ABSTRACT
Mycobacterium tuberculosis(Mtb) lipoproteins are known to facilitate bacterial survival by manipulating the host immune responses. Here, we have characterized a novel Mtb lipoprotein LprE(LprEMtb), and demonstrated its role in mycobacterial survival. LprEMtb acts by down-regulating the expression of cathelicidin, Cyp27B1, VDR and p38-MAPK via TLR-2 signaling pathway. Deletion of lprEMtb resulted in induction of cathelicidin and decreased survival in the host. Interestingly, LprEMtb was also found to inhibit autophagy mechanism to dampen host immune response. Episomal expression of LprEMtb in non-pathogenic Mycobacterium smegmatis(Msm) increased bacillary persistence by down-regulating the expression of cathelicidin and autophagy, while deletion of LprEMtb orthologue in Msm, had no effect on cathelicidin and autophagy expression. Moreover, LprEMtb blocked phago-lysosome fusion by suppressing the expression of EEA1, Rab7 and LAMP-1 endosomal markers by down-regulating IL-12 and IL-22 cytokines. Our results indicate that LprEMtb plays an important role in mycobacterial pathogenesis in the context of innate immunity.
INTRODUCTION
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a widespread disease that kills more than 1.5 million people every year worldwide [1]. Macrophages, the primary resident cells of Mtb, are important components of innate defense mechanisms of the host. Macrophages play a crucial role in recognition, phagocytosis, and killing of invaders. Non-pathogenic mycobacteria such as M. smegmatis (Msm) are readily killed by the macrophages, whereas pathogenic mycobacteria (Mtb) employ various strategies to survive inside the macrophages like prevention of phago-lysosome fusion [2], autophagy inhibition [3], modulation of host cytokine production [4], inhibition of reactive oxygen and nitrogen species [5] and the manipulation of antigen presentation to prevent or alter the quality of T-cell responses [6].
Among the key effector molecules responsible for bacterial killing are antimicrobial peptides such as cathelicidin and defensins that are expressed in different cells such as neutrophils, macrophages, monocytes and epithelial cells [7]. In contrast to the multiple defensins, only one cathelicidin gene, CAMP (cathelicidin antimicrobial peptide), has been found in humans [8, 9]. The gene product human cationic antimicrobial peptide-18 (hCAP18) is transcribed from the CAMP gene that contains vitamin D response elements (VDRE) in its promoter and requires enzymatic digestion by human neutrophil proteinase 3 (PR3) to produce mature LL-37 that exhibit broad antimicrobial activity [10]. LL-37 is an amphipathic α-helical peptide that binds to negatively charged groups of the bacterial outer membrane causing disruption of the bacterial cell wall [11]. Previously, we and other studies have shown that LL-37 is able to kill both non-pathogenic and pathogenic mycobacteria both in vitro and in vivo conditions [12, 13, 14].
Previously, it has been shown that vitamin D strongly up regulates the expression of cathelicidin [15]. The proposed model suggested that toll-like receptor-2/1 (TLR2/1) activation of macrophages induce the expression of CYP27B1 (25-hydroxyvitamin D-1α-hydroxylase) which, in turn, leads to the production of bioactive 1, 25(OH)2D from circulating inactive 25(OH) D [16]. The production of 1, 25(OH)2D activates CAMP gene expression through vitamin D receptor [17].
Bacterial lipoproteins are known to perform diverse functions such as host cell adhesion, cell invasion, nutrient transport, drug resistance and evasion of host defense mechanisms [18]. For example, Streptococcus pneumoniae PsaA lipoprotein is involved in the colonization of host cells and antibiotic resistance [19,20]. Borrelia burgdorfori VlsE lipoprotein is involved in bacterial persistence in host cells [21] and Haemophilus influenzae P6 lipoprotein activate host immune cells by inducing the secretion of pro-inflammatory cytokines through TLR-2 binding [22]. Similarly, bacterial lipoproteins have been shown to stimulate proliferation of B cells leading to increased immunoglobulin secretion [23]. Thus, bacterial lipoproteins are able to activate both the innate and adaptive wings of the immune system. Mycobacterium genome encodes for 99 lipoproteins, however, the functions of many of them are still unknown. Several of them have been shown to play a major role in virulence [16, 24, and 25]. Mtb PstS-1, a 38 kDa lipoprotein, is involved in phosphate transport as well as in inducing apoptosis [25]. Deletion mutants of Mtb lgt and lsp showed a significant reduction in virulence [26]. Similarly, an extensively studied Mtb 19-kDa lipoprotein (LpqH) has been shown to strongly induce the innate immunity by activating cathelicidin mediated autophagy in a TLR-2 dependent manner [24,16]. LpqH binds to TLR-2 that leads to activation of cytokines such as interleukin-12 (IL-12), interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α). Moreover, LpqH also plays a pivotal role in bacterial survival by inhibiting interferon-gamma (IFN-γ) response genes such as CIITA (Class II trans activator) that leads to reduced antigen presentation [24]. Another study has shown that a synthetic 19-kDa Mtb derived lipopeptide enhances the antimicrobial capacity of monocytes via TLR2/1 signaling, vitamin D and VDR-dependent pathway [27]. This involved induction of the CAMP gene and its protein [28]. Some of the lesser studied mycobacterial lipoprotein genes include LprG, LprA, LpqB, and LpqM.
In the present study, we have shown that one of the Mtb lipoproteins, LprE (LprEMtb), is involved in intracellular bacterial survival and evasion of host immune responses. We demonstrate that LprEMtb enhances bacillary survival by inhibiting the expression of CAMP via TLR-2-p38-Cyp27B1-VDR signaling pathway in human macrophages. We also demonstrate that LprEMtb downregulates the pathogen induced IL-1β pro-inflammatory response. Furthermore, mechanistic studies showed that LprEMtb inhibits autophagy and phago-lysosome fusion by down-regulating the expression of several endosomal markers such as Early Endosome Antigen 1 (EEA1), Rab7 and lysosomal-associated membrane protein 1 (LAMP-1), and IL-12 and IL-22 cytokines, thus aid bacterial survival in human macrophages. In summary, we have characterized a novel Mtb lipoprotein that aids bacterial survival in host macrophages by down-regulating the expression of antimicrobial peptide cathelicidin.
RESULTS
Genetic organization of LprE in M. tuberculosis genome
Initial annotation from the Tuberculist Web server suggested that Mtb H37Rv LprE (LprEMtb), encoded by a 609 bp Rv1252c gene, belongs to a previously uncharacterized Rv_dir301 operon (webTB.org database). This operon consists of yet another uncharacterized Rv1251c gene (Fig 1A). In Mtb CDC1551, a clinical isolate, LprE is encoded by an MT1291 gene (Fig 1B). Multiple sequence alignment and BLAST results showed 100% sequence homology between Mtb H37Rv and Mtb CDC1551 LprE genes (Suppl Fig 1).The tertiary structure of LprE is not available. Therefore, we predicted its structure using a Modeller program. The LprE sequence showed high structural homology (94.5%) with human mitochondrial RNA polymerase (PDB ID: 3SPA) (Fig 1C). Domain analysis by ProDom program (http://www.http://prodom.prabi.fr/) showed the presence of a conserved lipoprotein binding domain at amino acids 81-201 position (data not shown). To further confirm that LprE is a lipoprotein, we aligned LprEMtb with Escherichia coli outer membrane lipopolysaccharide transport lipoprotein LptE (PDB ID: 4NHR). We observed 100% homology between lipoprotein binding domains of LprEMtb and E. coli LptE proteins (Fig 1D).
Deletion of LprE in M. tuberculosis CDC1551 decreased bacterial survival in human macrophages
Pathogenic mycobacteria employ various strategies to survive and replicate inside the host cells. Earlier reports showed that Mtb lipoproteins play a crucial role in the intracellular survival of bacteria and that deletion of lipoproteins decrease Mtb virulence [26, 29]. Therefore, we investigated the role of LprEMtb in bacterial survival. Towards this, THP-1 cells were infected with Mtb, MtbΔLprE, and the complemented MtbΔLprE::LprE strains. Cells were lysed at 12, 24, 48 and 72 h post infection and the intracellular bacterial survival was determined. While Mtb and MtbΔLprE::LprE strains showed a time dependent increase in intracellular bacterial burden, significant decrease was observed when THP1 cells were infected with MtbΔLprE (Fig 2A). These results suggest a pivotal role for LprEMtb in the survival of Mtb inside macrophages.
To further confirm the role of LprE in bacterial survival, LprEMtb was episomally expressed in non-pathogenic Msm (Msm::LprE) with the help of pSMT3 shuttle vector. Several studies including our previous work have used Msm as a surrogate model to study the role of various Mtb proteins in pathogenesis [30–34]. The qRT-PCR analysis confirmed that LprE is ectopically expressed in Msm strain grown under in-vitro condition (Fig 2B). As shown lprEMtb level reached a maximum at 24 h followed by a gradual decrease in expression level upto 72 h. The transcript levels were normalized with Msm housekeeping sigA at 2 h and the fold changes were calculated.
Msm genome analysis showed presence of MSMEG_5043, an ortholog of LprEMtb [35]. To preclude the effect of MSMEG_5043, we first constructed MsmΔ5043 mutant by allelic exchange method (Fig 2C). Deletion of MSMEG_5043 was confirmed by PCR using gene specific and flanking region primers (Suppl Fig 2A). MsmΔ5043 strain was electroported with pSMT3-LprEMtb construct to generate a MsmΔ5043::LprE complementation strain. We did not observe significant difference in the in vitro growth pattern of Msm::pSMT3, Msm::LprE, MsmA.5043 and MsmΔ5043::LprE strains (Suppl Fig 2B). These results are consistant with the growth pattern observed for Mtb, MtbΔLprE, and MtbΔLprE::LprE strains (data not shown), suggesting that the presence or absence LprEMtb does not impact on the bacterial growth kinetics. In contrast, when compared with Msm::pSMT3 and MsmΔ5043, the intracellular survival of Msm::LprE and MsmΔ5043::LprE strains in THP1 cells was 10-15 fold higher after 24 and 48 h of postinfection (Fig 2D). However, intracellular count of MsmΔ5043::LprE was observed to be 3 fold less when compared to Msm::LprE 48 h post infection (p<0.0001; Fig 2D). To corroborate above results, we determined the intracellular survival of bacteria 24 h post infection with the help of flow cytometry. Cells were harvested 24 h post infection, lysed to release the intracellular bacteria and stained with propidium iodide (PI), a non-permeable nucleic acid binding dye that binds specifically to dead or with disintegrated cell wall bacteria. Flow cytometry analysis showed less PI stained Msm::LprE and MsmΔ5043:: LprE bacteria as compared with Msm::pSMT3 and MsmΔ5043 strains (Fig 2E), indicating less death in LprEMtb expressing Msm strains.
Consistent with above results Msm::LprE and MsmΔ5043::LprE also showed increased intracellular survival in human monocyte derived macrophages as compared to Msm::pSMT3 and MsmΔ5043 strains (Fig 2F). We did not observe any difference in the phagocytosis rate of Msm::pSMT3, Msm::LprE, MsmΔ5043 and MsmΔ5043::LprE strains (Fig 2G) indicating that the increased survival of Msm::LprE and MsmΔ5043::LprE strains is not due to the difference in the uptake of bacteria by macrophages.
LprEMtb inhibits cathelicidin expression in human macrophages
Macrophages exhibit antimicrobial response by inducing the expression of cationic antimicrobial peptide cathelicidin (hCAP18/LL-37) [36]. However, Mtb is known to down regulate the expression of cathelicidin to avoid killing by macrophages [37]. Previously, it has been shown that 19-kDa lipoprotein from Mtb interacts with TLR-2, which subsequently up-regulates the expression of Cyp27B1 hydroxylase, VDR translocation into the nucleus and finally the induction of cathelicidin expression [14]. Our previous study also showed that cathelicidin is able to kill both pathogenic and non-pathogenic mycobacteria under in vitro and ex vivo conditions [13]. Therefore, we first evaluated the expression of CAMP, which encodes human cathelicidin, in Mtb, MtbΔLprE, and MtbΔLprE::LprE infected THP-1 cells. We observed an ~4-fold increase in CAMP expression in cells infected with MtbΔLprE compared with Mtb and MtbΔLprE::LprE infected cells (Fig 3A). In contrast, Msm::LprE and MsmΔ5043::LprE infection of THP1 cells resulted significant down-regulation of CAMP expression when compared with Msm::pSMT3 and MsmΔ5043 infected cells (Fig 3B). These results suggest that LprEMtb is involved in the inhibition of cathelicidin expression, which in turn promotes the bacterial survival. To further validate the role of cathelicidin, we evaluated bacterial survival in THP-1 cells treated with 50 μg/ml of LL-37 2 h before (pre-treatment) and after (post-treatment) the infection. Earlier we have shown that treatment with cathelicidin (50 μg/ml) would eliminate Mtb under in vitro and ex vivo conditions without exhibiting any toxic effect on macrophages [13]. Furthermore we established that Msm elimination is dependent on cathelicidin expression [13]. In agreement with our previous data, we observed increased killing of Msm::LprE and MsmΔ5043::LprE under both pre and post-treated conditions in comparison with untreated cells (Fig 3C). However, more bacterial killing was observed under post-treated condition. As expected treatment with 50 μg/ml LL-37 did not show any cytotoxic effect on THP-1 cells (data not shown).
Vitamin D3 is a known inducer of cathelicidin, which results in increased bacterial killing in human macrophages [38]. As shown in Figure 3D, treatment with 20 nM Vitamin D3 further reduced Msm::pSMT3 and MsmΔ5043 survival compared with untreated cells (Fig 3D), whereas we observed no difference in the cfu’s of Msm::LprE and MsmΔ5043::LprE (Fig 3D), suggesting that LprEMtb impedes the vitamin D3 mediated response.
LpqHMtb and LprEMtb antagonostically regulate CAMP expression
Mycobacterium genome encodes for 90 lipoproteins. Among them, 19-KDa lipoprotein LpqHMtb has been shown to induce cathelicidin expression to restrict intracellular mycobacterial growth [16]. Therefore, we investigated the impact of LpqHMtb on the modulation of CAMP expression in presence of LprEMtb. Towards this, we co-expressed LpqHMtb and LprEMtb in Msm to generate a recombinant Msm::LprE-LpqH strain. Msm expressing LpqHMtb (Msm::LpqH) and LprEMtb (Msm::LprE) were used as control strains. In agreement with the previous report, we also observed increased CAMP expression in Msm::LpqH infected cells [16], whilst Msm::LprE infection inhibited CAMP expression. However, infection with LpqHMtb and LprEMtb co-expressing Msm strain (Msm::LprE-LpqH) showed the CAMP level in between LpqHMtb and LprEMtb expressing Msm strains (Fig 3E), suggesting that both LpqH and LprE are strong regulators of cathelicidin expression. Moreover, Msm::LpqH and Msm::LprE-LpqH strains showed decreased survival in comparison with Msm::LprE strain after 24(p<0.05) and 48 (p<0.0001) h of infection (Fig 3F).
LprEMtb inhibits cathelicidin via p38 MAPK pathway in macrophages
Based on above observations, our subsequent studies were focused on the investigation of the molecular mechanism(s) that are responsible for the down-regulation of CAMP expression by LprEMtb. It has been reported that LpqHMtb and TLR-2 interaction regulate CAMP expression via activation of downstream p38 MAPK signaling pathway [14]. Mycobacterial antigen-TLR 2 interaction activates p38 MAPK signaling pathway dependent modulation of 25-hydroxyvitamin D–1α-hydroxylase (CYP27B1), which convert inactive 25(OH) D to active form 1,25(OH)2D of vitamin D [27]. The latter then binds to vitamin D receptor (VDR) to activate the transcription of the CAMP gene (Fig 3G). We observed a significant increase in the expression of phospho-p38 in MtbΔLprE infected cells when compared with Mtb and MtbΔLprE::LprE at 6 h post-infection, however, no such significant difference was observed at 12 h post-infection (Fig 3H) indicating that LprEMtb modulates phospho-p38 during the early infection process. Similarly, phospho-p38 expression was found to be moderately decreased in Msm::LprE and MsmΔ5043::LprE infected macrophages as compared with MsmΔ5043 at 6 h post infection (Suppl Fig 3A). This is in agreement with the previous study that Mtb infection down-regulates phospho-p38 [39].
LprEMtb down-regulates CYP27B1 and VDR expression in macrophages
Next, we studied the expression of cathelicidin regulation pathway intermediates CYP27B1 and the VDR. Increased expression of CYP27B1 was observed at both transcriptional (Fig 3I) and translational levels (Fig 3J) in MtbΔLprE infected macrophages as compared to Mtb and MtbΔLprE::LprE infected cells. Of note, CYP27B1 expression was found to be increased both at 12 and 24 h post-infection (Fig 3J). On the other hand, the expression of CYP27B1 was found to be significantly down-regulated at both transcriptional (Suppl Fig 3B) and translational (Suppl Fig 3C) levels in Msm::LprE and MsmΔ5043::LprE infected cells relative to Msm::pSMT3 and MsmΔ5043 infected macrophages.
Similarly, the expression of VDR was found to be significantly up-regulated at both transcriptional (Fig 3K) and translational (Fig 3J) levels in MtbΔLprE infected macrophages as compared to Mtb and MtbΔLprE::LprE infected cells. In contrast, infection with Msm::LprE and MsmΔ5043::LprE strains down-regulated VDR expression at both transcriptional (Suppl Fig 3D) and translational (Suppl Fig 3E) levels. Altogether these results indicate that LprEMtb inhibits the expression of CAMP through down-regulation of p38-Cyp27B1-VDR signaling pathway to augment bacterial survival in macrophages.
LprEMtb down-regulate the expression of CAMP via TLR-2
Previous studies have shown that lipoproteins regulate cathelicidin expression through TLRs [40,41]. To identify the involvement of specific TLR in the regulation of CAMP expression, we first determined in-silico binding efficiency of LprEMtb with human TLR-1,2,4 and 6. For this, we first performed docking analysis of energy minimized LprEMtb protein with human TLR’s tertiary structure obtained from PDB database. LprEMtb showed strong binding efficiency with TLR-2 (-582.21) as compared to TLR-1 (-223.5), TLR-4 (2.89) and TLR-6 (-10.59) (Suppl Fig 4A), as determined by atomic contact energy (ACE) score obtained from PatchDock analysis [42].
To validate the in-silico data, we silenced TLR’s using siRNAs specific to human TLR-1,2,4 and 6 in THP-1 cells. Semi-quantitative RT-PCR analysis showed more than 80% knock-down efficiency in siRNA treated cells (Fig 4A). Then untreated, scrambled and TLR specific siRNA treated THP-1 cells were infected with Msm::pSMT3, MsmΔ5043 and MsmΔ5043::LprE bacteria and intracellular bacterial survival were determined. Interestingly, we observed a substantial decrease in the survival of MsmΔ5043::LprE in TLR-2 silenced macrophages as compared with TLR-1, 4 and 6 silenced macrophages (Fig 4B). Moreover, MsmΔ5043::LprE bacterial count was found to be approximately 1.5-fold less in TLR-1 siRNA treated cells in comparison with TLR-4 and 6 silenced macrophages (Fig 4B). This correlates well with the high ACE score observed in docking studies of LprEMtb with TLR-1 in comparison to TLR-4 and 6. We also checked the expression of CAMP, Cyp27B1 and VDR in TLR-1,4,2 and 6 silenced plus Msm::pSMT3, MsmΔ5043 and MsmΔ5043::LprE infected macrophages. We observed significant increase in the expression of CAMP, Cyp27B1 and VDR (Fig 4C-E) in MsmΔ5043::LprE infected samples when TLR-2 was silenced in comparison with scrambled siRNA treated and untreated cells. No difference in the expression of CAMP was observed in TLR-1,4 and 6 silenced macrophages (Suppl Fig 4B). Consistent with the above data MsmΔ5043::LprE infection still showed down-regulation of CAMP in TLR-1, 4 and 6 silenced macrophages (Suppl Fig 4B). These results unambiguously suggest that LprEMtb regulates CAMP expression through TLR-2 signaling pathway.
LprEMtb inhibits the production of pro-inflammatory cytokine IL-1β
IL-1β cytokine induces the expression of antimicrobial peptides through TLR signaling to clear the bacterial infection [43]. Active TB patients showed reduced levels of IL-1β suggesting that it may have a protective role in TB infection [44]. Next, we investigated the expression of IL-1β in Mtb, MtbΔLprE, and MtbΔLprE::LprE infected macrophages. qRT-PCR analysis showed significant up-regulation of IL-1β level in MtbΔLprE infected macrophages in comparison with Mtb and MtbΔLprE::LprE infected cells after 12 (p<0.0001) and 24 h (p<0.0001) of infection (Fig 5A). However, no difference in the expression levels of IL-1β was observed after 48 h of infection. In contrary, LprEMtb expressing Msm::LprE and MsmΔ5043::LprE infected cells showed reduced expression of IL-1β at both 12 and 24 h post-infection (Fig 5B).
In macrophages, processing and release of active IL-1β are dependent on caspase-1 activation [45]. Therefore, we investigated whether LprEMtb mediated IL-1β down-regulation is dependent on caspase activation. Indeed, western blot analysis showed an increased level of cleaved caspase-1 in MtbΔLprE infected macrophages when compared with Mtb and MtbΔLprE::LprE infected cells after 12 h of infection (Fig 5C). Together data suggests that LprEMtb suppresses caspase-1 dependent IL-1β production to facilitate bacterial persistence in macrophages.
LprEMtb inhibits autophagy to dampen host immune response
Autophagy is a known host defense mechanism that plays an important role in the restriction of Mtb growth [46. To investigate if LprEMtb modulates autophagy, we evaluated the expression of several autophagic markers such as microtubule-associated protein 1 light chain 3 (LC3), ATG-5 and Beclin-1 in Mtb, MtbΔLprE and MtbΔLprE::LprE infected THP-1 cells. Western blot analysis showed the increased conversion of LC3-I to a characteristic autophagic induction marker LC3-II in MtbΔLprE infected cells (Fig 6A).We also examined the expression of an autophagic flux marker p62(SQSTM1) [47]. As shown in Fig 6A, p62 level decreased in case of MtbΔLprE infected macrophages (Fig 6A). Moreover, the expression of other autophagic markers such as Atg-5 and Beclin-1, which are recruited to the phagosomal compartments during autophagic vesicle formation [48], were also found to be increased in MtbΔLprE infected cells as compared to Mtb and MtbΔLprE::LprE infected cells (Fig 6B).
To further confirm the role of LprEMtb in autophagy inhibition, we also checked the expression of LC3, p62, Atg-5 and Beclin-1 in Msm::pSMT3, Msm::LprE, MsmΔ5043 and MsmΔ5043::LprE infected macrophages. Consistent with above results, infection with both LprEMtb expressing Msm::LprE and MsmΔ5043::LprE strains significantly reduced the expression of LC3, Atg-5 and Beclin-5 (Suppl Fig 5A & B). On the other hand, p62 expression was found to be upregulated under similar conditions. Rapamycin is a known inducer of autophagy mechanism. Next, we checked if autophagy induction by rapamycin treatment exerts any impact on the outcome of bacterial survival. For this, THP-1 cells were pre-treated with rapamycin (50 nM) followed by infection with Msm::LprE and MsmΔ5043::LprE strains and the intracellular bacterial count was determined after 24 h of infection. Rapamycin treatment significantly reduced the survival of Msm::LprE and MsmΔ5043::LprE, which otherwise showed more survival by down-regulating the expression of autophagy, as compared to untreated cells (Fig 6C). We confirmed that rapamycin treatment indeed induced autophagy by determining the expression of LC3-II and Atg-5 by western blots analysis (Fig 6D).
LprEMtb blocks phago-lysosome fusion by down-regulating the expression of endosomal markers
Non-pathogenic mycobacteria containing phagosomes readily fuse with lysosomes leading to the elimination of pathogen; however, pathogenic mycobacteria inhibit the fusion of phagosomes with lysosomes to survive in macrophages for an extended period of time. Previously, we have shown that exogenous administration of cathelicidin increased the colocalization of M. bovis-BCG containing phagosomes with lysosomes resulting in increased bacterial killing [11]. Based on these observations, we extended our study to investigate whether LprEMtb also affects the phagosome maturation and subsequently phago-lysosome fusion. For this, we checked the expression of early endosomal antigen 1 (EEA1), Rab7 and lysosomal associated marker protein-1 (LAMP-1) proteins, which are the hallmarks of early, late and lysosomal compartments, in THP-1 cells infected with Mtb, MtbΔLprE, and MtbΔLprE::LprE strains. It has been shown that recruitment of these marker proteins on endocytic compartments is necessary for the sequential maturation of endosomes and phagolysosome fusion [49,50]. Western blot analysis showed a significant increase in the levels of EEA1, Rab7 and LAMP-1 in MtbΔLprE infected cells in comparison to Mtb and MtbΔLprE::LprE infected cells (Fig 7A &B). On the other hand, macrophages infected with LprEMtb expressing Msm strains showed decreased expression of these endosomal markers (Suppl Fig 6A).
LprEMtb arrest phago-lysosome maturation by down-regulating the expression of IL-12 and IL-22 cytokines
Upon mycobacterial infection cytokines strongly influence the outcome of infection. Cytokines individually or in combined fashion create a microenvironment that assists in the control of mycobacteria. Several cytokines have been shown to promote acidification of phagosomes to facilitate phago-lysosomal fusion and thus bacterial death. Other studies have shown that interleukin-12 (IL-12) and IL-22 cytokines play a critical role in phagosome maturation by promoting an acidification of the bacteria containing phagosomes [51,52]. IL-22 has been shown to enhance Rab7 expression in Mtb infected macrophages, thus enhancing phago-lysosomal fusion. Therefore, we investigated the expression of these two cytokines in infected THP-1 cells. As can be seen in Figure 7C transcript levels of IL-12 was elevated in a time dependent manner in macrophages infected with MtbΔLprE as compared to Mtb and MtbΔLprE::LprE strains. On the contrary, Msm::LprE infected cells showed reduced IL-12 expression as compared to vector control Msm::pSMT3 infected cells (Suppl Fig 6B). The IL-22 level also increased by more than 2-fold in macrophages infected with the MtbΔLprE (Fig 7D), while Msm::LprE and MsmΔLprE::LprE infected condition significantly reduced IL-22 transcript levels (Suppl Fig 6C). These results indicate that LprEMtb probably blocks phagosomal maturation by down-regulating the expression of IL-12 and IL-22 cytokines.
DISCUSSION
Mtb exhibits an extraordinary ability to survive inside the host cells. This is mainly attributed to the plethora of virulence factors produced by the bacterium. One of the most important classes of virulence factors produced by Mtb is lipoproteins. Of the 99 lipoproteins predicted in the mycobacterium genome, only a few of them have been functionally characterized. In this study, we have characterized one of the lipoproteins LprE, encoded by Rv1252c in H37Rv and MT1291 in CDC1551 strains, and investigated its role in mycobacterial virulence. Analysis of genetic organization of Mtb genome showed that Rv1252c is a part of a previously uncharacterized Rv_dir301 operon. Sequence analysis revealed 100% homology between lprE gene from H37Rv and CDC1551 strains. LprE structural analysis showed the presence of alpha helices, random coils and N-terminal lipoprotein binding domain between 81-201 amino acids. Further domain analysis showed a perfect match between the binding domain sequences of LprEMtb and LptE lipoprotein from E.coli. The previous study by Sutcliffe et.al. also predicted LprEMtb as a lipoprotein on the basis of the presence of G+Lpp pattern match, Lipobox and type II signal peptide sequence [53]. These data provide sufficient evidences that LprEMtb could be a lipoprotein. However further biochemical analysis is required to confirm that LprEMtb is indeed a lipoprotein.
Mtb is considered as one of the most successful pathogens due to its ability to evolve with numerous survival strategies. However, non-pathogenic Msm is readily killed by macrophages. Here, we employed two strategies to understand the role of LprE in Mtb pathogenesis. First, we used a Mtb LprE deletion mutant (MtbΔLprE). Secondly, we used Msm as a surrogate model to express LprEMtb (Msm::LprE). To ensure that the observed phenotype is indeed due to ectopic expression of LprE in Msm, we deleted MSMEG_5043, a LprEMtb orthologue present in Msm genome, to generate MsmΔ5043 strain. Infection assays with primary human macrophages and THP-1 cells showed that lprEMtb expression increased the survival of Msm, while deletion of LprE significantly reduced the Mtb survival in macrophages suggesting that LprEMtb is involved in bacterial persistence in macrophages.
Mtb employs various immune evasion strategies to survive inside the macrophages. The antimicrobial peptide cathelicidin/LL-37 is known to play a significant role in innate response against Mtb infection [14,54]. Gutierrez et al. (2008) have shown that Msm infected macrophages differentially expressed a set of genes including cationic AMPs [55]. Our previous study has shown that Msm infection up-regulated the expression of cathelicidin in macrophages that led to the intracellular killing of bacteria. Moreover, we and others have shown that cathelicidin is able to kill M. bovis-BCG and Mtb H37Rv under in-vitro and ex-vivo growth conditions [13]. We observed that Msm::LprE and the complemented MsmΔ5043::LprE strains survived better in human macrophages by down-regulating the expression of cathelicidin, while deletion of LprE de-repressed the cathelicidin expression thereby resulting in increased Mtb killing. External supplementation of purified LL-37 peptide decreased Msm::LprE and MsmΔ5043::LprE bacterial counts confirming that the increased survival was due to LprEMtb mediated down-regulation of cathelicidin levels. Transcriptional down regulation of CAMP during bacterial infection was suggested as a novel mechanism to escape the immune responses [56,57,58]. LprEMtb was also found to inhibit the expression of cathelicidin signaling pathway intermediates such as CYP27B1 and VDR to subvert the Vitamin D3 mediated bacterial killing. Together, these findings argue that LprEMtb is responsible for the down-regulation of cathelicidin thus promoting bacterial survival in macrophages. Unlike LprE, LpqHMtb was shown to induce the expression of cathelicidin [16]. Infection assays with Msm strain co-expressing both LpqHMtb and LprEMtb (Msm::LprE-LpqH) showed intermediate survival and cathelicidin expression indicating that both LpqHMtb and LprEMtb are antagonistic regulators of cathelicidin expression.
One of the proposed mechanisms for cathelicidin induction is that bacterial exposure leads to activation of downstream p38 MAPK signaling pathway [16,54], which subsequently up regulates CYP27B1 hydroxylase, VDR and finally cathelicidin [16]. Indeed, our results demonstrated that MtbΔLprE infection caused up-regulation of p38 MAPK, CYP27B1, and VDR. On the other hand infection with LprE expressing Mtb, MtbΔprE::LprE, Msm::LprE and MsmΔ5043::LprE strains down-regulated p38 MAPK, CYP27B1, and VDR. These results suggest that LprEMtb mediate cathelicidin suppression via inhibition of p38-Cyp27B1-VDR signaling pathway. Previous reports have shown that few intracellular bacteria have the ability to dysfunction the vitamin D receptor, thus make host cells more susceptible to bacterial infections [59,60]. We observed that LprEMtb inhibits VDR and that treatment with an active form of Vitamin D3 further increased the killing of LprEMtb expressing mycobacterial strains indicating a possible mechanism of immunosuppression employed by the bacteria for intracellular survival.
Pro-inflammatory cytokine IL-1β is known for a protective immune action against mycobacteria [61]. IL-1β functions in synergy with LL-37 to augment the host immune responses against pathogenic bacteria [62]. Expression of LprEMtb decreased IL-1β levels, while deletion of LprEMtb was found to de-repress IL-1ß level in caspase-1 dependent mechanism. Thus, our data indicate that LprEMtb modulates IL-1β levels possibly in tandem with LL-37 downregulation to provide a favorable niche for bacterial survival. However, further mechanistic studies are required to establish the correlation between cathelicidin and IL-1β modulation by LprE.
Lipoproteins are known to trigger innate immune responses by binding to pattern recognition receptors, specifically TLR’s [63,64,65]. LprA, LpqH, and LprG are the three mycobacterial lipoproteins known to interact with TLR-2. Our current study has provided sufficient evidences that LprEMtb has a high binding affinity towards TLR-2 in comparison to TLR-1, 4 and 6. To further confirm the involvement of TLR-2, we checked bacterial survival and the expression of CAMP, Cyp27B1 and VDR in TLR 1, 2, 4 and 6 silenced macrophages. We observed a significant increase in the expression of CAMP, Cyp27b1 and VDR that result in decreased survival of LprE expressing mycobacterial strains in TLR-2 silenced macrophages indicating that LprEMtb mediated inhibition of cathelicidin expression is a TLR-2 dependent phenomenon.
Many pathogenic bacteria, including Mtb, inhibit autophagy mechanism to facilitate its persistence in host cells [66]. Several bacterial virulence proteins were found to inhibit the autophagy. LpqHMtb activates autophagy, whereas EISMtb (enhance intracellular protein) protein suppresses autophagy [67]. Our previous work also showed that a Mtb phosphoribosyltransferase suppressed autophagy expression to promote intracellular bacterial survival [32]. In the current study, we found that LprEMtb is also responsible for the suppression of autophagy as observed by the significant up-regulation of autophagic markers LC3, Atg-5, and Beclin-1 in cells infected with LprEMtb deletion mutant. These results indicate that LprEMtb suppress Vitamin D3 responsive cathelicidin, which may subsequently cause autophagic inhibition. Previous studies have shown that cathelicidin plays a role in the regulation of autophagy [68]. Altogether, inhibition of these antibacterial effector molecules by Mtb aids bacterial persistence inside macrophages.
One of the prominent mechanisms employed by Mtb to survive in macrophages is to block the phagosome maturation by inhibiting the recruitment of EEA1, Rab7, and LAMP-1, which are essential for the development of early and late endosomes and finally phagolysosome fusion. We have previously shown that exogenous administration of LL-37 increases the co-localization of Msm and M. bovis-BCG containing phagosomes with lysosomes [13]. Here, we found that infection with LprEMtb expressing strain decreased the expression of EEA1, LAMP1, and Rab7 in THP-1 cells, while MtbΔLprE infection induced the expression of these proteins indicating that LprEMtb supports bacterial survival by preventing phago-lysosome fusion. L. monocytogenes lipoproteins have also been shown to play an important role in the phagosomal escape of bacteria [69].
Stimulation of macrophages with cytokines restricts the growth of microorganisms within endocytic compartments [70]. It is shown that treatment of human macrophages with IL-12 restrict the growth of M. bovis-BCG by facilitating the phago-lysososome fusion [51]. Similarly, treatment with IL-22 was shown to increase the expression of endocytic marker proteins and increased bacterial killing by inducing phago-lysosomal fusion [52]. In our study, we observed decreased levels of IL-12 and IL-22 in cells infected with LprEMtb expressing mycobacteria, while expression of these cytokines was found to be increased in MtbΔLprE infected cells. These results indicate that LprEMtb mediates phagosomal arrest by down-regulating the expression of IL-12 and IL-22 cytokines. Moreover, lower levels of cathelicidin and IL-1β also explain the inhibition of phagosome maturation and fusion with lysosomes.
Collectively, our findings identified a new Mtb lipoprotein that could be used by pathogenic mycobacteria to alter host immune responses thus presenting attractive targets for new drug therapies. Figure 8 shows a schematic representation of modulation of different host immune responses by LprEMtb leading to increased bacillary survival in macrophages.
MATERIALS AND METHODS
Ethics and Biosafety Statement
All experiments were approved by the Institutional Biosafety committee of KIIT University (vide DBT memorandum No-BT/BS/17/493/2012-PID). All the bacterial mutants were handled in adherence to experimental guidelines and procedures approved by the Institutional Biosafety Committee (IBSC) of School of Biotechnology, KIIT University (KIIT/3-12).
In-silico analysis
Bacterial gene sequences were obtained from Tuberculist (http://tuberculist.epfl.ch/) and Uniprot (http://www.uniprot.org/) databases. Protein secondary structure was predicted using SOPMA secondary structure based method (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html). For tertiary structure prediction, LprEMtb (Rv1252) sequence was BLAST against Protein Data Bank (PDB) to obtain a suitable template. Structure showing highest homology with human mitochondrial RNA polymerase (PDB ID: 3SPA) was chosen as a suitable template. Using this template, tertiary structure of LprEMtb was depicted using Modeller9.12 [71]. The modelled LprEMtb structure was energy minimized and validated using ModRefiner (http://zhanglab.ccmb.med.umich.edu/ModRefiner/) and RAMPAGE (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php) web based servers. LprEMtb domain analysis was done using Pfam (http://pfam.xfam.org/) database and visualized using PyMOL software v1.7.4 (http://www.pymol.org). Human toll like receptor-1,2, 4 and 6 structures were obtained from Protein Data bank (http://www.rcsb.org/pdb/home/home.do). Molecular docking of the structures was done using PatchDock server (http://www.bioinfo3d.cs.tau.ac.il/PatchDock/) and visualized using PyMOL. The Atomic surface energy was determined to analyze the protein-protein binding intensity.
Bacterial strains, cell lines, and reagents
M. tuberculosis CDC1551 (NR-13649) and M. tuberculosis CDC1551 LprE mutant (NR-17996) were obtained from BEI Resources, USA. All Mtb strains were grown in Middlebrook 7H9 broth or on Middlebrook 7H10 plates (Difco, Sparks, Maryland, USA) as described previously [32–34]. Escherichia coli XL-10 Gold (Stratagene, San Diego, California, USA) was grown in Luria-Bertani (LB) broth supplemented with 20 μg/ml tetracycline. pSMT3 vector was a kind gift from Dr. Rakesh Sharma (IGIB, Delhi). THP-1 cells were maintained in RPMI Glutamax (Gibco, Waltham, MA, USA) medium supplemented with 10% fetal bovine serum (FBS) (Gibco) and penicillin-streptomycin solution (Gibco) at 37 0C and 5% CO2.Vitamin D3 and tetracyline were purchased from Sigma Aldrich (USA). TRIzol was purchased from Invitrogen (Carlsbad, California, USA). Complementary DNA (cDNA) synthesis kit was procured from Fermentas (USA). Trypsin-EDTA, SDS, Tris-HCl, and NaCl were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). siRNA oligos were designed and manufactured by GeneX India Bioscience Pvt. Ltd (Chennai, India). All the cloning reagents and restriction enzymes were obtained from NEB (Ipswich, MA, USA).
Construction of M. tuberculosis CDC1551 LprE strain
For the construction of complemented strain, LprE was amplified from Mtb CDC1551 genomic DNA using gene specific primers (Table 1) and was cloned into NdeI-Hind III sites of the pVV16 vector [72] and pNiT vector [72]. The pVV16-LprE and pNit-LprE constructs were transformed into MtbΔLprE mutant to generate MtbΔLprE::LprE complemented strain.
Construction of recombinant M.smegmatis strain expressing LprEMtb and LpqHMtb
LprEMtb and LpqHMtb were PCR amplified using gene specific primers (Table 1) using genomic DNA as a template. The PCR amplified products were purified from the gel, double digested with PstI and HindIII and cloned into pSMT3 shuttle vector separately. The recombinant constructs were transformed into competent E. coli XL-10 gold. LB agar plates supplemented with 20 μg/ml tetracycline and 50 μg/ml hygromycin was used to select the positive colonies and were further confirmed by colony PCR and sequencing using gene specific primers. Finally, the recombinant constructs were transformed into electro competent Msm. The positive colonies were selected on 7H10 medium containing 50 μg/ml hygromycin. The positive transformants were confirmed by colony PCR and sequencing using gene specific primers (Table 1).Generation of recombinant Msm::LprE-LpqH strain was confirmed by sequencing using gene specific primers.
Construction of M. smegmatisΔ5043 mutant
The allelic exchange substrate (AES) for the generation of Msm 5043 mutant (MsmΔ5043) was produced as described previously [72]. Briefly, approximately 800 kb regions upstream and downstream of MSMEG_5043 loci were PCR amplified using specific primers (Table 1). The amplicons were digested with DraIII-HF (NEB) and ligated with two compatible fragments (OriE+cosλ and Hygres) from pYUB1474 [73] and pVV16 [72], respectively to generate allelic exchange substrate (AES). MSMEG_5043-AES was linearized with SnaBI to elute upstream-Hyg-downstream flank. AES was electroporated into electro-competent Msm cells. Hygromycin-resistant colonies were screened by PCR to confirm deletion of MSMEG_5043 at its genomic locus (MsmΔ5043). Then LprE-pSMT3 construct was electroporated into MsmΔ5043 mutant to create a complemented MsmΔ5043::LprE strain.
Growth kinetics
Msm::pSMT3 vector control, recombinant Msm expressing LprEMtb (Msm::LprE), MsmΔ5043 and MsmΔ5043::LprE strains were grown in 7H9 broth at 120 r.p.m at 370C with. The growth kinetics was assayed by measuring the O.D at 600 nm (OD600) at regular intervals till death phase was observed. Similarly growth kinetics of Mtb, MtbΔLprE and MtbΔLprE::LprE was determined by measuring OD600 at regular intervals till death phase was observed.
Intracellular survival assay
Mtb, MtbΔLprE, and MtbΔLprE::LprE, Msm::pSMT3, Msm::LprE, MsmΔ5043 and MsmΔ5043::LprE strains were grown to mid-exponential phase. The bacterial cultures were pelleted at 5000 g for 5 min, washed twice with 1X phosphate buffer saline (PBS) and resuspended in RPMI medium to adjust the OD600 to 0.1. THP-1 monocytes (2x105cells/well) were differentiated into macrophages by addition of 20 nM phorbol 12-myristate 13-acetate (PMA) and incubated for 24 h. After which the cells are washed with RPMI medium to remove PMA, and then the cells are allowed to differentiate for 3 days at 37 0C and 5% CO2. Cells were infected as described previously at a multiplicity of infection (MOI) 10 [74, 32]. The intracellular bacterial survival was determined by lysing the cells with chilled 0.5% Triton-X 100 (Sigma) at different time points and plating the serially diluted samples on 7H10 medium. Bacterial colonies were enumerated after 3 and 21 days for Msm and Mtb, respectively.
Flow cytometry analysis
THP-1 cells (2x105 cells/well) were seeded on a 24-well plate and treated with PMA as described above. Then cells were infected with Msm::pSMT3, Msm::LprE, MsmΔ5043, and MsmΔ5043::LprE at an MOI 10 as described above. After infection, cells were lysed by treating with 0.5% Triton X-100 for 2 min to release the intracellular bacteria. The cell suspension was centrifuged at 1000 g for 10 min to pellet down the lysed macrophage cells. Supernatants that contained released bacteria were again centrifuged at 12000 g for 10 min. The pellet was washed thrice with 1X PBS, bacterial cells were stained with 30 μM propidium iodide (PI) (Thermo Fisher, P3566) for 5 min in dark and the live cells were analyzed using BD FACSCantoII flow cytometer. Threshold (n=1000) was set on Side Scatter (SSC), and PMT voltages were set using an unstained sample of diluted bacteria grown to log phage. The bacterial population was positioned so that the entire population is on the scale on an FSC vs SSC plot. A total of 10,000 events are acquired and a gate was set according to the stained bacterial sample. Uninfected THP-1 cells stained with PI were also used to rule out cell contamination while analysis.
Phagocytosis assay
THP-1 cells (2x105 cells/well) were infected with Msm::pSMT3, Msm::LprE, MsmΔ5043, and MsmΔ5043::LprE at an MOI 10. 1 and 2 h post infection cells were washed rigorously with 1X PBS to remove un-internalized and membrane adhering bacteria. The cells were lysed with 0.5% Triton X-100 and released intracellular bacteria were plated onto 7H10 plates to count live bacteria. Bacterial preparations were plated before infection to ensure that an equal number of bacteria were used for infection assays.
Isolation of human peripheral blood mononuclear cells (PBMCs)
Blood was collected from healthy individuals, diluted (1:1 in PBS), layered onto an equal volume of Ficoll-Paque plus (GE Healthcare, 17-1440-02) and centrifuged at 100 g for 30 mins. The whitish buffy coat was washed twice with 1x PBS (100Xg, 10 mins). The cell pellet was resuspended in RPMI medium supplemented with 10% FBS. 1x105 cells were seeded onto 24-well for 24 h at 37 °C. Fresh RPMI media containing 5 ng/ml GM-CSF (R&D Systems, McKinley, MN, USA) was added per well and maintained at 37 °C for 72 hours. After differentiation, the cells were infected with mycobacterial strains as described above.
LL-37 treatment
LL-37 peptide (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) was synthesized (GeneXbio, Delhi, India) at a purity of >95%. The peptides were dissolved in 0.01% acetic acid and stored at -20°C until further use. THP-1 cells were treated with LL-37 (50μg/ml) 2 h before (pre-treated) and after (post-treated) infection as described previously [11].
TLR silencing
siRNA duplex against TLR-1, 2,4 and 6 were designed and synthesized (GeneX, New Delhi, India) at 10 nmol concentration. 20 nM siRNAs were electroporated into 106 THP-1 cells. Scrambled siRNA was used as a negative control. The electroporated cells were treated with 20 nM PMA and seeded onto 6 well plates for infection assays. Silencing efficiency was determined using gene specific primers.
Western blotting
All the antibodies except LC3 and CYP27B1 were purchased from Cell Signaling. The expression of phospho-p38 (Cat No-4511), total p38 (Cat No-9212), β actin (Cat No-4970),CYP27B1 (Thermo Scientific, Cat No-CA5 26065), VDR (Cat No-12550), Rab7 (Cat No-2094), EEA1(Cat No-3288), LAMP1 (Cat No-9091), LC3 (Sigma, L7543), SQSTM1/p-62 (Cat No-5114), Atg-5 (Cat No-8540) and Beclin-1 (Cat No-3495) was determined in THP-1 cells infected with Mtb, MtbALprE, MtbALprE::LprE, Msm::pSMT3, Msm:;LprE, MsmΔ5043 and MsmΔ5043::LprE strain as described above. Cells were harvested at the indicated time points by adding 80-100 μl lysis buffer (1M Tris, 2M Nacl, 0.1M EDTA, 100 mM DTT, 1% Triton X100, NasVO4'2H2O, 10% glycerol and PMSF) and stored in -80°C overnight. The lysates were thawed on ice, briefly vortexed for 30 secs for three times and centrifuged at 13,000 g. for 30 mins. The isolated proteins were estimated by Bradford assay, run on SDS-PAGE, electrophoresed and transferred onto PVDF membrane (GE healthcare, Chicago, Illinois, USA) for 90 mins at 50 mA. Then the blots were blocked with either 5% BSA or 5% skimmed milk for 2 h at room temperature. The membranes were then incubated with primary antibodies (1:1000) overnight at 4 °C. The membranes were then washed thrice with 1X PBS containing 1% Tween 20 (PBST) for 5 min. The washed membrane was then incubated with goat anti-rabbit IgG HRP secondary antibody (GeneI merck, 6.2114E14)) for 2 h at room temperature. The membranes were then washed with 1X PBST for three times 5 min each and developed on X-ray film using chemiluminescent solvents. The band densities were quantified by computer scanning of the films and normalizing the values for analysis. Image J2 (NIH, USA) was used for computer analysis of pixel intensity of bands on films. Relative band densities relative to respective loading control were determined.
Quantitative real time PCR
To check the expression of LprEMtb as a function of growth, Msm::pSMT3 and Msm::LprE strains were grown in 7H9 medium. Bacteria were harvested at different time points. Total RNA was isolated by using TRIzol reagent and cDNA was synthesized using Verso cDNA synthesis kit as per the manufacturer’s instructions (Thermo Scientific). qRT-PCR amplification was carried out using gene specific primers and the synthesized cDNA as a template. All reactions were performed in a total reaction volume of 10 μl using SYBR® Green PCR mastermix (KAPA Biosystems), and carried out in Insta Q96 (Himedia, India) with initial denaturation at 95°C for 10 min, final denaturation at 95°C for 30 secs, annealing at 55°C for 30 sec and extension at 72°C for 30 sec to generate 200 bp amplicons. Similarly, the expression of CAMP, Cyp27b1, and VDR, IL-12, IL-22 and IL-1β were measured by isolating RNA from infected THP-1 cells at mentioned time points post infection using gene specific primers (Table 1). The reaction conditions were as follows; initial denaturation at 95 °C for 10 min, final denaturation at 95°C for 30 secs, annealing at 55°C for 30 secs, and extension at 72°C for 30 sec. The mRNA levels were normalized to the transcript levels of sigA and GAPDH and the relative fold changes were calculated. All quantitative PCR experiments were performed for three replicates and the data represented for each sample were relative to the expression level of housekeeping gene calculated using the 2-ΔΔCT method.
Statistical analysis
Statistical analysis was performed with GraphPad Prism v 5.0 (GraphPad Software, La Jolla, California,USA, http://www.graphpad.com). Results are expressed as mean ± SD, unless otherwise mentioned. Significance was referred as *** for P < 0.0001, ** for P < 0.001, * for P < 0.05.
FUNDING INFORMATION
This work was supported by Department of Biotechnology and Indian Council of Medical Research grants, Government of India to Dr. Avinash Sonawane.
CONFLICT OF INTEREST
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
The Senior Research Fellowship (80/902/14-ECDI) awarded to Avinash Padhi from Indian Council of Medical Research (ICMR), Govt. of India is highly acknowledged. We are thankful to all members of Avinash Sonawane, Vinay Nandicoori and Pawan Gupta laboratory for valuable discussions.