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