1. Introduction
Tuberculosis (TB) caused by intracellular bacterium 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 et al. , 2009; Kumar et al. , 2014). The
Proline-Glutamate (PE)/ Proline-Proline-Glutamate (PPE) proteins encoded
within 7% of Mtb genome are exclusive to the pathogenicMycobacterium (Cole et al. , 1998; Gey Van Pittius et
al. , 2006; McGuire 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 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 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 et al. ,
2007) (Sharma 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 naïve 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 et al. , 2002), (Volkman et al. , 2004), (Saunders
and Britton, 2007), (Seimon et al. , 2010), (Ramakrishnan, 2012).
Mtb-infected lung granulomas have reported to contain apoptotic
macrophages. (Fayyazi et al. , 2000; Pan 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 et al. , 2018) (Sharma 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 (Chaiet 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 et al. , 2006), (Abdallahet al. , 2007), (Abdallah et al. , 2011), (Aguilo et
al. , 2013). PE_PGRS33 (Rv1818c) has been reported to trigger
mitochondria-mediated intrinsic apoptosis via TLR2-TNF-α signalling and
facilitates survival of Mycobacterium smegmatis expressing this
protein within host macrophages (Basu et al. , 2007) (Cadieuxet 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-α signalling pathway and favours
Mtb dissemination (Grover 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 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, 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. Preliminaryin-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 in-silico results,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-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-α 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.