein trigger the activation of Caspase9 in
THP-1 macrophages}

Initiator Caspase 9 activation is necessary for the development of the
apoptosome complex and the subsequent activation of executioner Caspases
3 and 7, which results in apoptotic cell death. To check the engagement
of initiator Caspase9 with respect to apoptosis induction by selected
recombinant protein, western blot assessment of the total cell lysate
from THP-1 macrophages post-stimulation with Rv2615c protein was done to
determine if initiator Caspase9 was activated for the induction of
apoptosis in response to Rv2615c. Levels of activated Caspase9 was
quantified using ImageJ and revealed to be increased in response to
Rv2615c stimulation relative to unstimulated cells and LPS stimulation
(Fig 2a and 2b).

\textbf{2.2.2. (f) Rv2615c-induced apoptotic cell death involves
activation of Caspase 3 and 7}

Classical intrinsic apoptosis includes the activation of Caspase 3 and
Caspase 7 as the ultimate executioner caspases following
APAF1-Caspase9-apoptosome formation. As a result, we investigated the
activation of Caspase 3 and Caspase 7 during Rv2615c-mediated cell
death. Rv2615c protein leads to activation of Caspase 3 and 7 at all the
time points shown by significant increase in the percentage of Caspase 3
and 7 positive cells (Fig 2c). Rv2615c-stimulation led to a significant
1.5-fold increase in Caspase 3 and 7 activations than unstimulated
cells. Staurosporine and LPS included as positive controls in the study
led to approximately 1.6-fold increase in percentage of Caspase 3 and 7
positive cells than the unstimulated cells at 48h. The percentage of
Caspase 3 and 7 positive cells was decreased by approximately 1.5 folds
when pre-treated with pan caspase inhibitor Z-VAD-fmk in cells
stimulated with Rv2615c protein and controls at 24h (Fig 2d).
Additionally, the percentage of AnnexinV/AnnexinV-PI dual positive cells
stimulated with Rv2615c protein decreased by 1.3-fold when pre-treated
with pan Caspase inhibitor Z-VAD-fmk at 24h (Fig 2e). These results
depicted that the selected Rv2615c protein induces Caspase 3 and 7
dependent apoptotic cell death in THP-1 macrophages.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1. Flow cytometry analysis of AnnexinV and TUNEL positive
cells expression in THP-1 macrophages following protein stimulation at
different time points}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

THP-1 macrophages were left unstimulated or stimulated with
control/Rv2615c protein for 16h, 24h and 48h. Annexin V-FITC and PI/
TUNEL assay was performed in Flow Cytometer. 1(a) Graphs were plotted
for different time points with a) \% of annexinV + annexinV/PI dual
positive cells and c) \% of TUNEL positive cells on y axis and
control/Rv2615c protein on x axis. Statistical analysis was done with
Student's t test and results are Mean \selectlanguage{ngerman}± SEM of three independent
experiments where * represents comparison with unstimulated cells and
*P\textless{}0.05, **P\textless{}0.01, ***P\textless{}0.001,
****P\textless{}0.0001. 1(b) Dot plot showing gating strategy for cells
stained with AnnexinV-FITC and PI staining which were analysed by Flow
Cytometer. Plots were generated as annexinV fluorescence (FL1) on x axis
and PI fluorescence (FL3) on y axis. Total 10000 events were acquired
for each sample and threshold was set at 80000. Cells in lower right
quadrant coloured in green depicts percentage of annexinV single
positive (early apoptotic cells); cells in upper right quadrant colours
in red depicts percentage of annexinV /PI dual positive (late apoptotic
cells); cells in upper left quadrant depicts percentage of PI single
positive (necrotic cells). 1d) Dot plot showing gating strategy for
percentage of TUNEL positive cells following staining with FITC-dUTP and
PI to label the DNA breaks. Plots were generated as FITC-dUTP (FL1) on x
axis and PI fluorescence (FL2) on y axis. Total 10000 events were
acquired for each sample and threshold was set at 80000. Cells in gate
P3 shows the total percentage of TUNEL positive cells or cells with DNA
breaks.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 2. Western blot and graphs depicting activation of
Caspase9 and Caspase 3 and 7 respectively following protein stimulation
of THP-1 macrophages}

Activated Caspase9, 3 and 7 estimation experiments were carried out on
THP-1 macrophages that had either been left unstimulated or stimulated
with control/Rv2615c protein. (a) Western blot image showing bands for
activated Caspase9. Proteins were transferred onto the PVDF membrane
after cell lysate was prepared and separated on SDS-PAGE post 24 hours
stimulation. The polyclonal anti-Caspase9 antibody was used to measure
the amounts of activated Caspase9, and an internal loading control,
GAPDH, was employed at a dilution of 1:1000. b) ImageJ software was used
to quantify the size of each western blot band. Results are shown as the
meanSEM values of three separate experiments and were examined by
showing the ratio of each band area with respect to unstimulated cells.
Student's t test was performed where * depicts the comparison with
unstimulated cells for Caspase9 and GAPDH separately.
(*P\textless{}0.05, **P\textless{}0.01). c) Unstimulated/control/Rv2615c
protein stimulated THP-1 macrophages were incubated for different time
points (16h, 24h and 48h) and activation of Caspase 3 and 7 was
estimated which is a characteristic of intrinsic apoptosis.

Each western blot band was measured using ImageJ software. The ratio of
each band area with respect to unstimulated cells was investigated, and
the results are presented as them Mean\selectlanguage{ngerman}±SEM values of three distinct
experiments. The comparison of Caspase9 and GAPDH individually with
unstimulated cells was shown using the Student's t test, where *.
(*P\textless{}0.05, **P\textless{}0.01). c) THP-1 macrophages that were
left unstimulated or stimulated with control/Rv2615c protein were
incubated for different time points (16 hours, 24 hours, and 48 hours),
and the activation of caspase 3 and 7 was estimated. THP-1 cells were
also blocked with z-VAD-fmk Caspase inhibitor prior to stimulation and
evaluated for d) levels of Caspases 3 and 7 and e) expression of
AnnexinV/AnnexinV-PI dual positive cell population at 24h. Graphs were
plotted for different time points with \% of positive cells on y axis
and control/Rv2615c on x axis. Statistical analysis was done with
Student's t test and results are Mean ± SEM of three independent
experiments where * represents comparison with unstimulated cells and
*P\textless{}0.05, **P\textless{}0.01, ***P\textless{}0.001.

\textbf{2.3. Rv2615c protein enhances the expression of TLR4, HLA-DR and
downstream effector molecules to modulate the host immune response}

\textbf{2.3. (a) Docking studies revealed preferential binding of
Rv2615c protein with TLR4 than TLR2}

TLRs in particular are essential for the recognition of the pathogenic
Mtb proteins. The interaction of PE\_PGRS proteins with TLRs has been
shown in a number of studies (Abdallah \emph{et al.} , 2007;
Bansal\emph{et al.} , 2010; Ramakrishnan, 2012; Grover \emph{et al.} ,
2018; Liu\emph{et al.} , 2020). I-TASSER homology modelled Rv2615c
protein docking experiments with TLR2 and TLR4 demonstrated Rv2615c's
strong affinity for TLR4 binding (Fig 3a). HADDOCK predicted a score of
-141.3±13.8 for Rv2615c-TLR4 docked complex while -109.5±5.8 for
Rv2615c-TLR2 docked complex. The TLR2-Rv2615c complex had a total of 46
structures, which were grouped into 9 clusters, whereas the TLR4-Rv2615c
complex had 67 structures, which were grouped into 9 clusters. TLR2 and
Rv2615c complexes were predicted to have the best Z scores, with -2 for
TLR2 and -1.7 for Rv2615c.

\textbf{2.3. (b) Rv2615c leads to up-regulation of TLR4 and HLA-DR
expression}

Expression profile of TLR2 and TLR4 was estimated by flow cytometry in
response to Rv2615c-stimulation in THP-1 macrophages. Both Rv2615c and
LPS-a TLR4 agonist resulted in significant 1.27-fold increase in TLR4
percent positive cells at 24h than unstimulated control. There was no
up-regulation of TLR4 expression observed for cells stimulated with cell
wall fraction of Mtb. When THP-1 cells were blocked with Anti-TLR4
antibody prior to stimulation with Rv2615c or controls; the percentage
of TLR4 positive cells significantly decreased by 1.4-fold implicating
that the function of Rv2615c is TLR4 mediated. Further, there was no
up-regulation in TLR2 expression in response to both LPS and
Rv2615c-stimulation at all-time points. Cell wall fraction which was
included as positive control for TLR2 expression showed a significant
1.2-fold increase in TLR2 expression at all-time points (Fig 3b).

For determining the effect of Rv2615c-stimulation on TLR-associated
antigen presenting molecule HLA-DR, we performed flow cytometry analysis
to study the expression of HLA-DR. Rv2615c showed significant increase
in percentage of HLA-DR positive cells at 24h and 48h. At 24h, there was
significant 2-fold increase in percentage of HLA DR positive cells
induced by Rv2615c in comparison to unstimulated cells (Fig 3b).

In comparison to unstimulated cells, LPS and CWF significantly
(\textasciitilde{}1.5-fold ) raised the levels of HLA-DR positive cells.
We examined the HLA-DR levels in cells inhibited with anti-TLR4 antibody
prior to stimulation to determine the TLR4-dependent up-regulation of
Rv2615c-induced HLA-DR expression. In comparison to unstimulated cells,
we observed a 2.8-fold decrease in HLA-DR expression in control (LPS and
CWF) and Rv2615c-stimulated THP-1 macrophages (Fig 3c and 3d).

\selectlanguage{romanian}\textbf{2.3. (c) Rv2615c leads to up-regulation of downstream effector
molecule- MyD88 and NF-ƙB}

RT-PCR evaluation of cDNA from isolated mRNA of unstimulated and Rv2615c
protein/LPS stimulated THP-1 macrophages revealed an increase in fold
change in MyD88 gene expression after 24h of stimulation and in NF-ƙB
gene expression after 24 hours and 48 hours of stimulation. When
compared to unstimulated cells, Rv2615c protein stimulation led to
approximately \textasciitilde{}2.6-fold increase and LPS stimulation led
to approximately \textasciitilde{}3-fold increase in the expression of
MyD88 gene respectively (Fig 3d). Expression of NF\selectlanguage{greek}κ\selectlanguage{english}B was observed to be
approximately \textasciitilde{}4-fold increase in response to Rv2615c
and approximately \textasciitilde{}2 to 4-fold increase in response to
LPS after 24 and 48hrs of stimulation as compared to unstimulated cells
(Fig 3e).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\selectlanguage{romanian}\textbf{Figure 3. Expression profile of TLRs, HLA-DR, MyD88 and NF-ƙB in
Rv2615c-stimulated THP-1 macrophages}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}

a) Predicted docked complex of Rv2615c (yellow) bound to TLR2 (green)
and TLR4 (blue) using HADDOCK server. b) THP-1 macrophages were
stimulated with control/Rv2615c protein for 24h and 48h. Expression
profile of TLR2, TLR4 and HLA-DR was evaluated at 24h and 48h of
stimulation. The percentage of TLR/HLA-DR positive cells was plotted on
the y axis and control/Rv2615c protein on the x axis of graphs for
various time points. The findings of the statistical study, which used
the Student's t test, are shown as Mean\selectlanguage{ngerman}±SEM for three separate
experiments where * represents comparison with unstimulated cells and
*P\textless{}0.05, **P\textless{}0.01, ***P\textless{}0.001. c) THP-1
cells were blocked with Anti-TLR4 antibody prior to stimulation and
evaluated for levels of TLR4 and HLA-DR at 24h. Isolated mRNA from
unstimulated or Rv2615c/LPS stimulated macrophages were synthesized into
cDNA and RT-PCR was performed to estimate the levels of d) MyD88 and e)
NF-\selectlanguage{greek}κ\selectlanguage{english}B gene expression using 2\textsuperscript{-[?][?]} CT method. MyD88 and
NF-\selectlanguage{greek}κ\selectlanguage{english}B gene expression in unstimulated cells was considered 1 and with
respect to unstimulated cells fold change in MyD88 and NF-\selectlanguage{greek}κ\selectlanguage{english}B gene
expression in LPS and Rv2615c was plotted. Graphs were plotted for
different time points with fold change in MyD88/NF-\selectlanguage{greek}κ\selectlanguage{english}B expression on y
axis and LPS/Rv2615c protein on x axis. Results are expressed as Mean \selectlanguage{ngerman}±
SEM of three independent experiments.

\selectlanguage{greek}\textbf{2.3 (d) Rv2615c leads to up-regulation of pro-inflammatory
cytokine TNF-α but down-regulation of IL-1β}

\selectlanguage{english}A plethora of pro-inflammatory cytokines are secreted when an immune
response is triggered. Additionally, pro-inflammatory immune response
activation is also involved in various cell death mechanisms that take
place at the host-pathogen interface. TLR4-NF-\selectlanguage{romanian}ƙB signaling is a widely
studied cascade known to activate key pro-inflammatory cytokines such as
TNF-\selectlanguage{greek}α. \selectlanguage{english}We found significantly up-regulated levels of TNF-\selectlanguage{greek}α \selectlanguage{english}at all the
time points in response to LPS and Rv2615c protein stimulated THP-1
macrophages as compared to unstimulated cells (Fig 4a). Both Rv2615c and
LPS showed approximately \textasciitilde{} 3 to 4-fold increase in TNF-\selectlanguage{greek}α
\selectlanguage{english}expression till 48h of stimulation. Another pro-inflammatory cytokine,
IL-1\selectlanguage{greek}β, \selectlanguage{english}is also reported to be released as a result of TLR4-NF-\selectlanguage{romanian}ƙB
signalling. IL-1 \selectlanguage{greek}β \selectlanguage{english}is a modulator of extrinsic apoptosis via Caspase 8
activation and inflammasome activation. Interestingly, IL-1\selectlanguage{greek}β \selectlanguage{english}levels in
Rv2615c-stimulated macrophages were either equivalent to or lower than
in unstimulated cells at all time periods. (Fig 4b). IL-1\selectlanguage{greek}β \selectlanguage{english}expression in
response to LPS stimulation was found to be significantly 2 to 3-fold
up-regulated than unstimulated cells till 24h.

To further ensure that the release of TNF-\selectlanguage{greek}α \selectlanguage{english}in response to Rv2615c
protein stimulation was mediated by TLR4; we evaluated the expression of
TNF-\selectlanguage{greek}α \selectlanguage{english}after blocking the expression of TLR4. There was a 2-fold decrease
in TNF-\selectlanguage{greek}α \selectlanguage{english}release in response to Rv2615c and control stimulated THP-1
macrophages when blocked with Anti-TLR4 antibody prior to stimulation
(Fig 4c).

\selectlanguage{greek}\textbf{Figure 4. Cytokine ELISA to determine the levels of
pro-inflammatory cytokines- TNF-α and IL-1β}

\selectlanguage{english}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/imagf0/imagf0}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image10/image10}

\end{center}
\end{figure}

THP-1 macrophages were unstimulated or stimulated with LPS/Rv2615c
protein and for 16h, 24h and 48h were analysed for release of a) TNF-\selectlanguage{greek}α
\selectlanguage{english}and b) IL-1\selectlanguage{greek}β \selectlanguage{english}in cell culture supernatants c) TNF-\selectlanguage{greek}α \selectlanguage{english}release was also
estimated in supernatant blocked with Anti-TLR4 antibody prior to
stimulation. ELISA was done according to manufacturer's instructions.
Graphs were plotted for different time points with cytokine release in
pg/ml on y axis, controls, and the protein Rv2615c on the x axis. The
findings of the statistical analysis, which used the Student's t test,
are shown as Mean \selectlanguage{ngerman}± SEM for three separate experiments where *
represents comparison with unstimulated cells and *P\textless{}0.05,
**P\textless{}0.01, ***P\textless{}0.001.

\textbf{2.4 Rv2615c has CARD-like domain similar to CARD domains in
APAF1 and Caspase9}

\emph{In-silico} clues pointed the similarity of Rv2615c protein with
apoptosome complex and prediction of molecular function in activation of
cysteine-type endopeptidase/Caspases during apoptosis. In-vitro studies
substantiated the role of Rv2615c protein in inducing Caspase-mediated
host macrophage apoptosis. APAF1-Apoptosome complex initiates the
activation of Caspase9 and active Caspase9 is recruited via its CARD
domain onto the CARD domain of APAF1. Our findings of Rv2615c led us to
hypothesize- whether Rv2615c possess a CARD-like domain similar to CARD
domains present in APAF1 and Caspase9 that facilitates interaction and
activation of cysteine type endo-peptidase activity of Caspase9.
Multiple sequence alignment of Rv2615c protein with CARD domains of
APAF1 and Caspase9 revealed many conserved and highly similar amino acid
residues (Fig 5b and 5c). CARD domains of both APAF1 and Caspase9 are
known to contain some highly conserved mostly hydrophobic and crucial
residues through which protein-protein interactions occur (Fig 5a). We
observed 14 crucial residues of APAF1-CARD and 9 crucial residues of
Caspase9-CARD either totally conserved or highly similar in Rv2615c
protein positioned from 60 to 230 amino acids which we denoted as
CARD-like domain (Fig 5b, 5c and S4a). Interestingly, the MINNOU server
predicted that the CARD-like domain in Rv2615c is mostly hydrophobic in
nature similar to the CARD domains of APAF1 and Caspase9 (Fig S4b). The
secondary structure characterization reveals that the CARD-like domain
in Rv2615c contains highly disordered random coiled region and fewer
alpha-helixes (Fig S4b). Structural superimposition showed helical
region of CARD-like domain in Rv2615c mostly aligned with CARD-APAF1 and
CARD-Caspase9 (Fig S5). Coiled regions are highly flexible and unstable
which facilitate dynamic protein-protein interactions.

\textbf{Figure 5. Multiple sequence alignment of CARD domains}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image11/image11}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image14/image14}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image15/image15}

\end{center}
\end{figure}

Multiple sequence alignment of 8(a) CARD-APAF1 and CARD-Caspase9 8(b)
Rv2615c and CARD-Caspase9 8(c) Rv2615c and CARD-APAF1. All the crucial
conserved or similar residues are denoted by asterisk (*). The common
Leucine116 and Isoleucine117 residues in Rv2615c protein which were
mutated to Alanine are denoted by red arrow.

\textbf{2.4.1 CARD-like domain of Rv2615c has strong binding affinity
for CARD domains of APAF1 and Caspase9}

Docking studies with whole Rv2615c protein revealed that its CARD-like
domain could bind strongly with CARD-APAF1 (Hex energy score: -655.54
Kcal/mol) and with CARD-Caspase9 (Hex energy score: -694.29 Kcal/mol)
(Fig 6). Rv2615c formed 25 bonds (3 electrostatic, 7 hydrogen and 15
hydrophobic bonds) with CARD-APAF1 while 24 bonds (6 electrostatic, 6
hydrogen and 12 hydrophobic bonds) with CARD-Caspase9 through its
CARD-like domain (Fig S6). These Hex energy scores were much higher than
the Hex scores of positive controls included in the study i.e., complex
of CARD-APAF1 with CARD-Caspase9 (PDB ID 3YGS- Hex score: -463.25
Kcal/mol) (Fig 6). However, Hex score for Rv2615c docked with APAF1-ADP
was very less (-110.48 Kcal/mol) compared to the positive control. This
could possibly be because of low affinity of Rv2615c for inactive form
of APAF1 protein that is not bound to Apoptosome complex.

We observed two crucial adjacent hydrophobic residues i.e., Leucine-116
and Isoleucine-117 in CARD-like domain of Rv2615c which were common,
totally conserved or similar and aligned with conserved residues of
CARD-APAF1 (Methionine-29 and Isoleucine-30) and CARD-Caspase9
(Leucine-58 and Isoleucine-59). These residues (Leucine-116 and
Isoleucine-117) within Rv2615c were also involved in bond formation with
CARD-APAF1 and CARD-Caspase9 (Fig S6). Mutant of Rv2615c in which
Leucine-116 and Isoleucine-117 within the CARD-like domain were mutated
to Alanine was less stable. The binding affinity of this mutant Rv2615c
with CARD-APAF1 and with CARD-Caspase9 was also decreased. Mutant
Rv2615c protein bound to APAF1 CARD with energy score of 594.43 Kcal/mol
and with Caspase9 CARD with energy score of -646.87 Kcal/mol.

\textbf{2.1.4 Validation of docking results through MD simulation}

The RMSD and RMSF plots obtained after the extensive (100 ns) MD
simulation confirmed the structural stability of the protein-protein
complexes (Fig 7). The RMSD value of backbone for all the complexes
increased for initial 10 to 20ns following which it became stable with
an average value of 1nm to 1.2nm for Rv2615c-APAF1 CARD and
Rv2615c-Caspase9 CARD till 100ns. The RMSD value for mutant
Rv2615c-APAF1 CARD was comparable to that of Rv2615c- APAF1 CARD while
the RMSD value of mutant Rv2615c-Caspase9 CARD was higher in the range
of 1.4nm. The average RMSD value of control structure was 0.70nm. RMSF
analysis revealed that the docked complexes of Rv2615c protein with
APAF1-CARD and Caspase9-CARD and mutant Rv2615c with Caspase9-CARD
displayed average RMSF value of 0.80nm. Mutant Rv2615c with APAF1-CARD
showed high fluctuations with average RMSF value in range of 1.20nm. The
average RMSF value of control structure was 0.30nm. Overall, MD
simulation results revealed that the docked complexes of Rv2615c showed
a comparatively higher RMSD and RMSF value than control structure
(APAF1-CARD with Caspase9-CARD) but all values are in acceptable range.
One reason for this higher RMSD and RMSF values of Rv2615c docked
structures could be large size of Rv2615c protein (461 residues) in
comparison to the APAF1-CARD (95 residues) and Caspase9-CARD structures
(97 residues).

\textbf{Figure 6. Molecular docking of Rv2615c protein with CARD domains
of APAF1 and Caspase9}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image16/image16}

\end{center}
\end{figure}

Rv2615c protein (pink color) showed high affinity for CARD domain of
both APAF1 (orange color) and Caspase9 (blue color) and establishes
interaction via its CARD-like domain. The binding affinity of Rv2615c
for CARD domain of both APAF1 and Caspase9 was much higher than the
positive control included in the study involving interaction of APAF1
CARD and Caspase9 CARD. However, Rv2615c showed low binding affinity for
inactive form of APAF1 ADP implicating its preference for active APAF1
Apoptosome complex.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image18/image18}

\end{center}
\end{figure}

\textbf{Figure 7. MD Simulation of docked structures}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image19/image19}

\end{center}
\end{figure}

MD simulations for 100 ns was performed for Rv2615c protein docked with
APAF1 CARD, Caspase9 CARD and mutant Rv2615c protein docked with APAF1
CARD, Caspase9 CARD. Docked complex of APAF1 CARD and Caspase9 CARD was
included as positive (a) Root mean square deviation for complex backbone
with x axis denoting time in ns and y axis denoting RMSD in nm (b) and
(c) Root mean square fluctuation of backbone for control (APAF1 CARD and
Caspase9 CARD) and other complexes with x axis denoting atom index of
complex and y axis denoting RMSF in nm.

\textbf{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,
\emph{in-vitro}experiments 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 \emph{et al.} , 2010), (Chen \emph{et
al.} , 2013)(Grover \emph{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 \emph{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
(Ur\selectlanguage{ngerman}án~Landaburu \emph{et al.} , 2020).\emph{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 \emph{et al.,}where the Mtb-induced apoptosis was
blocked by Caspase inhibitors (Augenstreich \emph{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 \emph{et al.} , 2010; Grover \emph{et al.} , 2018;
Liu\emph{et al.} , 2020). Our \emph{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-\selectlanguage{romanian}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 \emph{et al.} ,
2007; Chen\emph{et 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 \emph{et al.} , 2018; Sharma \emph{et al.} ,
2021; Medha, Priyanka, \emph{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 (S\selectlanguage{ngerman}ánchez \emph{et al.} , 2010). A
PE/PPE family protein- PE13 functions by engagement of TLR4 mediated
up-regulation of NF-\selectlanguage{romanian}ƙB and implicated in modulation of the macrophage
activation and apoptosis (Li \emph{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 \emph{et al.} , 2002; Yamamoto \emph{et al.} , 2002).
Activation of TLR4-MyD88-NF-ƙB cascade is also responsible for
production of proinflammatory cytokines TNF-\selectlanguage{greek}α \selectlanguage{english}and IL-1\selectlanguage{greek}β
\selectlanguage{english}(Yamamoto\emph{et al.} , 2002; Amaral and Andrade, 2017). In our study,
levels of soluble TNF-\selectlanguage{greek}α \selectlanguage{english}were observed to be up-regulated in
Rv2615c-stimulated macrophages whereas no significant release of IL-1\selectlanguage{greek}β
\selectlanguage{english}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-\selectlanguage{romanian}ƙB signalling and increased levels of pro-inflammatory
cytokines are prerequisite for activation of macrophages (Saraav
\emph{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 (Pr\selectlanguage{ngerman}óchnicki and Latz, 2017).
IL-1\selectlanguage{greek}β \selectlanguage{english}is a mediator of inflammasome activation and plethora of studies
have reported its heterogenous regulation (Mishra \emph{et al.} , 2010;
Hackett \emph{et al.} , 2020). The transcriptional regulation of IL-1\selectlanguage{greek}β
\selectlanguage{english}is not only limited to TLR4-NF\selectlanguage{greek}κ\selectlanguage{english}B axis but is also controlled by
macrophage immune-metabolic responses (Gleeson \emph{et al.} , 2016).
Recent findings established that persistent Mtb infection of macrophages
was associated with suppression of glycolysis and reduced production of
IL-1\selectlanguage{greek}β, \selectlanguage{english}despite upregulated TNF-\selectlanguage{greek}α \selectlanguage{english}levels (Pr\selectlanguage{ngerman}óchnicki and Latz, 2017).
Therefore, our observation of selective pro-inflammatory cytokine
secretion of only TNF-\selectlanguage{greek}α \selectlanguage{english}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 \emph{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-\selectlanguage{greek}α \selectlanguage{english}expression which
suggests that the~macrophage activation~in response to Rv2615c could be
TLR4 mediated. Observations of selective release of pro-inflammatory
cytokine TNF-\selectlanguage{greek}α, \selectlanguage{english}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 \emph{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 \emph{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-\selectlanguage{romanian}ƙB-TNF-\selectlanguage{greek}α \selectlanguage{english}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.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image21/image21}

\end{center}
\end{figure}

\textbf{Figure 8. Schematic representation of immuno-modulatory role
played by Rv2615c}

Rv2615c can interact and activate the host immune receptor TLR4 leading
to activation of downstream effectors such as MyD88, NF-\selectlanguage{romanian}ƙB and TNF-\selectlanguage{greek}α
\selectlanguage{english}along with up-regulation of antigen presenting molecules HLA-DR. By
possessing a eukaryotic CARD-like domain, Rv2615c could activate the
initiator Caspase9 activity and formation of apoptosome complex which
further activates executioner Caspase 3 and 7 and induces apoptosis.
These observations implicate that Rv2615c could be a late stage
expressing protein of Mtb facilitating cell-to spread of infection,
activation and recruitment of immune cells at granuloma site and
ultimately pathogen persistence.

\textbf{4. Materials and Methods:}

\textbf{4.1 \emph{In-silico} characterization of Rv2615c}

\textbf{4.1.1 Homology modelling and structure-based functional
prediction of Rv2615c protein}

The sequence of Rv2615c protein was retrieved from Mycobrowser and
subjected to various servers for structure and function-based
predictions. Antigenicity of Rv2615c was estimated in Vaxijen 2.0 server
with a set threshold default value of 0.4 (Doytchinova and Flower,
2007).

The secondary structure and the structure-based function of Rv2615c
protein was predicted using I-TASSER (Iterative Threading ASSEmbly
Refinement) server. Structural analogs, Gene ontology (GO) template
analogs and consensus GO terms for biological process and molecular
functions were predicted (Roy, Kucukural and Zhang, 2010). Five
homology-based models were predicted for Rv2615c. Chiron server was used
for energy minimization of models (Ramachandran \emph{et al.} , 2011).
The output models were validated for their stereo-chemical quality
through PROCHECK, VERIFY3D and ERRAT server (L\selectlanguage{ngerman}üthy, Bowie and Eisenberg,
1992; Laskowski \emph{et al.} , 1996). PROCHECK confirms the quality of
protein structure by Ramachandran plot where \textless{}5 residues in
the disallowed region are considered to constitute a good structure. The
best model of protein structure was selected with fewer residues in
disallowed region of Ramachandran plot, high score in VERIFY3D and
ERRAT.

\textbf{4.2 In vitro assessment of Rv2615c potential for inducing
apoptosis}

\textbf{4.2.1 Cloning, expression and purification of recombinant
Rv2615c protein}

Cloning, expression and purification of Rv2615c protein was performed as
described in Medha \emph{et al} ., (Medha, Joshi, \emph{et al.} , 2022)
(Medha, Priyanka, \emph{et al.} , 2022).

Briefly, the Rv2615c gene was directly cloned in the pGEM-T Easy vector
and its successful sequencing was verified. The full-length gene was
then sub-cloned using BamHI and HindIII restriction enzymes in the
expression vector pET-28a (+) in frame with a 6x histidine tag at the
N-terminus. The target gene containing recombinant plasmid was
introduced into \emph{Escherichia coli (E. coli)} BL21 \emph{DE3} for
protein over-expression after being induced with 1mM IPTG and maintained
at 37° 250 rpm for a couple of hours, then at 20° overnight. Culture was
pelleted and re-suspended in 20-30 ml of 1XPBS+1mM PMSF followed by
sonication (duty cycle 30; 5-6 cycles). Pellet was washed step wise with
10 ml 2\%, 1\% TritonX in PBS followed by 10ml 1X PBS and finally
dissolved in Buffer (50mM NaH2PO4, 300 mMNacl, 8M Urea) and kept for
binding at 4° with Ni-NTA matrix in column. Column was washed thrice
with 1X cold PBS and wash fractions collected. Elution was performed
with different gradients of immidazole. Elution buffer constituents:
50mM NaH2PO4, 300mM Nacl, 10mM TrisCl, 1mM PMSF, 10\% glycerol, \selectlanguage{greek}β\selectlanguage{english}Me, 8mM
urea. SDS-PAGE was used to analyse the recombinant protein's purity,
which was then validated by Coomassie Blue staining and immuno-blotting
using anti-His antibodies. To remove immidazole and urea, purified
protein was dialyzed against 1XPBS and decreasing urea concentrations.
Purified recombinant proteins were then incubated for 1 h at 4\selectlanguage{ngerman}°C with
polymyxinB-agarose beads to remove endotoxin contamination. Limulus
Amoebocyte Lysate (LAL) assay was performed using Peirce LAL assay kit
following manufacturer's instructions to ensure negligible endotoxin
levels in the purified protein (Pierce LAL Chromogenic Endotoxin
Quantitation Kit, Cat no. 88282). The BCA protein assay was used to
determine the protein content. Up to usage, the purified protein was
kept in tiny aliquots at 80 °C.

\textbf{4.2.2 Human Cell Culture: THP-1 cells stimulation with
recombinant Rv2615c protein}

In tissue culture plates, the human monocytic cell line THP-1 (NCCS,
Pune) was cultured (1X10\textsuperscript{5}cells/ml cells per ml) in
RPMI 1640, 10\% foetal calf serum and an antibiotic cocktail that
included penicillin (10,000 units/ml) and streptomycin (10,000 g/ml).
For the purpose of differentiating the monocytic cells into macrophages,
40ng/ml Phorbol-Myristate Acetate (PMA; Sigma) was added to the
monocytic cells and left overnight.

\textbf{4.2.2 (a) Detection of Cell Viability Using MTT and Cell Titer
blue}

For dose dependent viability assessment, cells were stimulated with
different concentration (5\selectlanguage{greek}µ\selectlanguage{english}g, 10\selectlanguage{greek}µ\selectlanguage{english}g, 15\selectlanguage{greek}µ\selectlanguage{english}g) of purified Rv2615c protein
for 24h. For checking the viability in time dependent manner,
unstimulated cells and cells stimulated with (10\selectlanguage{greek}µ\selectlanguage{english}g/ml) Rv2615c/(40ng/ml)
LPS/(2\selectlanguage{greek}μ\selectlanguage{english}M) Staurosporine were incubated for 16h, 24h and 48h.
Staurosporine~is an alkaloid derivative from the
bacterium~\emph{Streptomyces staurosporeus} which has been extensively
been used as an inducer of apoptosis because it inhibits many different
kinases.

Cells were exposed to either CellTiter Blue Reagent (20 mL/well) or MTT
(5 mg/ml) and incubated for 4 hours at 37 \selectlanguage{ngerman}°C and 5\% CO2. Tetrazolium
rings can be transformed into purple, insoluble formazan crystals by
active dehydrogenases found in live cells. The crystals were dissolved
in 100 \selectlanguage{greek}µ\selectlanguage{english}L of DMSO and the overall activity of bio-reductase enzyme was
measured spectrophotometrically at (570-690) nm. The CellTiter-Blue
Reagent constitutes of resazurin in a buffer solution. Resorufin (dark
pink), which is extremely fluorescent and whose absorbance may be
measured spectrophotometrically at (570--600) nm, can be reduced from
resazurin (dark blue) by viable cells. The percentage of cell viability
was calculated using the acquired absorbance values. Cells cultured in
medium alone were thought to be completely viable.

\textbf{4.2.2 (b) Lactate Dehydrogenase (LDH) release assay to identify
cell cytotoxicity}

Lactate dehydrogenase is a cytosolic enzyme which is released into the
cell culture media with plasma membrane damage which can be measured.
Levels of LDH released in the media are an indicator whether the cells
are undergoing necrosis. High levels of LDH in the media are
proportional to cell death via necrosis. Plates with unstimulated cells
and Rv2615c or control (LPS/Staurosporine) stimulated cells were
centrifuged and 20\selectlanguage{greek}µ\selectlanguage{english}l of media from each well and 20\selectlanguage{greek}µ\selectlanguage{english}l reaction buffer
were added to fresh wells. Plates were incubated for 30 min at room
temperature following addition of 20\selectlanguage{greek}µ\selectlanguage{english}l stop solution. Absorbance reading
was recorded at 490nm.

\textbf{4.2.2 (c) Detection of Phosphatidyl-serine Exposure by Annexin
V-FITC Staining}

Phosphatidylserine exposure, an early step of apoptosis induction in
protein-stimulated THP-1 macrophages was estimated by Annexin V-FITC
apoptosis detection kit (Thermo Scientific). Unstimulated and cells
stimulated with Rv2615c and controls- (LPS/Staurosporine) were stained
with AnnexinV-FITC and Propidium Iodide (PI) following manufacturer's
protocol and analysed using flow cytometer (BD AccuriC6). Following
stimulation time points of 16h, 24h and 48h; macrophages were collected
after cold PBS washing and detached by scraping. After washing twice,
the cells were re-suspended in binding buffer. An aliquot of 200 \selectlanguage{greek}µ\selectlanguage{english}l was
mixed with 5 \selectlanguage{greek}µ\selectlanguage{english}l of Annexin V-FITC and 5 \selectlanguage{greek}µ\selectlanguage{english}l of PI. The mixture was
incubated for 10 min at room temperature in the dark. Finally, the cells
were analysed by flow cytometry.

\textbf{4.2.2 (d) DNA Fragmentation analysis by TUNEL Assay}

Unstimulated and cells stimulated with Rv2615c or controls-
(LPS/Staurosporine) were incubated for different time points. Harvested
cells were fixed in 1\% paraformaldehyde and incubated for 60 minutes on
ice. Fixed cells were washed first with ice-cold PBS followed by 70\%
ethanol and initially incubated for 30 minutes on ice and later on
stored at -20 \selectlanguage{ngerman}°C for 18h. Finally, for labelling DNA breaks to detect
apoptosis; according to the manufacturer's instructions, cells were
stained with the APO-Direct kit (BD Pharmingen) and analysed using the
BD accuri C6 flow cytometer. Briefly, Cells were stained in reaction
buffer constituting of Terminal de-oxynucleotidyltransferase (TdT)
enzyme and fluoresceinated-dUTP (FITC-dUTP).

\textbf{4.2.2 (e) Detection of activated initiator Caspase9}

Total protein was extracted after 24 hours of protein stimulation by
lysing THP-1 macrophages using RIPA lysis buffer and a protease
inhibitor cocktail (Santa Cruz Biotechnology Ltd.). Equal concentrations
of samples were analysed through SDS-PAGE before being transferred onto
nitrocellulose membrane. The total protein content of the entire cell
lysate was measured. Primary polyclonal antibody to Caspase9
(PAA627Hu03, Cloud-Clone Corp.), as well as GAPDH (Thermo Scientific) as
an internal control, were incubated overnight in membranes after
blocking for 1h in 5\% skimmed milk dissolved in Tris-buffered saline
with Tween 20 (TBST) buffer. Membranes were washed in TBST solution
before being incubated for 1 hour with a secondary antibody that has
been HRP-conjugated. To create the blot, an ECL Chemiluminescence Kit
was employed (Thermo Scientific). Using ImageJ software, Western
Blotting pictures were also quantified. Each sample's developed band
area was examined, and graphs were drawn as a ratio to the Unstimulated
sample.

\textbf{4.2.2 (f)} \textbf{Detection of activated Caspases 3 and 7}

For detection of activation of Caspase 3 and 7 which is a distinctive
feature of early stages of apoptosis, CellEvent Caspase-3/7 Green Flow
Cytometry Assay kit (Invitrogen) was used. Following incubation,
unstimulated cells and cells stimulated with either Rv2615c or controls-
(LPS/Staurosporine) were harvested and 500nM cell event Caspase 3/7
reagent was added to 1ml cell suspension in 1xPBS. Cells were incubated
for 30 mins at 37°C. During final 5 mins of staining, 1\selectlanguage{greek}µ\selectlanguage{english}M SYTOX dead
cell stain was added and samples were analysed in BD accuri C6 flow
cytometer.

To confirm the observed apoptosis in response to Rv2615c to be Caspase
dependent, Caspase inhibition studies were done. For inhibition study,
cells were incubated with 20\selectlanguage{greek}μ\selectlanguage{english}M total Caspase inhibitor (Z-VAD-fmk;
Promega) for 1 h, followed by treatment with respective test
protein/controls and incubated for 24 h. Percentage of Caspase 3 and 7
activation and percentage of AnnexinV and AnnexinV/PI dual positive cell
population were analysed using flow cytometry.

\textbf{4.3. Role of Rv2615c in immune response modulation via
expression of TLR, HLA-DR and downstream effector molecules}

\textbf{4.3 (a) Predicting the identity of the TLR involved in Rv2615c
interaction}

The human TLR4 (PDB ID 3FXI) and TLR2 (PDB ID: 2Z7X) protein structures
were obtained from the RCSB protein data bank. Rv2615c interactions with
TLR4 and TLR2 were compared using HADDOCK 2.2 web server (van
Zundert\emph{et al.} , 2016). The active sites within the TLR and
Rv2615c molecules were predicted using CastP server (Tian \emph{et al.}
, 2018). Site directed docking was performed and docked complex were
evaluated on the basis of Z scores.

\textbf{4.3 (b) Expression profile of TLRs and HLA-DR in response to
Rv2615c}

The expression profile of TLR2 and TLR4 was evaluated through flow
cytometry following 24h stimulation of THP-1 macrophages with the
10\selectlanguage{greek}µ\selectlanguage{english}g/ml Rv2615c protein. The controls included were TLR4 agonist- LPS
(40ng/ml) and TLR2 agonist- Cell Wall Fraction of H37Rv (CWF) (5\selectlanguage{greek}μ\selectlanguage{english}g/ml)
(BEI Resources) (Man\selectlanguage{polish}č\selectlanguage{english}ek-Keber and Jerala, 2015). Following incubation,
cells were harvested and washed in PBS and stained with APC labelled
anti-human TLR2 (CD282) antibody (Thermo Scientific) or APC labelled
anti-human TLR4 (CD284) antibody (Thermo Scientific) and PE labelled
anti-human HLA-DR antibody (Thermo Scientific). Cells were analysed in
BD Accuri C6 flow Cytometer. Results were analysed on the basis of
percentage of positively stained cells.

For inhibitor assay, 1h prior to stimulation; cells were blocked with
Anti-TLR4 monoclonal antibody (Thermo Scientific) and analysed for
expression of TLR4 and HLA-DR after 24h using flow cytometry.

\selectlanguage{romanian}\textbf{4.3 (c) Expression profile of downstream effectors- MyD88 and
NF-ƙB using RT-PCR}

mRNA was isolated from unstimulated and either Rv2615c (10\selectlanguage{greek}μ\selectlanguage{english}g/ml) or LPS
(40ng/ml) stimulated THP-1 macrophages after 16h, 24h and 48h using mRNA
synthesis kit (Promega Corporation) as per manufactures' protocol. 1\selectlanguage{greek}µ\selectlanguage{english}g
of mRNA was converted to cDNA (Promega Corporation) using cDNA synthesis
kit. mRNA and cDNA were checked by setting up PCR of endogenous control
i.e., housekeeping gene GAPDH. Quantitative real time PCR for downstream
effector genes- MyD88 and NF-\selectlanguage{romanian}ƙB was performed by relative quantification
in which the target concentration is expressed as ratio of target vs.
reference gene (housekeeping gene GAPDH). The expression of MyD88 was
evaluated following 24h of protein stimulation and the expression of
NF-ƙB was evaluated following 24h and 48h of protein stimulation. The
primers used were Forward Primer 5' ATG GCT TCT ATG AGG CTG AG 3' and
Reverse Primer 5' GTT GTTGTT GGT CTG GAT GC 3'. Equal concentration of
cDNA for each sample was added to SYBR PCR master mix along with
respective gene primers. 40 cycles of amplification followed by data
acquisition and analysis was done. Data was calculated using the
2\selectlanguage{english}\textsuperscript{-[?][?]} CT method and are presented as fold induction.
Fold change was normalized to GAPDH expression levels. Experiments were
repeated thrice.

\textbf{4.3 (d) Study of pro-inflammatory cytokine profile in response
to Rv2615c}

THP-1 macrophages were left unstimulated or stimulated with 10ug/ml of
Rv2615c or control- LPS {[}40ng/ml{]} for 16h, 24h and 48h were analysed
for secretion of TNF\selectlanguage{greek}α \selectlanguage{english}(Thermo Scientific) and IL-1\selectlanguage{greek}β \selectlanguage{english}(Biolegend) in cell
culture supernatants. ELISA was done according to manufacturer's
instructions.

ELISA was also performed for estimation of TNF\selectlanguage{greek}α \selectlanguage{english}and IL-1\selectlanguage{greek}β \selectlanguage{english}in
supernatants blocked with Anti-TLR4 monoclonal antibody prior to
stimulation with Rv2615c/controls till 24 h.

\textbf{Statistical analysis}

All the experiments were performed in triplicates. The results of all
the experimental data are presented as Mean\selectlanguage{ngerman}±SEM. GraphPad Prism version
5.02 was used to do statistical comparisons employing Student's t-test
(San Diego, CA, USA). Mean differences were analysed and * was used to
represent the comparison with unstimulated control. Differences were
considered significant at *P\textless{}0.05, **P\textless{}0.01,
***P\textless{}0.001.

\textbf{4.4 Sequence scanning of Rv2615c protein}

Predictions of I-TASSER strongly suggested the similarity of Rv2615c
with APAF1-apoptosome structure involved in Caspase activation and cell
death pathways followed by \emph{in-vitro} experiments which further
confirmed the apoptogenic role played by Rv2615c protein. It is reported
that the APAF1-apoptosome activates the Caspase9 via CARD-CARD
interaction between APAF1-Caspase9. Therefore, multiple sequence
alignment of Rv2615c was performed with CARD-APAF1 and CARD-Caspase9.
The alignment was checked for various conserved and similar residues.
Secondary structure of predicted CARD-like domain in Rv2615c was
analysed with MINNOU server (Cao \emph{et al.} , 2006). Structural
superimposition of predicted CARD-like domain in Rv2615c with CARD-APAF1
and CARD-Caspase9 was carried out in Pymol software.

\textbf{4.4.1 Evaluating the binding affinity of CARD-like domain of
Rv2615c with CARD-APAF1 and CARD-Caspase9}

Molecular docking of Rv2615c protein was performed to estimate the
binding affinity of CARD-like domain of Rv2615c with CARD-APAF1 and
CARD-Caspase9 using PatchDock server (Schneidman-Duhovny \emph{et al.} ,
2005). Top ten docked structures were further refined in FireDock server
(Mashiach \emph{et al.} , 2008). The best model was evaluated based on
number of bonds formed and the energy score generated in HEX 8.0.0.
Visualization was done in Discovery studio Visualizer 4.1 (Accelyrs
Inc., USA). The complex of CARD-APAF1 with CARD-Caspase9 (PDB ID: 3YGS)
was included as positive control and the inactivated independent
APAF-ADP complex (PDB ID: 1Z6T) was included as negative control in the
study.

We observed Leucine-116 and Isoleucine-117 were two common residues
within CARD-like domain of Rv2615c which aligned with conserved residues
Methionine-29 and Isoleucine-30 of CARD-APAF1 and Leucine-58 and
Isoleucine-59 of CARD-Caspase9. Therefore, site directed mutation was
incorporated changing Leucine-116 and Isoleucine-117 to the most
preferred Alanine substitution in CARD-like domain of Rv2615c and the
mutant structure was checked for its stability using I-Mutant 2.0 server
(Capriotti, Fariselli and Casadio, 2005). Furthermore, binding affinity
of this mutant Rv2615c protein with CARD-APAF1 and with CARD-Caspase9
was evaluated through docking studies as described previously.

\textbf{4.4.2 MD simulation of docked complexes}

MD simulation for 100 nanoseconds (ns) using Gromacs software 2020.5 was
performed to evaluate the stability of docked complexes of whole Rv2615c
protein with CARD-APAF1 and CARD-Caspase9 separately and mutant Rv2615c
with CARD-APAF1 and CARD-Caspase9 separately. The docked structure of
APAF1-CARD and Caspase9-CARD (PDB ID: 3YGS) was included as positive
control in the simulation studies. The docked complex was solvated by
TIP3P {[}transferable intermolecular potential with 3 points{]} water
model using CHARMM36 force field. The final MD simulation run was
performed at 300K temperature and standard pressure of 1.01 bar using
NPT (number of particle {[}N{]}, system pressure {[}P{]} and temperature
{[}T{]}) and NVT (number of particle {[}N{]}, system volume {[}V{]} and
temperature {[}T{]}) ensemble. Backbone Root Mean Square Deviation
(RMSD) and Root Mean Square Fluctuation (RMSF) of the entire complex was
calculated and plotted.

\textbf{Acknowledgement}

We acknowledge Department of Science and Technology (DST), Government of
India, for providing financial assistance under Grant Sanction No.
EMR/2016/006774. The Council of Scientific and Industrial Research
{[}CSIR{]}, Government of India, awarded Senior Research Fellowships to
Medha and Priyanka. Professor Vikas Jain of the Indian Institute of
Science Education and Research {[}IISER{]}, Bhopal, is sincerely
acknowledged for his direction and assistance in carrying out MD
simulation research.

\textbf{Author's Contributions}

Monika Sharma led in conceptualization and funding acquisition. Medha
contributed in conceptualization, execution of all the
\emph{in-silico}studies, and writing of the manuscript. Medha and
Priyanka conducted all the experiments. Monika Sharma, Medha and Sadhna
Sharma contributed in reviewing and editing of the manuscript.

\textbf{Conflict of interest}

The authors have no competing interests to declare.

\textbf{Data Availability Statement}

The datasets generated during and/or analysed during the current study
are included in this article (and its supplementary information files).
A descriptive dataset can be provided by the corresponding author upon
reasonable request.

\textbf{Compliance with Ethical Standards}

This article does not contain any studies with human participants or
animals performed by any of the authors.

\textbf{References}

Abdallah, A.M. \emph{et al.} (2007) `Type VII secretion--mycobacteria
show the way.', \emph{Nature reviews. Microbiology} , 5(11), pp.
883--891. Available at: https://doi.org/10.1038/nrmicro1773.

Abdallah, A.M. \emph{et al.} (2011) `Mycobacterial Secretion Systems
ESX-1 and ESX-5 Play Distinct Roles in Host Cell Death and Inflammasome
Activation', \emph{The Journal of Immunology} , 187(9), pp. 4744 LP --
4753. Available at: https://doi.org/10.4049/jimmunol.1101457.

Aguilo, J.I. \emph{et al.} (2013) `ESX-1-induced apoptosis is involved
in cell-to-cell spread of Mycobacterium tuberculosis', \emph{Cellular
Microbiology} , 15(12), pp. 1994--2005. Available at:
https://doi.org/10.1111/cmi.12169.

Amaral, E.P. and Andrade, B.B. (2017) `Nuclear Factor \selectlanguage{greek}κ \selectlanguage{english}B Activation
Pathways During Mycobacterium tuberculosis Infection'. Available at:
https://doi.org/10.1177/1179568917695833.

Augenstreich, J. \emph{et al.} (2017) `ESX-1 and phthiocerol
dimycocerosates of Mycobacterium tuberculosis act in concert to cause
phagosomal rupture and host cell apoptosis', \emph{Cellular
Microbiology} , 19(7), p. e12726. Available at:
https://doi.org/10.1111/cmi.12726.

Bansal, K. \emph{et al.} (2010) `PE\_PGRS Antigens of Mycobacterium
tuberculosis Induce Maturation and Activation of Human Dendritic
Cells',\emph{The Journal of Immunology} , 184(7), pp. 3495 LP -- 3504.
Available at: https://doi.org/10.4049/jimmunol.0903299.

Basu, S. \emph{et al.} (2007) `Execution of macrophage apoptosis by
PE\_PGRS33 of Mycobacterium tuberculosis is mediated by toll-like
receptor 2-dependent release of tumor necrosis factor-\selectlanguage{greek}α\selectlanguage{english}', \emph{Journal
of Biological Chemistry} , 282(2), pp. 1039--1050. Available at:
https://doi.org/10.1074/jbc.M604379200.

Brennan, M.J. (2017) `The Enigmatic PE/PPE Multigene Family of
Mycobacteria and Tuberculosis Vaccination', \emph{Infection and
Immunity} . Edited by A.T. Maurelli, 85(6), pp. e00969-16. Available at:
https://doi.org/10.1128/IAI.00969-16.

Cadieux, N. \emph{et al.} (2011) `Induction of cell death after
localization to the host cell mitochondria by the Mycobacterium
tuberculosis PE\_PGRS33 protein', \emph{Microbiology} , 157(3), pp.
793--804. Available at: https://doi.org/10.1099/mic.0.041996-0.

Cao, B. \emph{et al.} (2006) `Enhanced recognition of protein
transmembrane domains with prediction-based structural
profiles.',\emph{Bioinformatics (Oxford, England)} , 22(3), pp.
303--309. Available at: https://doi.org/10.1093/bioinformatics/bti784.

Capriotti, E., Fariselli, P. and Casadio, R. (2005) `I-Mutant2.0:
predicting stability changes upon mutation from the protein sequence or
structure.', \emph{Nucleic acids research} , 33(Web Server issue), pp.
W306-10. Available at: https://doi.org/10.1093/nar/gki375.

Chai, Q. \emph{et al.} (2019) `A Mycobacterium tuberculosis surface
protein recruits ubiquitin to trigger host xenophagy.', \emph{Nature
communications} , 10(1), p. 1973. Available at:
https://doi.org/10.1038/s41467-019-09955-8.

Chai, Q., Wang, L. and Liu, C.H. (2020) `New insights into the evasion
of host innate immunity by Mycobacterium tuberculosis', \emph{Cellular
\& Molecular Immunology} {[}Preprint{]}, (July). Available at:
https://doi.org/10.1038/s41423-020-0502-z.

Chen, T. \emph{et al.} (2013) `Mycobacterium tuberculosis PE\_PGRS17
promotes the death of host cell and cytokines secretion via Erk kinase
accompanying with enhanced survival of recombinant Mycobacterium
smegmatis.', \emph{Journal of interferon \& cytokine research : the
official journal of the International Society for Interferon and
Cytokine Research} , 33(8), pp. 452--458. Available at:
https://doi.org/10.1089/jir.2012.0083.

Chmiela, M. and Gonciarz, W. (2017) `Molecular mimicry in Helicobacter
pylori infections', \emph{World journal of gastroenterology} , 23(22),
pp. 3964--3977. Available at: https://doi.org/10.3748/wjg.v23.i22.3964.

Cole, S.T. \emph{et al.} (1998) `Deciphering the biology of
Mycobacterium tuberculosis from the complete genome
sequence',\emph{Nature} , 393(6685), pp. 537--544. Available at:
https://doi.org/10.1038/31159.

Davis, J.M. \emph{et al.} (2002) `Real-time visualization of
mycobacterium-macrophage interactions leading to initiation of granuloma
formation in zebrafish embryos', \emph{Immunity} , 17(6), pp. 693--702.

Desler, C. \emph{et al.} (2009) `In Silico screening for functional
candidates amongst hypothetical proteins', \emph{BMC Bioinformatics} ,
10(1), p. 289. Available at: https://doi.org/10.1186/1471-2105-10-289.

Dorhoi, A. and Kaufmann, S.H.E. (2014) `Tumor necrosis factor alpha in
mycobacterial infection', \emph{Seminars in Immunology} , 26(3), pp.
203--209. Available at:
https://doi.org/https://doi.org/10.1016/j.smim.2014.04.003.

Doytchinova, I.A. and Flower, D.R. (2007) `VaxiJen: a server for
prediction of protective antigens, tumour antigens and subunit
vaccines.', \emph{BMC bioinformatics} , 8, p. 4. Available at:
https://doi.org/10.1186/1471-2105-8-4.

Fayyazi, A. \emph{et al.} (2000) `Apoptosis of macrophages and T cells
in tuberculosis associated caseous necrosis', \emph{The Journal of
pathology} , 191(4), pp. 417--425.

Gey Van Pittius, N.C. \emph{et al.} (2006) \emph{Evolution and expansion
of the Mycobacterium tuberculosis PE and PPE multigene families and
their association with the duplication of the ESAT-6 (esx) gene cluster
regions} , \emph{BMC Evolutionary Biology} . Available at:
https://doi.org/10.1186/1471-2148-6-95.

Gleeson, L.E. \emph{et al.} (2016) `Cutting Edge: Mycobacterium
tuberculosis Induces Aerobic Glycolysis in Human Alveolar Macrophages
That Is Required for Control of Intracellular Bacillary
Replication',\emph{The Journal of Immunology} , 196(6), pp. 2444 LP --
2449. Available at: https://doi.org/10.4049/jimmunol.1501612.

Grover, S. \emph{et al.} (2018) `The PGRS domain of Mycobacterium
tuberculosis PE\_PGRS protein Rv0297 is involved in Endoplasmic
reticulum stress-mediated apoptosis through toll-like receptor
4',\emph{mBio} , 9(3). Available at:
https://doi.org/10.1128/mBio.01017-18.

Hackett, E.E. \emph{et al.} (2020) `Mycobacterium tuberculosis Limits
Host Glycolysis and IL-1\selectlanguage{greek}β \selectlanguage{english}by Restriction of PFK-M via
MicroRNA-21',\emph{Cell Reports} , 30(1), pp. 124-136.e4. Available at:
https://doi.org/https://doi.org/10.1016/j.celrep.2019.12.015.

Hasan, S. \emph{et al.} (2006) `Prioritizing Genomic Drug Targets in
Pathogens: Application to Mycobacterium tuberculosis', \emph{PLOS
Computational Biology} , 2(6), p. e61. Available at:
https://doi.org/10.1371/journal.pcbi.0020061.

Horng, T. \emph{et al.} (2002) `The adaptor molecule TIRAP provides
signalling specificity for Toll-like receptors', \emph{Nature} ,
420(6913), pp. 329--333. Available at:
https://doi.org/10.1038/nature01180.

Hu, Q. \emph{et al.} (2014) `Molecular determinants of caspase-9
activation by the Apaf-1 apoptosome', \emph{Proceedings of the National
Academy of Sciences of the United States of America} . 2014/10/13,
111(46), pp. 16254--16261. Available at:
https://doi.org/10.1073/pnas.1418000111.

Kruh, N.A. \emph{et al.} (2010) `Portrait of a Pathogen: The
Mycobacterium tuberculosis Proteome In Vivo', \emph{PLOS ONE} , 5(11),
p. e13938. Available at: https://doi.org/10.1371/journal.pone.0013938.

Kumar, K. \emph{et al.} (2014) `Functional annotation of putative
hypothetical proteins from Candida dubliniensis', \emph{Gene} , 543(1),
pp. 93--100. Available at:
https://doi.org/https://doi.org/10.1016/j.gene.2014.03.060.

Laskowski, R.A. \emph{et al.} (1996) `AQUA and PROCHECK-NMR: programs
for checking the quality of protein structures solved by
NMR.',\emph{Journal of biomolecular NMR} , 8(4), pp. 477--486. Available
at: https://doi.org/10.1007/BF00228148.

Li, H. \emph{et al.} (2016) `Mycobacterium tuberculosis PE13 (Rv1195)
manipulates the host cell fate via p38-ERK-NF-\selectlanguage{greek}κ\selectlanguage{english}B axis and
apoptosis',\emph{Apoptosis : an international journal on programmed cell
death} , 21(7), pp. 795--808. Available at:
https://doi.org/10.1007/s10495-016-1249-y.

Liu, S. \emph{et al.} (2020) `PE\_PGRS31-S100A9 Interaction Promotes
Mycobacterial Survival in Macrophages Through the Regulation of
NF-\selectlanguage{greek}κ\selectlanguage{english}B-TNF-\selectlanguage{greek}α \selectlanguage{english}Signaling and Arachidonic Acid Metabolism.', \emph{Frontiers
in microbiology} , 11, p. 845. Available at:
https://doi.org/10.3389/fmicb.2020.00845.

L\selectlanguage{ngerman}üthy, R., Bowie, J.U. and Eisenberg, D. (1992) `Assessment of protein
models with three-dimensional profiles.', \emph{Nature} , 356(6364), pp.
83--85. Available at: https://doi.org/10.1038/356083a0.

Man\selectlanguage{polish}č\selectlanguage{english}ek-Keber, M. and Jerala, R. (2015) `Postulates for validating TLR4
agonists', \emph{European Journal of Immunology} , 45(2), pp. 356--370.
Available at: https://doi.org/https://doi.org/10.1002/eji.201444462.

Mashiach, E. \emph{et al.} (2008) `FireDock: a web server for fast
interaction refinement in molecular docking', \emph{Nucleic acids
research} . 2008/04/19, 36(Web Server issue), pp. W229--W232. Available
at: https://doi.org/10.1093/nar/gkn186.

McGuire, A.M. \emph{et al.} (2012) `Comparative analysis of
mycobacterium and related actinomycetes yields insight into the
evolution of mycobacterium tuberculosis pathogenesis', \emph{BMC
Genomics} , 13(1). Available at:
https://doi.org/10.1186/1471-2164-13-120.

Medha, Joshi, H., \emph{et al.} (2022) `Elucidating the function of
hypothetical PE\_PGRS45 protein of Mycobacterium tuberculosis as an
oxido-reductase: a potential target for drug repurposing for the
treatment of tuberculosis', \emph{Journal of Biomolecular Structure and
Dynamics} , pp. 1--17.

Medha, Priyanka, \emph{et al.} (2022) `Role of C-terminal domain of
Mycobacterium tuberculosis PE6 (Rv0335c) protein in host mitochondrial
stress and macrophage apoptosis.', \emph{Apoptosis : an international
journal on programmed cell death} , pp. 1--30. Available at:
https://doi.org/10.1007/s10495-022-01778-1.

Medha, Sharma, S. and Sharma, M. (2021) `Acta Tropica Proline-Glutamate
/ Proline-Proline-Glutamate ( PE / PPE ) proteins of Mycobacterium
tuberculosis : The multifaceted immune-modulators', \emph{Acta Tropica}
, 222(April), p. 106035. Available at:
https://doi.org/10.1016/j.actatropica.2021.106035.

Mishra, B.B. \emph{et al.} (2010) `Mycobacterium tuberculosis protein
ESAT-6 is a potent activator of the NLRP3/ASC
inflammasome',\emph{Cellular Microbiology} , 12(8), pp. 1046--1063.
Available at:
https://doi.org/https://doi.org/10.1111/j.1462-5822.2010.01450.x.

Murphy, D.J. and Brown, J.R. (2007) `Identification of gene targets
against dormant phase Mycobacterium tuberculosis infections', \emph{BMC
Infectious Diseases} , 7(1), p. 84. Available at:
https://doi.org/10.1186/1471-2334-7-84.

Pan, H. \emph{et al.} (2005) `Ipr1 gene mediates innate immunity to
tuberculosis', \emph{Nature} , 434(7034), pp. 767--772.

Pr\selectlanguage{ngerman}óchnicki, T. and Latz, E. (2017) `Inflammasomes on the Crossroads of
Innate Immune Recognition and Metabolic Control', \emph{Cell Metabolism}
, 26(1), pp. 71--93. Available at:
https://doi.org/https://doi.org/10.1016/j.cmet.2017.06.018.

Ramachandran, S. \emph{et al.} (2011) `Automated minimization of steric
clashes in protein structures.', \emph{Proteins} , 79(1), pp. 261--270.
Available at: https://doi.org/10.1002/prot.22879.

Ramakrishnan, L. (2012) `Revisiting the role of the granuloma in
tuberculosis', \emph{Nature Reviews Immunology} , 12(5), pp. 352--366.

Roy, A., Kucukural, A. and Zhang, Y. (2010) `I-TASSER: a unified
platform for automated protein structure and function
prediction',\emph{Nature Protocols} , 5(4), pp. 725--738. Available at:
https://doi.org/10.1038/nprot.2010.5.

Saini, N.K. \emph{et al.} (2016) `Suppression of autophagy and antigen
presentation by Mycobacterium tuberculosis PE\_PGRS47', \emph{Nature
Microbiology} , 1(9), p. 16133. Available at:
https://doi.org/10.1038/nmicrobiol.2016.133.

Sánchez, D. \emph{et al.} (2010) `Role of TLR2- and TLR4-mediated
signaling in Mycobacterium tuberculosis-induced macrophage
death',\emph{Cellular Immunology} , 260(2), pp. 128--136. Available at:
https://doi.org/https://doi.org/10.1016/j.cellimm.2009.10.007.

Saraav, I. \emph{et al.} (2017) `Mycobacterium tuberculosis MymA is a
TLR2 agonist that activate macrophages and a TH1
response',\emph{Tuberculosis} , 106, pp. 16--24. Available at:
https://doi.org/https://doi.org/10.1016/j.tube.2017.05.005.

Saunders, B.M. and Britton, W.J. (2007) `Life and death in the
granuloma: immunopathology of tuberculosis', \emph{Immunology \& Cell
Biology} , 85(2), pp. 103--111. Available at:
https://doi.org/10.1038/sj.icb.7100027.

Schneidman-Duhovny, D. \emph{et al.} (2005) `PatchDock and SymmDock:
servers for rigid and symmetric docking', \emph{Nucleic acids research}
, 33(Web Server issue), pp. W363--W367. Available at:
https://doi.org/10.1093/nar/gki481.

Seimon, T.A. \emph{et al.} (2010) `Induction of ER Stress in Macrophages
of Tuberculosis Granulomas', \emph{PLOS ONE} , 5(9), p. e12772.
Available at: https://doi.org/10.1371/journal.pone.0012772.

Sharma, N. \emph{et al.} (2021) `Mycobacterium~tuberculosis~Protein PE6
(Rv0335c), a Novel TLR4 Agonist, Evokes an Inflammatory Response and
Modulates the Cell Death Pathways in Macrophages to Enhance
Intracellular Survival', \emph{Frontiers in immunology} , 12, p. 696491.
Available at: https://doi.org/10.3389/fimmu.2021.696491.

Sonia, M. \emph{et al.} (2021) `Molecular Mimicry: a Paradigm of
Host-Microbe Coevolution Illustrated by Legionella', \emph{mBio} ,
11(5), pp. e01201-20. Available at:
https://doi.org/10.1128/mBio.01201-20.

Tian, W. \emph{et al.} (2018) `CASTp 3.0: computed atlas of surface
topography of proteins.', \emph{Nucleic acids research} , 46(W1), pp.
W363--W367. Available at: https://doi.org/10.1093/nar/gky473.

Tiwari, B., Ramakrishnan, U.M. and Raghunand, T.R. (2015) `The
Mycobacterium tuberculosis protein pair PE9 (Rv1088)--PE10 (Rv1089)
forms heterodimers and induces macrophage apoptosis through Toll-like
receptor 4', \emph{Cellular Microbiology} , 17(11), pp. 1653--1669.
Available at: https://doi.org/10.1111/cmi.12462.

Urán~Landaburu, L. \emph{et al.} (2020) `TDR Targets 6: driving drug
discovery for human pathogens through intensive chemogenomic data
integration', \emph{Nucleic Acids Research} , 48(D1), pp. D992--D1005.
Available at: https://doi.org/10.1093/nar/gkz999.

Volkman, H.E. \emph{et al.} (2004) `Tuberculous Granuloma Formation Is
Enhanced by a Mycobacterium Virulence Determinant', \emph{PLOS Biology}
, 2(11), p. e367. Available at:
https://doi.org/10.1371/journal.pbio.0020367.

WHO (2021) \emph{WHO Global TB Report} . World Health Organization.

Yamamoto, M. \emph{et al.} (2002) `Essential role for TIRAP in
activation of the signalling cascade shared by TLR2 and
TLR4',\emph{Nature} , 420(6913), pp. 324--329. Available at:
https://doi.org/10.1038/nature01182.

van Zundert, G.C.P. \emph{et al.} (2016) `The HADDOCK2.2 Web Server:
User-Friendly Integrative Modeling of Biomolecular
Complexes.',\emph{Journal of molecular biology} , 428(4), pp. 720--725.
Available at: https://doi.org/10.1016/j.jmb.2015.09.014.

\par\null

\selectlanguage{english}
\FloatBarrier
\end{document}

