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.