Apoptosis: Implications in Viral and Mycobacterium tuberculosis infections

Apoptosis is a form of programmed cell death leading to genetically controlled self-destruction of cells. It is essential in the development, maintenance, and regulation of cells during physiological as well as pathological conditions. Deregulation of apoptotic mechanisms is associated with various pathological diseases including cancer, autoimmune disorders, viral and bacterial infections. Virus and Mycobacterium tuberculosis elicit host cell apoptosis as a part of host immune defense or pathogen dissemination. They inhibit both extrinsic and intrinsic pathways of apoptotic mechanisms facilitating pathogen survival and escape from host immune defense.


Introduction
Apoptosis is a form of programmed cell death which is the most common form of physiological cell death in eukaryotes, evolutionarily conserved from yeast to humans.It leads to the genetically controlled sequence of events that eventually give rise to spatially and temporally regulated selfdestruction of cells [1,2].Apoptotic mode of cell death is an active process, critical in the development of multicellular organisms and the maintenance and regulation of cell populations during physiological and pathological conditions [3,4].Deregulation of apoptosis leads to various pathological conditions including cancer, autoimmune disorders, and spreading of viral infections while AIDS, Neurodegenerative disorders, and ischemic diseases are caused or enhanced by accelerated apoptosis [3,[5][6][7][8].Both viral and Mycobacterium tuberculosis (Mtb) infections modulate host cell apoptosis for their benefits [6,[9][10][11].This review briefly summarizes the mechanisms of apoptotic deaths and their regulation and significances in viral and mycobacterial infections.

Apoptosis
Various extracellular and intracellular stimuli trigger apoptosis.Ligation of cell surface receptors, DNA damage (because of defects in DNA repair mechanism, cytotoxic drugs, or irradiation), lack of survival signals, contradictory cell cycle signaling or developmental death signals are some of the signals evoking apoptosis [1].Apoptosis depends on the activation of a proteolytic cascade of pro-caspases into active caspases.These caspases are synthesized in cells as inactive zymogens called as pro-caspases.Procaspases are cleaved by pre active caspases at one or two specific aspartic acids splitting them into two subunits, one small and another large.The assembly of two heterodimers of small and large subunits results in the formation of active caspases.The pro-caspases fall into two classesinitiator and executioner [12,13].Apoptotic stimuli trigger activation of initiator caspases (caspases 2, 8, 9, 10) which in turn cleave and activate the executioner caspases (caspases 3, 6, 7) [14].The executioner caspases cleave thousands of substrates responsible for the characteristic morphological and biochemical features of apoptotic cells [14].The three main established routes of apoptosis in mammals are extrinsic, intrinsic and perforin/granzyme pathways [2,15].Irrespective of the death stimuli or apoptotic paths, all the three routes lead to the activation of executioner caspases 3, 6 and 7 (Figure 1).

Extrinsic pathway of apoptosis
External apoptotic signaling mediates the activation of transmembrane death receptors that transmit apoptotic signals after binding to extracellular death ligands such as FasL or tumor necrosis factor-α (TNFα) [16].Death receptors belong to tumor necrosis factor receptor (TNFR) superfamily including TNFR-1, Fas/CD95 and TNF receptor-related apoptosis-inducing ligand (TRAIL) receptors DR-4 and DR-5 [17].Proteins of TNFR family result in trimerization and activation of intracellular death domain after ligand binding.Adaptor proteins like FADD or TRADD get recruited through their death domains to the death domains of activated death receptors forming death inducing silencing complex (DISC).Death effector domains of FADD or TRADD recruit pro-caspase 8 leading to their autocatalytic activation and release of active caspase 8. Activated caspase 8 then cleaves and activates downstream executioner caspases 3 and 7.In some cases, the extrinsic death signals can crosstalk with an intrinsic pathway through caspase 8-mediated proteolysis of the BH3-only protein Bid.Truncated Bid can translocate to mitochondria and induce the release of cytochrome c and assembly of apoptosome triggering activation of pro-caspase 9 [18][19][20] (Figure 1).

Intrinsic pathway of apoptosis
Intracellular death signals such as DNA damage, oxidative stress, starvation and others trigger intracellular apoptotic pathway.All of these stimuli cause changes in inner mitochondrial membrane resulting in the opening of the mitochondrial permeability transition (MPT) pore, loss of the mitochondrial transmembrane potential (ΔΨm) and release of two groups of pro-apoptotic proteins from the intermembrane space into the cytosol [21].The first group of released proteins constitutes cytochrome c, Smac/DIABLO, and the serine protease HtrA2/Omi that promotes caspase-dependent mitochondrial pathway [22][23][24].Cytochrome c binds and activates Apaf-1 (apoptosis protease activating factor 1) which hydrolyzes bound dATP to dADP.Replacement of dADP with dATP/ATP leads to Apaf-1cytochrome c complex to oligomerize into a wheel like a heptamer called apoptosome.Pro-caspase 9 gets recruited in the apoptosome through its caspase recruitment domain (CARD) [25] and gets activated and cleaved which then triggers activation of downstream executioner caspases (Figure 1) [25].Smac/DIABLO and the serine protease HtrA2/Omi, on the other hand promote apoptosis by inhibiting IAP (inhibitors of apoptosis) proteins [23,24].The second group of released proteins includes AIF, endonuclease G, and CAD which translocate to the nucleus and cause DNA fragmentation and condensation of peripheral nuclear chromatin [26][27][28].In addition to the release of mitochondrial factors, the loss of the ΔΨm leads to regulation of biochemical homeostasis of the cell viz.ATP synthesis gets stopped, redox molecules like NADH, NADPH and glutathione are oxidized, and reactive oxygen species are enhanced [29][30][31][32].

Perforin/ Granzyme pathway of apoptosis
Cytotoxic T lymphocytes (CTL) or natural killer (NK) cells can exert their cytotoxic effects on tumor cells and virus-infected cells by secretion of the transmembrane pore-forming molecule perforin with the subsequent release of cytoplasmic granules through the pore into the target cell [33].These granules constitute the serine proteases granzyme A and B. Granzyme B can cleave proteins at aspartate residues and thus activate pro-caspase 8 and Bid.Direct activation of pro-caspase 3 and cleavage of ICAD could also be the results of granzyme B. Thus granzyme B dependent routes of apoptosis may be mitochondrial or direct [26].Granzyme A activates caspase-independent apoptosis [34] (Figure 1).Inside the cell, it enables DNA degradation by DNase NM23-H1.Granzyme A cleaves SET complex (nucleosome assembly protein that usually inhibits DNase NM23-H1 gene) thereby releasing the inhibition of DNase NM23-H1 leading to DNA degradation [34].

Virus-mediated modulation of apoptosis
In most cases of viral infections, immune and inflammatory responses, as well as apoptosis of the infected host cell, are triggered.Meanwhile, some viruses utilize apoptosis as a mechanism of killing cells and spreading virus by targeting a variety of crucial steps in the pathways that block or delay apoptosis.Thus viral infection elicits host cell apoptosis as a part of host immune defense or viral survival component [41].

Virus modulates the extrinsic pathway of apoptosis
Many viruses can efficiently modulate the extrinsic pathway of apoptosis.Adenovirus proteins E3-10.4K and E3-14.5Kreduce the presentation of Fas molecules on the surface of the cells that results in resistance to Fas-mediated cell death [42].These proteins also resist TNFmediated apoptosis [43].Epstein-Barr virus LMP-1 (latent membrane-1) protein acts like constitutively activated TNF receptor which interacts with TNF receptor-associated death domain (TRADD) protein [44].The myxoma virus protein M-T2, a viral mimic protein of TNF receptor, Cowpox virus cytokine response modifying (CRM) proteins and vaccinia virus protein A53R inhibits TNF-mediated apoptosis [45,46,47].Membrane-bound HIV-1 gp120 induces apoptosis through syncytia formation while it triggers apoptosis by various mechanisms like upregulation of Fas, FasL, and TNFα expression, upregulation of TRAIL receptors DR4 and DR5, and acting as a molecular mimic of Fas [48].HIV-1 Nef protein downregulates the expression of CD4 and MHC I molecules but heightens the membrane expression of TNF and related cytokines [49].HIV-1 Tat mediates apoptotic resistance in the infected cells by decreasing susceptibility to TRAIL, TNFα, and Fas, but it reconciles apoptosis in uninfected bystander cells by upregulation of FasL [50].Various herpes viruses encode viral FLICE-like inhibitory proteins (FLIPs), which contain death effector domain but lack caspase activity, inhibit extrinsic apoptotic pathway at the point of DISC formation [51].The human cytomegalovirus encodes vICA, which associates with caspase 8 and blocks its activation [52] (Table 1).

Virus modulates the intrinsic pathway of apoptosis
Many viruses alter apoptosis utilizing the tumor suppressor p53.SV40 virus large T antigen and West Nile capsid protein binds to p53 and sequesters it in an inactive complex [53,54].Moreover, Human papillomavirus E6 protein and adenovirus E1B-55K protein promote ubiquitin mediated degradation of p53 [55,56] and Hepatitis B virus pX protein binds and inactivates p53 [57].Virus-encoded orthologs of anti-apoptotic Bcl2 proteins are also crucial players in the modulation of apoptosis.Adenovirus E1B-19K is similar to Bcl2 which binds to Bak preventing Bax-Bak oligomerization [58].Human herpes viruses, Epstein-Barr virus and Kaposi's sarcoma-associated γ-herpes virus use Bcl-2 orthologs to block the mitochondrial release of cytochrome c [59,60].Although human cytomegalovirus (CMV) protein vMIA shares no sequence homology to Bcl2, it is functionally similar to Bcl-2 and inhibits Fas-mediated apoptosis [61,62].Some viruses use IAP orthologs that can inhibit caspases.For example, Poxviruses serpin CrmA suppresses caspase 1 and 8 and inhibits TNF and  1).

Mtb modulates the extrinsic pathway of apoptosis
Gene expression profiling study suggests that numerous apoptosis-related genes are downregulated in active tuberculosis patients compared to latently infected subjects.

Figure 1 .
Figure 1.Diagram showing general apoptosis process through three main pathways: Extrinsic (death receptormediated viz.FasL, Fas, Trail-R1, and R2) pathway, intrinsic (mitochondria-dependent) pathway and perforin (granzyme)-mediated pathway.The extrinsic pathway starts with the binding of death receptor ligand (DR ligand) to the cell surface death receptors including tissue necrosis factor (TNF) receptor superfamily include CD95 and TNFrelated apoptosis-inducing ligand (TRAIL)-R1/-R2, with the rapid activation of the initiator caspase 8.In the intrinsic pathway, stress (reactive oxygen species, ROS, UV, genotoxic stress, etc.) results in the perturbation of mitochondria membrane permeability, release of the proteins such as cytochrome c from the inner mitochondrial membrane space.The release is regulated in part by Bcl2 family members, with anti-apoptotic (Bcl2/Bcl-XL/Mcl1) and pro-apoptotic (Bax, Bak, and tBid).Once released, cytochrome c binds to apoptotic protease-activating factor 1 (Apaf1), which results in the formation of the Apaf1-caspase 9 apoptosome complex and activation of the initiator caspase 9.The activated initiator Caspases 8 and 9 then activate the effector caspase 3, 6 and7 with normal cell apoptosis or another T-cell effector mechanism.Cytotoxic T lymphocytes (CTL) or natural killer (NK) cells secrete the transmembrane poreforming molecule perforin and release cytoplasmic granules (Granzyme A/B) into tumor cells or virus-infected cells.Granzyme A activates DNA degradation by DNase NM23-H1while granzyme B cleaves pro-caspase 8, pro-caspase 3 or Bid.
Fasmediated apoptosis [63].Likewise, African swine flu virus produces vIAP that inhibits caspase 3 and Baculovirus protein p35 is another vIAP with a potential to inhibit caspases 1, 3, 6, 7, 8 and 10 [64,65,66].HIV-1 gp120 triggers apoptosis by reduced expression of Bcl2, phosphorylation of mTOR and p53, increased expression of proapoptotic protein PUMA and activation of p38 [67].HIV-1 Tat inhibits apoptosis in infected cells by upregulation of Bcl2 and c-FLIP expression [68,69] and downregulation of caspase 10 expression [70].The same protein triggers apoptosis in bystander cells by upregulation of Bax, caspase 8 and RCAS-1 expression [71,72], and Bim-mediated intrinsic apoptosis [73].Matrix protein Z of some arenaviruses (New World arenavirus, Tacarible virus (TCRV), and the attenuated vaccine strain of Junín virus (JUNV) Candid #1) activates caspase 9 thereby triggering the intrinsic apoptotic pathway [74,75].Though the exact molecular mechanism of viral protein Zmediated apoptosis is still not clear, in vitro experiments suggest a direct activation of BH3only proteins and an indirect interaction with proteins like p53 and PI3K/Akt through cellular oncoprotein promyelocyte leukemia protein (PML) [74-76].The Old World arenaviruses, the lymphocytic choriomeningitis virus (LCMB) and Lassa virus (LASV) do not cause apoptosis of infected cells [77,78].Caspase-mediated cleavage of nucleoproteins (NPs) of Old World areanviruses generates multiple truncated isoforms of NPs [74,79].A decoy function of NPs has been proposed in which the cleavage of highly expressed NPs within the cell suppresses the cellular targets of caspases thereby inhibiting the apoptosis of the infected cell [74].Enterovirus 71 2B protein directly interacts with and activates the proapoptotic protein Bax leading to the activation of mitochondrial pathway of apoptosis [80,81] (Table Bacterial pathogens are known to have antiapoptotic mechanisms.Mycobacterium tuberculosis (Mtb) causes persistent infection indicating that it employs effective mechanisms to inhibit host cell death [82].Published studies highlight both proapoptotic as well as anti-apoptotic capabilities of virulent Mtb [83,84], however the underlining molecular mechanisms are still not well understood.Though there is a lack of published data favoring Mtb-mediated apoptosis of host cells, increased apoptosis of primary human macrophages or human macrophage-like cell lines (U937 and THP1) were reported upon infection with virulent Mtb in vitro [85-87].Human alveolar macrophage-derived from bronchoalveolar lavage of tuberculosis patients also showed increased apoptotic death compared to healthy subjects [88,89].Apoptosis of Mtb infected cells accompanied by the recruitment of uninfected macrophages through upregulation of MMP9 on epithelial cells surrounding the granuloma helps in the dissemination of the bacteria [90].In the studies involving the zebrafish and mouse lung models, the pro-apoptotic nuoG Mtb mutant induced enhance innate response, longer survival and rapid dissemination of the bacteria [91,92].Thus, evidence suggests that host cell apoptosis is crucial for host resistance to Mtb infection.Considerable less apoptosis of human alveolar macrophage or macrophage-like cell lines when infected with virulent Mtb compared to infection with less virulent strains was reported [93-96].Furthermore, fact that inhibition of apoptosis of human and murine macrophages by Apoptosisinducing species M. kansaii after over-expression of Mtb-nuoG/SecA2/PknE [97-99] and resistance to FasL and TNFα-mediated apoptosis of Mtb ©NJB, Biotechnology Society of Nepal 52 Nepjol.info/index.php/njbinfected cells provide the evidence that Mtb inhibits host cell apoptosis [100].

Table 1 :
List of viral and Mtb proteins involved in apoptosis deregulation