1. Microbiology and Infectious Disease
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Mycobacterium tuberculosis exploits host ATM kinase for survival advantage through SecA2 secretome

  1. Savita Lochab
  2. Yogendra Singh
  3. Sagar Sengupta
  4. Vinay Kumar Nandicoori  Is a corresponding author
  1. National Institute of Immunology, India
  2. Department of Zoology, University of Delhi, India
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Cite this article as: eLife 2020;9:e51466 doi: 10.7554/eLife.51466

Abstract

(Mtb) produces inflections in the host signaling networks to create a favorable milieu for survival. The virulent Mtb strain, Rv caused double strand breaks (DSBs), whereas the non-virulent Ra strain triggered single-stranded DNA generation. The effectors secreted by SecA2 pathway were essential and adequate for the genesis of DSBs. Accumulation of DSBs mediated through Rv activates ATM-Chk2 pathway of DNA damage response (DDR) signaling, resulting in altered cell cycle. Instead of the classical ATM-Chk2 DDR, Mtb gains survival advantage through ATM-Akt signaling cascade. Notably, in vivo infection with Mtb led to sustained DSBs and ATM activation during chronic phase of tuberculosis. Addition of ATM inhibitor enhances isoniazid mediated Mtb clearance in macrophages as well as in murine infection model, suggesting its utility for host directed adjunct therapy. Collectively, data suggests that DSBs inflicted by SecA2 secretome of Mtb provides survival niche through activation of ATM kinase.

Introduction

In response to the damage, the host activates an intricate and indispensable signaling cascade entitled ‘DNA damage response’ (DDR), which not only detects and repairs the damaged lesions in DNA but also regulates the activation of effectors that determine the fate of the cell. Ataxia telangiectasia mutated (ATM), ATM- and Rad3-related protein (ATR) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are three drivers of DDR which belong to the family of phosphoinositide 3-kinase like kinases (PIKKs). PIKKs respond to the DNA damage by activating their downstream substrates leading to cell cycle delay/arrest and DNA repair and depending on the extent of damage can also lead to apoptosis. ATM is activated through autophosphorylation of S1981 residue and is subsequently recruited to the double strand breaks (DSBs) through a sensor complex, MRN (MRE11, NBS1 and Rad50) (Lee and Paull, 2005). DSBs are also the site of recruitment and activation of DNA-PKcs, facilitated by a DSB-bound heterodimer Ku70-80 (Gottlieb and Jackson, 1993; Spagnolo et al., 2006). ATR-ATRIP complex is recruited in response to RPA-coated ssDNA (Ball et al., 2005). Even though all the three kinases coordinate their functions independently, crosstalk among these kinases is known to exist, such as ATM-dependent activation of ATR (Shiotani and Zou, 2009); ATM and ATR-dependent phosphorylation of DNA-PKcs (Chen et al., 2007; Hirokawa et al., 1992) and DNA-PKcs-mediated modulation of ATM (Peng et al., 2005; Zhou et al., 2017). Moreover, these kinases also share substrates such as p53 (Canman et al., 1998; Lakin et al., 1999; Lees-Miller et al., 1990). Phosphorylation of H2AX at the serine 139 (termed γH2AX), at the chromatin regions flanking the damage site is considered as a marker for DNA damage (Burma et al., 2001; Rogakou et al., 1998). While phosphorylation of H2AX is predominantly modulated by ATM, ATR and DNA-PKcs also contribute either partially or entirely (Burma et al., 2001Royo et al., 2013; Wang et al., 2005). Phosphorylation of H2AX acts as the foundation for recruitment of other DDR mediator proteins eventually leading to DNA repair (Ciccia and Elledge, 2010). Thus, DDR is an indispensable mechanism that restores the genomic integrity and regulates the cellular response by modulating DNA repair, cell cycle progression, apoptosis, or senescence in response to DSBs in the cell. Thus, DDR is also a preferred target of pathogenic bacteria.

Pathogenic Escherichia coli and Helicobacter pylori are capable of imparting notable DNA damage to the host and subsequently impair the DDR to avoid premature cell death (Cuevas-Ramos et al., 2010; Toller et al., 2011). Chlamydia trachomatis triggers DSBs in the host, and γH2AX induction but simultaneously it impairs the DDR by inhibiting the recruitment of 53BP1, ensuing inadequate signal amplification (Chumduri et al., 2013). Listeriolysin O (LLO) secreted by Listeria monocytogenes induces degradation of crucial DNA damage sensor, MRE11. This results in impaired DDR, curtailing the host ability to halt cell cycle, thus successfully promoting multiplication and survival of the pathogen (Samba-Louaka et al., 2014). H. pylori impedes nucleotide repair by downregulating the proteins and their respective transcripts that are involved in mismatch and base excision repair (Kim et al., 2002; Machado et al., 2009). Neisseria gonorrhoeae, H. pylori and C. trachomatis also downregulate p53 levels to promote host cell survival and inhibit apoptosis (Buti et al., 2011; Wei et al., 2010; Vielfort et al., 2013).

Since ancient times Mycobacterium tuberculosis (Mtb) has been steadily evolving sophisticated tactics to dodge the defense responses of the host. One of the remarkable survival strategies of Mtb is to intervene with the fundamental signaling events of the host cell (Koul et al., 2004) and to facilitate these manipulations Mtb secretes an enormous number of characterized and uncharacterized effectors inside the host. These effectors modulate host cellular processes such as phagosome maturation, apoptosis, autophagy, calcium homeostasis, activation of pro-inflammatory responses and TLR, TNFα, MAPK signaling pathways (Dey and Bishai, 2014). However, till date, the role of ATM kinase in the survival of Mtb inside the host has not been investigated.

In this study, we demonstrate that Mtb causes DSBs and determine its impact on the activation of host DDR. SecA2 secretome is necessary and sufficient for inflicting DSBs in the host. We show that instead of classical ATM-Chk2 pathway, Mtb gains survival advantage through activation of ATM-Akt signaling cascade that results in the inhibition of apoptosis. In a chronic mice infection model, Mtb infected lungs showed significant DSBs and activation of ATM. Combining ATM inhibitor, KU55933 with INH resulted in better clearance of Mtb compared with INH treatment alone in the lungs and spleen of infected mice. This study reveals novel exploitation mechanism utilized by Mtb, wherein the pathogen inflicts persistent DSBs in the host to activate ATM-Akt signaling pathway and thereby inhibiting apoptosis and accentuating cell growth.

Results

Mtb inflicts DNA damage in the host cell

To address the question of whether Mtb infection leads to the damage of the host DNA we used PMA differentiated THP-1, RAW264.7 (RAW) macrophages and primary murine peritoneal macrophages (PΦ). The cells were infected with the virulent Mtb strain, H37Rv (Rv) and the γH2AX levels, the hallmark of DNA damage, was evaluated. Results showed considerable DNA damage in the infected cells compared to the corresponding uninfected control and these observations were consistent across all the three cell types (Figure 1a–c). The damage could be observed as early as 1 hr post infection (p.i) (Figure 1b) and persisted even at 72 hr (Figure 1a). We assessed if the observed DNA damage is dependent on the presence of live bacteria by infecting cells with live or heat killed Rv. Results showed that (Figure 1d) only the live bacilli could cause damage to the host genome. To evaluate the role of Mtb virulence in inflicting genotoxicity, we performed infection experiments with Rv or its avirulent counterpart H37Ra (Ra). Based on its characteristics such as reduced survival under anaerobic conditions, inability to produce persistent infection in mice and guinea pigs, Ra is considered as attenuated, avirulent strain of Mtb (Alsaadi and Smith, 1973; North and Izzo, 1993). While both Ra and Rv infected THP1 cells showed considerably higher γH2AX levels compared with the uninfected control, there was consistent and noticeably higher levels of γH2AX levels in Rv compared with the Ra infected cells (Figure 1e; compare 4, 24 and 48 hr time points). However, in case of RAW cells the higher γH2AX levels in Rv was apparent only in the early time points (Figure 1f and g: up to 4 hr). Subsequent to DNA damage, H2AX gets phosphorylated on the chromatin flanking the site of damage, which appears as foci in the nucleus. We performed immunofluorescence experiments using antibodies specific for γH2AX and observed significantly higher numbers of γH2AX foci at both 4 and 24 hr p.i (Figure 1h) compared with the uninfected controls. Consistent results were observed in infected THP1 and PΦ cells (Figure 1—figure supplement 1a and b). Collectively, the data suggests that both Rv and Ra possess genotoxic characteristics and virulent Rv strain triggers rapid damage post infection.

Figure 1 with 1 supplement see all
Mtb inflicts DNA damage in the host cell.

(a) PMA differentiated THP-1 cells were infected with Rv for 4 hr. Cells were washed thrice with 1XPBS to remove extracellular bacilli and supplemented with fresh media for further time points which were calculated with respect to 4 hr point. (b and c) RAW264.7 macrophages (RAW) (b) and murine peritoneal macrophages (PΦ) (c) were infected with Rv as described in (a) except that the extracellular bacilli were washed off after 1 and 2 hr post-infection (p.i) For the remaining time points, 4 hr serves as the reference point. (d) RAW264.7 macrophages were infected with Rv or heat-killed Rv (HK-Rv) and cell lysates were prepared at 4 and 24 hr p.i (e–g) THP-1 or RAW264.7 cells were infected with Rv and Ra. Cell lysates were prepared as indicated above after 1, 2, 4, 24, 48 or 72 hr p.i. (a–g) Whole cell lysates (WCL) were resolved on SDS-PAGE, transferred to nitrocellulose membrane and probed with α-γH2AX(S139) and α-β-actin/α-β-tubulin antibodies. (h) Representative immunofluorescence image showing γH2AX foci (AlexaFluor594-Red) and nucleus (DAPI-blue) in RAW264.7 infected with GFP expressing Rv and Ra (Green) independently at 4 and 24 hr p.i. Images were captured at 63X magnification with scale bar of 5 µm.

Host cell activates DNA damage response

Subsequent to the challenge to genome integrity, eukaryotic cells activate the DDR, which initiates either cell survival or cell death signals contingent upon the magnitude of damage. Depending on the type of damage, - DSBs or single-stranded DNA (ssDNA; due to replication fork stalling) either ATM-Chk2 or ATR-Chk1 pathways, respectively, are activated (Figure 2aLee and Paull, 2007; Zou and Elledge, 2003). To determine the type of DDR involved in Ra and Rv inflicted damage, lysates prepared at 4 and 24 hr p.i were examined for the phosphorylated forms of ATM, Chk2, ATR and Chk1, indicative of their activation. Infection by Ra led to the formation of distinct nuclear pRPA2 (Figure 2c) and RPA2 (Figure 2—figure supplement 1a) foci and robust activation of ATR-Chk1 pathway (Figure 2b; right panel), suggesting that Ra induces generation of ssDNA. On the other hand, infection by Rv led to significant activation of ATM-Chk2 pathway in both RAW and PΦ cells, suggesting occurrence of DSBs in the host nuclei (Figure 2b, left panel and 2d). These results were corroborated by immunofluorescence experiments, wherein we observed distinct pATM foci upon Rv infection (Figure 2e). Furthermore, we assessed the transmission of DNA damage signals by evaluating the expression and activation levels of sensors, transducers and mediators upon Rv infection. While there were no significant perturbation in the protein levels, enhanced phosphorylation of Nbs1, MDC1, 53BP1, representing the activated forms, was evident upon Rv infection (Figure 2—figure supplement 1b and cShiloh, 2003). To corroborate these findings, we evaluated γH2AX foci for the presence of mediator proteins 53BP1, which accumulates at the DNA damage site. Distinct focal pattern and colocalization of γH2AX and 53BP1 foci in Rv infected PΦ supported the argument that infection with Rv induces DSBs (Figure 2f).

Figure 2 with 1 supplement see all
Host cell activates DNA damage response.

(a) Schematic outline of ATM-Chk2 and ATR-Chk1 DDRs. Double strand breaks (DSBs) mediate the activation of ATM and its corresponding effector, Chk2 whereas ssDNA originated due to replication stalling leads to the activation of ATR and the downstream effector Chk1. Chk2 and Chk1 further activate cell cycle checkpoints to delay or arrest cell cycle progression. Cells with extensive DNA damage exit the route of cell cycle and undergo apoptosis. Phosphorylation status of proteins is depicted with P in a circle (b) To determine the signalling cascade coordinating the DNA damage, RAW264.7 macrophages infected with Rv, Ra or HK-Rv. WCL prepared at 4 and 24 h p.i. were subjected to immunoblotting with α-γH2AX, α-pATM(S1981), α-ATM, α-pCHK2(T68), α-CHK2 (left panel) and α-β-actin, α-ATR(S428), α-ATR, α-pCHK1(S345), α-CHK1 (right panel) antibodies. (c) Representative immunofluorescence showing presence of pRPA2-S4/8 foci (AlexaFluor594-Red) in uninfected or GFP-Rv/GFP Ra (Green) infected RAW264.7 macrophages at 24 hr (d) WCL prepared from PΦ infected with Rv for 4 and 24 hr were immunoblotted with α-γH2AX, α- pATM(S1981), α-ATM, α-pCHK2(T68), α-CHK2 and α-β-actin antibodies. (e–f). Representative images of immunofluorescence showing (e) pATM(S1981) (AlexaFluor594-Red) and (f) γH2AX, 53BP1 foci formation (AlexaFluor594-Red and AlexaFluor647-yellow) in PΦ infected with GFP expressing Rv (Green) for 24 hr. Nuclei were stained with DAPI. Images were captured at 100X magnification with 2.9 X optical zoom in LSM510 Meta System (Zeiss, Germany) confocal microscope. Scale bar: 2 µm.

Intracellular Mtb causes continuous DSBs

Mtb infection not only elicits early DNA damage (Figure 1f) but that also persists at higher levels for prolonged duration (Figure 1a, e and Figure 3a left panel). DDR is necessary for sensing, amplifying and eventually repairing the damage. However, persistent DSBs indicate either inefficient DDR or continued presence of the factors released by Rv that are responsible for the damage. To determine whether there is a direct correlation between the levels of γH2AX and Rv load, we evaluated γH2AX levels in the absence or presence of isoniazid (INH), the front line anti-tuberculosis antibiotic (Nuermberger and Grosset, 2004). Analysis of colony-forming units (CFUs) at different time points post Rv infection ascertained efficient clearance of intracellular pathogen in the presence with INH (Figure 3b and Figure 3—source data 1). Presence or absence of INH did not influence the extent of γH2AX levels in uninfected control cells (Figure 3a; right panel). While γH2AX levels continued to persist in Rv infected cells, treatment of INH led to time dependent reduction in γH2AX, pATM and pChk2 levels (Figure 3c), concomitant with the clearance of the pathogen (Figure 3b). Thus, the data suggests that even though DDR is active, inefficient repair is due to continuous generation of DSBs during prolonged Rv infection.

Intracellular Mtb causes continuous DSBs.

(a) Left panel: RAW264.7 cells were infected with Rv. Cells were harvested at indicated time points to prepare WCLs which were then probed with α-γH2AX and α-β-actin antibodies. Right panel: RAW cells were treated with 1 µg/mL (7.3 µM) INH for indicated time points. WCL were prepared and immunoblotted with α-γH2AX and α-β-actin. (b and c) RAW264.7 cells were infected with Rv. After 4 hr, extracellular Rv were washed with PBS and supplemented with fresh media with or without 7.3 μM isoniazid (INH) (b). Cells at indicated time points were lysed with 0.05% SDS, serially diluted and plated on 7H11 plates to enumerate bacillary load. Bar graph on y-axis represents Mean CFU (log10) ± SD of one of the three independent experiments performed in triplicates. **p≤0.005, ***p≤0.0005, (c). WCL were prepared at indicated time points were probed with α-γH2AX, α-pATM(S1981), α-ATM, α-pChk2(T68), α-Chk2 and α-β-actin antibodies. (d) Schematic diagram illustrating the experimental approach for the cell cycle analysis using flow cytometry. RAW264.7 cells were infected with GFP expressing Rv. FACS profile showing GFP+ve population containing Rv and GFP-ve population that lacks intracellular Rv (bystander uninfected cells). Uninfected control cells are GFP-ve. (e) Pie chart showing percentage of uninfected and GFP+ve infected cells in different phases of cell cycle. Infection was performed at the indicated timepoints. (f) Schematic representation of signaling activated due to DSB occurrence in the host cell. ATM phosphorylates its self and H2AX. Subsequently it activates the downstream effector, Chk2 at T68. Activated Chk2 stabilizes p53 which in turn elevates p21 levels. Upregulation of p53 can divert cells toward apoptosis while p21 leads to cell cycle arrest (g). WCLs prepared from RAW264.7 infected with GFP expressing Rv for indicated time points were subjected to immunoblotting with α-γH2AX, α-p21, α-p53, α-pChk2(T68), α-Chk2 and α-β-actin antibodies.

Apart from activating signals for repair, the DDR stalls the DNA replication and cell division by activating the cell cycle checkpoints (Smith et al., 2010). To examine the consequence of Rv-induced DSBs on the host cell cycle progression, RAW cells were infected with GFP-expressing Rv and the populations of infected and uninfected cells were analyzed through flow cytometry (Figure 3d). Flow cytometry profile of uninfected control cells would show single population, whereas Rv-infected RAW cells would have two populations: GFP+ve cells harboring intracellular GFP-Rv and GFP-ve uninfected bystander cells that lack intracellular GFP-Rv (Figure 3d). Interestingly, compared with the uninfected cell population, Rv infected GFP+ve population showed decreased accumulation of cells in G1 phase, soon after the infection (4 h p.i). Lowered accumulation of cells in the G1 phase suggest alterations in the progression of cell cycle in the cells harboring intracellular bacilli (Figure 3e). Importantly, at late stages of infection we observed concomitant increase in the sub-G1 cell population, indicative of cell death (Figure 3e). DDR activation maintains the genomic stability by delaying the occurrence of cell division till DNA is repaired, else in case of irreversible damages; key regulators such as p53 mediates apoptosis (Figure 3f). To corroborate flow cytometry results, we evaluated key molecular markers that modulate the transition of cells through different cell cycle phases (Abraham, 2001; Matsuoka et al., 1998). Elevated levels of p53 and p21 upon Rv infection are reflective of molecular level alterations in the cell cycle leading to eventual cell death indicated as sub-G1 population (Figure 3g). Taken together, presence of Rv inside host causes constant DNA damages followed by cell cycle perturbations.

Mtb SecA2 secretome is necessary and sufficient for DNA damage

Pathogenic bacteria often release genotoxins and cyclomodulins to modulate host cellular processes (Nougayrède et al., 2005). Whi, WhiB3, a redox regulator in Mtb has been shown to regulate the production of specific polyketides and lipid cyclomodulins in the host (Cumming et al., 2017). We speculated that the Mtb secretome might play a role in promoting DNA damage. Culture filtrate (CF) of Mtb contain proteins that are translocated into the extracellular milieu (Figure 4a). Addition of Mtb CF resulted in robust γH2AX induction, even higher than those observed upon Rv infection (Figure 4b). Importantly as little as 1 µg/ml CF treatment induced significant γH2AX (Figure 4c). In Mtb, proteins are secreted using both classical secretory pathways such as TAT and SecA1, which are essential for in vitro growth; and accessory secretion pathways such as SecA2 and Type VII secretion systems, which are necessary for survival and virulence in the host (Feltcher et al., 2010; Ligon et al., 2012; Miller et al., 2017). The TypeVII secretion system and its effectors are encoded by Region of difference 1 (RD1) in Mtb. Host cells infected with Rv or RvΔRD1(Rv carrying RD1 deletion) or RvΔCE (Rv strain with CFP10-ESAT6 deletion) had comparable γH2AX levels (Figure 4d & f). On the other hand, expression of γH2AX observed upon infection with RvΔsecA2 (SecA2 exporter mutant) was significantly lower at the later time points (Figure 4e & f). However, since the deletion of secA2 compromises the survival of Mtb in the host (Braunstein et al., 2003; Kurtz et al., 2006; Miller et al., 2017), we were unable to rule out the possibility that the decreased γH2AX levels could be due to better clearance of the pathogen. To address this issue, cells were treated with CFs prepared from either Rv or RvΔRD1 or RvΔsecA2 and the γH2AX levels were evaluated. While the γH2AX levels in cells treated with CF from RvΔRD1 were comparable to those observed upon Rv-CF treatment; cells treated with RvΔsecA2-CF showed negligible γH2AX levels (Figure 4g). Treatment of cells either with Rv-CF or RvΔRD1-CF led to the activation of DDR players, ATM and Chk2. However, treatment with RvΔsecA2-CF cells did not show ATM-Chk2 activation. Analogous results were obtained when the experiment was performed with PΦ (Figure 4h). Together, results suggest that effectors secreted by SecA2 pathway are necessary and sufficient for inflicting genotoxic stress in the host.

Mtb SecA2 secretome is necessary and sufficient for DNA damage.

(a) Schematic depiction of culture filtrate (CF) preparation (described in Materials and methods). (b) RAW264.7 cells were either uninfected or infected with Rv or treated with 20 µg/ml of CF obtained from Rv (Rv-CF). (c) RAW264.7 cells were either left untreated or treated with 1 µg/ml of Rv-CF for 4, 24 and 48 hr. (d and e) RAW264.7 cells were infected with Rv or (d) RvΔRD1 (deletion mutant of RD1 region) or (e) RvΔsecA2 (deletion mutant of SecA2) for indicated time points. (f) PΦ cells were infected with Rv or RvΔCE or RvΔsecA2 for indicated time points (g and h). 1 µg/ml of CF obtained from Rv (Rv-CF) or RvΔRD1 (ΔRD1-CF) or RvΔsecA2 (ΔsecA2-CF) was used to treat (g) RAW264.7 or (h) PΦ for indicated time points. (b–h) WCL from all the above conditions were subjected to immunoblotting with indicated antibodies.

ATM activation confers survival advantages to Mtb

DNA damage mediates the activation of ATM kinase, which in addition to activating DDR is also known to modulate other cellular processes such as apoptosis, DNA repair, cell division, autophagy and inflammatory responses (Kastan and Lim, 2000). We assessed the impact of host ATM kinase activation in regulating the survival of Mtb with the help of KU55933 (ATM-I), a specific inhibitor of ATM activation (Hickson et al., 2004). Treatment of infected cells with ATM-I inhibitor resulted in substantial decrease in the γH2AX levels and abrogation of ATM and Chk2 activation (Figure 5a). This was also reflected in disappearance of pATM foci upon ATM-I treatment in Rv-infected cells (Figure 5b). Notably, ATM-I treatment of Rv-infected cells resulted in reduced survival of the pathogen in a dose dependent manner (Figure 5c and Figure 5—source data 1). However, ATM-I did not impact in vitro Mtb growth suggesting that the clearance of the bacilli is mediated through inhibition of ATM activation (Figure 5—figure supplement 1). Next, we examined if the compromised Mtb survival upon ATM-I treatment is contingent on inhibition of DDR pathway. Toward this, we treated the cells with the inhibitor for immediate downstream effector Chk2. Interestingly, treatment of infected cells with Chk2 inhibitor (Chk2-I) had negligible effect on Mtb survival (Figure 5d & e; Figure 5—source data 1), suggesting that while ATM activation is essential; Mtb survival is not dependent on the activation of downstream effectors. Thus, we hypothesized that ATM activation upon infection may be channeling survival signals through alternate pathways.

Figure 5 with 1 supplement see all
ATM activation provides survival advantages to Mtb.

(a) PΦ were pre-treated with 10 µM of KU55933 (ATM-I) for 1 hr prior to Rv infection. 4 h p.i, extracellular bacilli were washed off and cells were replenished with fresh media containing ATM-I for 24 or 48 hr. Cells were lysed with RIPA to prepare WCL which were then subjected to immunoblotting against indicated antibodies. (b) Representative images of immunofluorescence showing pATM(S1981) foci formation (AlexaFluor594-Red) in PΦ infected with GFP-Rv (Green) in the presence or absence of ATM-I for 24 hr p.i. Nuclei were stained with DAPI. Images were captured at 100X magnification. Scale bar: 2 µm. (c) PΦ were infected with Rv and treated with 1, 5 or 10 µM of ATM-I as described above. Cells were lysed with 0.05% SDS to release the intracellular Mtb bacilli and CFUs were enumerated. (d–e) PΦ were pre-treated with 10 µM ATM-I or Chk1/2 inhibitor (Chk2-I) or DMSO (vehicle). Cells were lysed either with RIPA or 0.05% SDS. (d) RIPA WCL were subjected to immunoblotting using α-pATM, α-ATM, α-pChk2(T68), α-Chk2 and α-GAPDH antibodies. (e) SDS lysates were serially diluted and plated on 7H11 plates to determine CFUs.

Inhibition of ATM-Akt axis hypersensitizes host cells to apoptosis

In addition to ATM and ATR, H2AX can also be phosphorylated by DNA-PK catalytic subunit (DNA-PKcs), the third PIKK that also coordinates DDR. DNA-PKcs is recruited to the DSBs at the heterodimer complex of Ku70/80 to form the holoenzyme DNA-PK, wherein it initiates the repair. Infection of PΦ with Rv resulted in the activation of DNA-PKcs, albeit to much lower levels and at later time points compared with the ATM (Figure 6a). Results in Figure 5a showed presence of residual γH2AX despite ATM-I treatment, thus we sought to assess the effect of combinatorial inhibition of ATM and DNA-PK or ATR on the levels γH2AX. PΦ treated with ATM-I or ATM-I + DNA-PK-I (NU7441; inhibitor of DNA-PK) or ATM-I + ATR-I(VE-281; inhibitor of ATR) were infected with Rv and the extent of H2AX phosphorylation was evaluated (Figure 6b). PΦ irradiated at 2Gy was used as the positive control (Figure 6—figure supplement 1). While the addition of ATR-I to ATM-I did not alter the residual levels of γH2AX, addition of DNA-PK-I to ATM-I resulted in further decrease in the levels of γH2AX, corroborating data in Figure 6a. Next, we assessed the role of DNA-PK and ATR in the survival of pathogen inside the host. Toward this, host cells treated with inhibitors individually were infected with Rv and were enumerated for CFUs (Figure 6c and Figure 6—source data 1). It is evident that addition of DNA-PK-I or ATR-I did not alter the CFUs, suggesting that even though DNA-PK seems to be activated upon Mtb infection, it does not influence the survival of the pathogen (Figure 6c and Figure 6—source data 1). Taken together, the data suggests that ATM might be feeding in to some parallel pathway which is necessary for the intracellular survival of Mtb.

Figure 6 with 2 supplements see all
Inhibition of ATM-Akt axis hypersensitizes host cells to apoptosis.

(a) To determine the activation of DNA-PKcs upon Rv infection, PΦ were infected with Rv as indicated and WCL were immunoblotted with α-pDNA-PKcs-S2056, α-DNA-PKcs, α- pATM(S1981), α-ATM, and α-β-actin. WCLs prepared from PΦ irradiated at 2Gy was used as positive control. (b) PΦ were pre-treated with 10 µM ATM-I or 10 µM ATM-I + 1 µM DNA-PK-I or 10 µM ATM-I + 10 µM ATR-I for 1 hr prior to Rv infection. 4 h p.i, extracellular bacilli were washed off and cells were replenished with fresh media containing above inhibitors with indicated combinations for 24 and 48 hr. Cells were lysed with RIPA to prepare WCL which were then subjected to immunoblotting against α-γH2AX and α-β-actin antibodies. (c) PΦ were infected with Rv and treated with DMSO or 10 µM ATM-I or 1 µM DNA-PK-I or 10 µM ATR-I individually as described above. Cells were harvested at indicated time points and CFUs were enumerated as described previously. (d) WCL from PΦ infected with Rv for indicated time points were immunoblotted with α-pAkt (S473), α-Akt antibodies (e–f) PΦ were pre-treated with either 10 µM of ATM-I or Akt inhibitor (Akt-I) or ATM-I + Akt-I for 1 hr prior to Rv infection. 4 hr p.i., extracellular bacilli were washed off and cells were replenished with fresh media containing inhibitors. At defined time points, cells were lysed either with RIPA or 0.05% SDS. (e) WCL were subjected to immunoblotting with α-pATM(S1981), α-ATM, α-pAkt (S473), α-Akt and α-β-actin antibodies. (f) Serially diluted SDS lysates were plated on 7H11 to determine CFU. (g) At 48 hr time point cells were scraped in PBS and subsequently stained with Annexin V-FITC and 7AAD. Cells were analyzed by flow cytometry to determine the percentage of live cells (upper panel) or cells undergoing early/late apoptosis (lower panel). Inhibitor treatments were as described above. Readings are average of three biological replicates. Error bar, SD. *, p≤0.05; **, p≤0.005; ***, p≤0.0005. Significance was calculated with respect to Rv.

An established survival strategy employed by Mtb is to circumvent host apoptosis and upregulate pro-survival signals (Srinivasan et al., 2014). PI3K/Akt pathway, which upon activation inhibits apoptosis and promotes cell survival, has been shown to be up regulated during Mtb infection (Figure 6dLachmandas et al., 2016). Western blot analysis showed that treatment with either ATM-I or Akt-I inhibited the activation of ATM and Akt, respectively (Figure 6e). Data suggests that administration of Akt inhibitor (Akt-I) alone or in combination with ATM-I significantly reduced the intracellular survival of Mtb (Figure 6f). Treatment with a combination of ATM-I + Akt-I showed survival defects similar to Akt-I alone indicating that ATM mediated signals are channeling through Akt (Figure 6f and Figure 6—source data 1). While treatment of cells with ATM-I resulted in reduced activation of Akt, addition of Akt-I did not impact activation of ATM, suggesting that ATM activation is upstream of Akt activation (Figure 6e). Interestingly, the impact of Akt inhibitor on the survival of Rv was higher compared with ATM-I (Figure 6f; Figure 6—source data 1). Moreover, while the addition of ATM-I decreased activation of Akt, the reduction with Akt-I was higher, suggesting that other signaling pathways could also be involved in activating Akt (Figure 6e).

There exists a possibility that the survival differences observed could be due to reduced viability of host cells in the presence of inhibitor. To negate the cytotoxic effects of inhibitors, we evaluated the survival of uninfected and Rv infected PΦ in the presence of different concentrations of ATM-I or Akt-I or Chk2-I (Figure 6—figure supplement 2). Treatment of uninfected PΦ with ATM-I or Akt-I showed reduced viability, however, infection of PΦ with Rv marginally decreased the survival, compared with the UI cells, suggesting that the modulation of pathogen survival is not due to cytotoxicity (Figure 6f and Figure 6—figure supplement 2). To analyze the impact of ATM-I or Akt-I or ATM-I + Akt-I on the apoptosis, we performed flow cytometry analysis of UI and Rv infected cells in the absence and presence of inhibitors using Annexin V/7-AAD staining. While the addition of inhibitors did not impact the percent live cells (Figure 6g; top panel and Figure 6—source data 1) in UI, their presence decreased the percent live population in Rv-infected cells (Figure 6g; top panel and Figure 6—source data 1). We observed concomitant increase in the early and late apoptotic cells in the presence of ATM-I or Akt-I or ATM-I + Akt-I (Figure 6g; bottom panel and Figure 6—source data 1). Thus, the data suggests that Rv inflicts DSBs to activate ATM which in turn activates Akt resulting in anti-apoptotic and pro-survival signals which favors Mtb survival (Figure 7g).

Figure 7 with 2 supplements see all
ATM kinase is a potential therapeutic target for host directed TB therapy.

(a) Graphical representation depicting Rv load at day 1 (n = 4) and day 56 (n = 5) of infection in lungs and spleen of mice. Lung homogenates prepared at indicated time points were plated on 7H11 plates to enumerate CFU per lung or per spleen (b) Lung and spleen lysates were prepared from uninfected and Rv infected mice 56 days p.i. 100 µg lung lysates were subjected to immunoblotting with α-γH2AX, α-pATM(S1981) α-ATM, α-pCHK2(T68), α-CHK2, α-pAkt (S473), α-Akt, α-p53 and α-β-actin antibodies. (c) PΦ were infected with Rv at high MOI (1:10) as described above. 24 h p.i, cells were treated with either 3.6 µM isoniazid (INH) or 10 µM ATM-I alone or INH + ATM-I together. CFUs were enumerated at 24, 48 and 72 hr post treatment. (d) Schematic representation of the mice infection and drug treatment protocol used. (e–f) CFUs were enumerated in lungs of mice at Day 1, and in the lungs (e) and spleen (f) on day 15 and 30 of infection. Number of mice in each batch was 6 (n = 6) except in Rv at Day 1(n = 5). Error bar, SD. *, p≤0.05; **, p≤0.005; ***, p≤0.0005. (g) Model depicting the findings. Rv induces genotoxicity and causes deleterious DSBs in the host genome through SecA2 secretome. Host cell in response to the occurrence of DSBs activate ATM kinase and is recruited at the site of damage by the sensor, MRN complex. Activated ATM autophosphorylates itself and phosphorylates H2AX in the chromatin flanking the sites of DNA damage, which becomes the foundation for the recruitment of mediator protein MDC1, thus amplifying DDR. pATM promote recruitment of 53BP1 at the damage site. pATM as a part of DDR also activates downstream effectors, Chk2 and p53, which are responsible for alterations in the host cell cycle. pATM in a parallel pathway also activates Akt, which is known inhibit apoptosis and promote cell survival. Activation of ATM and Akt and subsequent inhibition of apoptosis provides survival advantage to Rv. Inhibition of ATM or Akt activation through inhibitors, ATM-I or Akt-I, respectively, promote host cell apoptosis, which impedes the bacilli growth. Phosphorylation status of proteins is depicted with a P in a circle.

ATM kinase is a potential therapeutic target for host directed TB therapy

Persistent DNA damage drives genomic instability (Tubbs and Nussenzweig, 2017). Continuous DNA damage triggered during prolonged Mtb infection in established and primary cell lines encouraged us to examine the occurrence of possible DSBs in Mtb infected mice lungs. BALB/c mice challenged with Rv strain through aerosolic route were evaluated for bacillary load at 1- and 56 days p.i (Figure 7a and Figure 7—source data 1). We observed appearance of granulomatous lesions and significant bacillary load at 56 days p.i suggesting that Mtb has established chronic infection in the lungs (Figure 7—figure supplement 1a). Subsequently, tissue lysates prepared from uninfected and Rv infected mice lungs were subjected to western blotting to evaluate the expression of molecular markers corresponding to DNA damage and DDR. While the levels of γH2AX, pATM, pChk2, and pAkt was negligible in the uninfected mice lungs, we observed significantly elevated levels of γH2AX, pATM, pChk2 and pAkt in the lungs of Rv infected mice, suggesting that Mtb infection produces sustained DSBs and Akt mediated proliferation (Figure 7b and Figure 7—figure supplement 1b). In a similar vein, we also observed increased levels of γH2AX in the lysates obtained from spleen of infected mice (Figure 7b; bottom panels). CFUs in spleen after 56 days of infection suggests dissemination of Rv from lungs (Figure 7a). Collectively the results suggest that pathogenic Mtb induces genotoxicity both ex vivo and in vivo resulting in deleterious DSBs in the host genome.

DSBs activate ATM, which in turn phosphorylates H2AX to mark the damaged site and activates Chk2 and p53 to alter the cell cycle progression. In parallel, it also activates Akt to delay cell death and promote survival of the pathogen (Figure 7b). We hypothesized that Mtb mediated genotoxicity provides a survival niche through activation of ATM kinase (Figure 7b). We sought to explore the possibility of utilizing ATM inhibitor ATM-I as a possible candidate that can be used in combination with INH towards adjunct host directed therapy (HDT) (Rayasam and Balganesh, 2015). To investigate this possibility, PΦ were infected with Rv and the infection was allowed to establish for 24 hr. The cells were then treated with either vehicle or INH or ATM-I or INH +ATM-I and the CFUs were enumerated at 24, 48 and 72 hr post treatment. The treatment of Rv infected cells with ATM-I or INH alone decreased the survival of pathogen by ~2 or 4-fold, respectively (Figure 7c and Figure 7—source data 1). Notably, treatment of Rv infected cells with ATM-I + INH decreased the pathogen survival by ~11 fold. This encouraged us to investigate the possibility of utilizing ATM inhibitor toward adjunct HDT for TB with the help of murine infection model. Mice were aerosolically infected with Rv and 15 days p.i mice were treated with vehicle or INH or ATM-I or INH+ATM-I for the next 15 days followed by CFU enumeration in both lungs and spleen (Figure 7d). While treatment with INH alone resulted ~1 log fold reduction in the Mtb load, treatment with ATM-I alone did not show any difference. Notably, combination of ATM-I + INH treatment resulted in ~1 log fold better clearance compared with INH treatment alone (Figure 7e and Figure 7—source data 1). The impact of ATM-I+INH treatment compared with INH treatment alone was more evident in the spleen (Figure 7f and Figure 7—source data 1), suggesting combined therapy might have compromised dissemination of the pathogen. Taken together, we propose that ATM inhibitor is a potential candidate for HDT.

Discussion

The present study was designed to address the following questions: i) Does Mtb target the genomic integrity of the host? If so, do virulent and avirulent Mtb target the genomic integrity of the host differentially, and in that case, how? ii). How the host cell responds to the DNA damage mediated through Mtb? iii). What is the role of the Mtb secretome, if any, in imparting DNA damage? iv). What is the role of ATM kinase activation and subsequent downstream signaling in the survival of pathogen within the host? vi). Is this Mtb mediated DNA damage manifested in the mice model of in vivo infection?

Results show that Mtb infection promotes γH2AX upregulation in ex vivo models of murine/human origin and murine primary macrophages. The induction of γH2AX and subsequent formation of foci could also be detected by immunofluorescence (Figure 1a–h). These results are in accordance with earlier studies wherein induction of γH2AX foci and genomic instability was detected through immunofluorescence and in situ hybridization, respectively, in the ex vivo model of Mtb infection (Castro-Garza et al., 2018; Mohanty et al., 2016). While the induction of γH2AX is dependent on the presence of the live bacteria, Ra a non-pathogenic counterpart of Rv could also induce γH2AX levels, albeit at comparatively slower rates (Figure 1d–g). In addition to temporal differences, Ra and Rv appear to inflict distinct types of damages resulting in the activation of ATR and ATM pathways, respectively (Figure 2b). The infection with Rv results in lingering γH2AX levels even at later time points, suggesting the sustained presence of pathogenic Mtb continues to inflict DNA damage (Figure 3a). In consonance with this hypothesis, elimination of intracellular Mtb drastically reduced γH2AX levels/DNA damage, suggesting Mtb may be inducing DNA damage at a faster frequency relative to the host’s ability to repair them (Figure 3c).

In response to the DSBs generated upon pathogenic E. coli infection, host activates phosphorylation of Cdc25 through ATM-Chk2 pathway to hamper cell cycle progression. However, at higher MOI, host cell undergoes p53 SUMOylation through SENP1 downregulation to promote senescence (Cougnoux et al., 2014). On the other hand, N. gonorrhoeae decelerate the host cell cycle by reducing p53 and concurrent elevation of p21 and p27 (Vielfort et al., 2013). L. monocytogenes in addition to impairing host MRE11 also activates DDR in ATM/ATR independent fashion eventually resulting in cell cycle delay (Leitão et al., 2014). Taken together, it is evident that pathogens utilize diverse and distinct mechanisms to perturb the host cell cycle to avoid untimely death. We observed activation of ATM-Chk2 pathway (Figure 2b) and subsequent decrease in the cells in G1 phase (Figure 3e), an indicator of differed host cell cycle at the early stages of Mtb infection. However, at the later time points, infected cells showed increased cell death, most likely an effort by the host to curb the transmission of genotoxic effects (Figure 3e). Interestingly, cell cycle perturbations were limited to the cells possessing intracellular bacilli but not extended to bystander-uninfected cells (Figure 3e). Thus, our results demonstrate that Mtb mediated DNA damage disturbs the homeostatic coordination among the cellular components participating in cell cycle.

Toxins like Cytolethal distending toxin (CDT), typhoid toxin and colibactin from pathogenic bacteria are examples of characterized toxins that are reported to block the host cell cycle progression after inducing DNA damage in the host (El-Aouar Filho et al., 2017). Persistent presence of DSBs and modulation of host cell cycle in cells harboring bacilli, propelled us to examine the role of Mtb secretory factors in imparting genotoxicity. The proteins exported from Mtb are considered a source of virulence, however not many secreted proteins have been individually characterized. Mtb secretes >300 secretory factors with the help of secretory systems such as classical TAT and SecA1; accessory SecA2 and TypeVII. Results presented in Figure 4e–h suggest that SecA2 mediated secretory factors are principally responsible for the DSBs in the host. Mass spectrometry-based study has identified the secretion of 37 proteins, including Mce1, Mce4 family and multiple solute binding lipoproteins to be dependent on the SecA2 pathway. In addition to these, secretion of protein kinase G (PknG), superoxide dismutase (SodA) and phosphatase SapM proteins are also through SecA2 pathway (Feltcher et al., 2015; Miller et al., 2017; Sullivan et al., 2012; Zulauf et al., 2018). As polyketides and lipids from mycobacteria were suggested to be cyclomodulins; therefore, we speculate that one or more lipoproteins secreted through the SecA2 pathway could be critical for genotoxicity (Cumming et al., 2017; George et al., 1998). Macrophages generate reactive oxidation species as an anti-microbial activity to combat the survival of Mtb intracellularly. Mice lacking p47 and gp91 subunit of NADPH oxidase (gp91phox-/-) are susceptible to Mtb as the macrophages in these mice fail to generate ROS (Cooper et al., 2000). While the primary purpose of generated ROS is to clear the intracellular bacilli, it can however damage its own DNA as exemplified in case of C. trachomatis infection (Chumduri et al., 2013; Yu and Anderson, 1997). On the contrary, H. pylori, L. monocytogenes mediated DSBs are ROS-independent. SodAMtb, which is secreted through SecA2 pathway plays an important role in detoxification of ROS generated inside the host thus partially neutralizing the anti-microbial property of ROS (Chan et al., 1992; Lau et al., 1998; Piddington et al., 2001). In RvΔSecA2 mutant SodAMtb secretion is expected to be compromised and hence one would expect higher levels of host ROS upon infection. The fact that RvΔSecA2 mutant fails to inflict DSBs suggest that Mtb mediated damage is unlikely to be ROS-dependent. In agreement with this, addition of N-acetyl cysteine (NAC), a scavenger of ROS, to the infected cells did not alter the γH2AX, suggesting that DNA damage is not mediated through ROS (Figure 7—figure supplement 1c). Our future study would be aimed towards identifying the specific effector(s) and the mechanism of how the effector brings about DSBs in the host.

Mtb is known to modulate multiple host signaling pathways to establish favorable environment in the host for its survival. Moreover, a previous study (Hinchey et al., 2007) showed that RvΔsecA2 strongly induced markers of apoptosis in both human and murine macrophages suggesting a critical role played by SecA2 secretome in preventing apoptosis. Thus, we investigated the role of Mtb induced host DNA damage and subsequent activation of ATM-Chk2 pathway in facilitating and enhancing its survival. While presence of ATM inhibitor significantly compromised the Mtb survival, addition of inhibitor for one of the downstream effectors, Chk2 which is associated to cell cycle checkpoints activation, failed to do so (Figure 5e). These results suggest that while the survival signals for Mtb are dependent on activation of ATM, it is not channeled through the classical DDR. High-throughput study revealed that following DSBs, ATM-dependent phosphorylation of ~700 substrates regulate multitude of cellular processes such as cell cycle, cellular differentiation etc., emphasizing its global regulatory role. Importantly, DDR was suggested to crosstalk with the IGF-1/PI3K/Akt pathway to elicit cell survival signals, which is also supported by data that demonstrated ATM-dependent phosphorylation of Akt at S473 (Khalil et al., 2011; Matsuoka et al., 2007). Previous reports suggested that inhibiting Akt diminished Mtb survival inside host cells and proposed it to be a novel target for host directed therapy (Kuijl et al., 2007). Thus, we hypothesized a probable divergence of ATM signaling towards Akt activation may play a role in influencing Mtb survival. We observed that addition of Akt-I compromises the survival of Mtb (Figure 6f). Furthermore, combinatorial addition of both ATM and Akt inhibitors did not show either additive or synergistic impact on the Mtb survival, suggesting that survival signals are routed through ATM-Akt axis (Figure 6f).

Mtb infection of the host cells can induce host cell death through both apoptotic and non-apoptotic pathways. Apoptotic death is considered to be advantageous for the host as it results in the elimination of pathogen (Fratazzi et al., 1999; Gan et al., 2008; Molloy et al., 1994). Induction of non-apoptotic death of the host cells is beneficial for the bacilli as it fails to eliminate the pathogen (Dobos et al., 2000; Keane et al., 2000; Lee and Paull, 2005; Martin et al., 2012; Park et al., 2006). Recently, it was reported that internalization of Mtb aggregates by the host cell results in killing of the host and there was a direct correlation between the size of aggregates and cell death. However, the death in such cases was through non-apoptotic pathway (Mahamed et al., 2017). In our experiments we kept MOI at one to circumvent aggregation and non-apoptotic cell death of the host. We observed significantly increased apoptosis upon the addition of inhibitors for either ATM or Akt or both (Figure 6g). These results are in line with the previous findings, wherein bacterial pathogens such as Salmonella typhimurium and Coxiella burnetii have been shown to get survival advantage through Akt activation (Kuijl et al., 2007; Voth and Heinzen, 2009).

We hypothesize that persistent DSBs caused by Mtb could be a means to constitutively activate ATM, which in turn would activate Akt and inhibit apoptosis thus facilitating the survival of the pathogen. We corroborated our finding in the murine model of chronic tuberculosis infection, wherein the infected lungs showed the activation of γH2AX and ATM-Chk2 signaling (Figure 7a–b). Addition of ATM inhibitor to the INH regime substantially reduced the bacilli load in the host establishing ATM as a potent target for HDT (Figure 7c–f). In conclusion, our study demonstrates that Mtb through SecA2 secretome challenges the host fitness by damaging the genome (Figure 7g). In an effort to repair the damage, the host cell activates the ATM kinase mediated signaling cascade, which is exploited by Mtb to survive. We believe we have identified a novel survival mechanism utilized by Mtb, wherein the pathogen constantly challenges the host genome leading to the activation of pro-survival ATM-Akt signals (Figure 7g). Hence, we propose the use of ATM inhibitors as adjunct for HDT in the treatment of tuberculosis.

Materials and methods

Materials

Fine chemicals used in the study were purchased from Sigma-Merck. Bacterial media components were purchased from Difco-BD biosciences. All cell culture reagents were procured from Thermo fisher scientific Inc or Hyclone. α-γH2AX (2577S), α-ATM (2873S), α-pATR-S428 (2853S), α-pChk1-S345 (2348S), α-pChk2-T68 (2661S), α-Chk2 (2662S), α-CHK1(2360), α-ATR (13934), α-pAkt-S473 (9271S), α-Akt (9272S), α-p53BP1-T543 (3428S), α-MRE11(4895S), α-pNbs1-S343 and α-Nbs1 (3002S) antibodies were procured from Cell Signalling Technologies. α-pATM-S1891(ab81292) α−53BP1 (ab36823), α-pMDC1-T4 (ab35967), α-RPA2 (ab2175) and α-p53 (ab28) antibody was procured from Abcam. α-p21 (sc-817), α-β-actin (sc-47778), α-β-tubulin (sc-55529) and α-GAPDH (sc-25778) antibodies were purchase from Santacruz Biotechnology. α-DNA-PKcs (SAB4502385), α-pDNA-PKcs-S2056 (SAB4504169) were purchased from Sigma. α-MDC1 and α-pRPA2-S4/S8 was purchased from Novus Biologicals and HRP conjugated secondary antibodies were purchased from Jackson laboratories. α-MDC1 was purchased from Novus Biologicals. AlaxaFluor594 and AlexaFuor647 fluorophore conjugated secondary antibodies were procured from Thermo fisher scientific Inc.

Mtb strains, cell culture and Mtb infection

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Mtb strains, H37Rv (Rv) and H37Ra (Ra), RvΔRD1 and RvΔSecA2 were propagated in Middlebrook 7H9 broth (Difco) supplemented with 10% ADC (Albumin-Dextrose-Catalase complex), 0.2% glycerol (v/v), 0.05% (v/v) Tween-80 with shaking at 100 rpm at 37°C. Rv and Ra strains were electroporated with pSC-301-GFP plasmid (Cowley and Av-Gay, 2001) to generate GFP expressing strains. Human acute monocytic leukemia cell line, THP-1 (ATCC) and RAW264.7 (ATCC), peritoneal macrophages (PΦ) derived from Balb/c mice were grown in the cell culture media (RPMI-1640 constituted with 1X Anti-Anti and 10% heat inactivated (HI)-fetal bovine serum) at 37°C. THP-1 cells were treated with 10 ng/mL PMA for 24 hr followed by 12 hr resting without PMA before Mtb infection. RAW264.7 cells were seeded 12–15 hr before Mtb infection. PΦ were extracted from thioglycolate injected Balb/c mice as described earlier (Zhang et al., 2008). PΦ isolated from the peritoneal cavity were resuspended in cell culture media and infected 12–15 hr post plating with Mtb. THP1 cells used in the study have been authenticated by STR profiling. All the cell lines used were free of mycoplasma contamination. Single-cell suspensions of actively growing Mtb strains were prepared and all infections were performed at MOI of 1, else indicated. Four hours p.i extracellular bacilli were removed by washing thrice with PBS and the cells were supplemented with fresh cell culture media for defined periods of time. For the inhibitor studies, RAW or PΦ were pre-treated with different doses (1, 5 or 10 µM) of ATM kinase inhibitor (KU55933, SML1109, Sigma; S1092, Selleckchem) or Chk2 inhibitor (C3742, Sigma) or Akt1/2 kinase inhibitor (A6730, Sigma), ATR inhibitor (VE-281, SML1415, Sigma), DNA-PK inhibitor (NU7441, 18003649897, Cayman chemical company). ATM-I, DNA-PK-I and ATR-I were used at reported dose of 10 µM, 1 µM and 10 µM, respectively. 4 hr p.i cells were supplemented with fresh media containing inhibitors. For the clearance of intracellular Mtb from infected RAW cells, cells were treated with 1 µg/ml (7.3 µM) of isoniazid (INH). Colony forming units (CFUs) were enumerated at 24, 48 and 72 hr p.i to evaluate the bacterial load. RAW/PΦ were lysed in 0.05% SDS and different dilutions of the lysates were plated on 7H11 agar plates supplemented with 10% OADC (oleic acid- Albumin-Dextrose-Catalase complex). After 21 days, CFUs of Mtb were enumerated.

Preparation of lysates, western blotting, and immunofluorescence

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Cell lines were lysed and snap-frozen mice lungs and spleen were macerated in cold RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1.0% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS, freshly supplemented with PhosSTOP and Complete protease inhibitor (Roche). The lysates were resolved in 10–15% SDS-PAGE followed by transfer to nitrocellulose membrane (Biorad). Antibody dilutions, incubation time and temperature conditions were according to the manufacturers’ protocol. Blots were developed on autoradiograms using chemiluminescent HRP substrate (ECL, Millipore). For immunofluorescence assay, 5 × 105 cells were seeded on sterile coverslips in 6-well tissue culture plates and infected with GFP expressing Rv/Ra. At defined time points, cells were fixed with 2% PFA and immunostaining was performed according to the manufacturers’ protocol provided by respective antibody. Coverslips were mounted on slides with DAPI containing Vectashield mountant (H-1200, Vector Labs). Images were captured in Carl Zeiss LSM510 Meta confocal microscope.

Animal infection

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Actively growing Rv cultures were used for aerosolically infecting mice to implant 200 CFU/lung of Balb/c mice (6–8 weeks old of either sex) inside Madison Aerosol Chamber (University of Wisconsin, Madison, WI). Infection load in lungs and spleen was determined at day 1 and 56 p.i to evaluate deposition and commencement of chronic infection of Mtb, respectively. One lung and part of spleen of infected mice were homogenized and plated on 7H11 plates supplemented with PANTA (BD Bioscience) to enumerate CFUs. Second lung and part of the spleen from infected and uninfected mice were used preparation of tissue lysates. In another experimental set up, Balb/c mice were infected with Rv as stated above. Post 15 days of infection, mice were either kept untreated or treated with Vehicle (5% DMSO in sterile PBS), INH (prepared in sterile water, 25 mg/kg/dose through oral gavage), KU55933 (prepared in DMSO and further diluted in PBS such that DMSO is 5% (v/v) in PBS before injecting in the peritoneal cavity; administered every third day at 10 mg/Kg/dose [Batey et al., 2013]) or INH+ATM-I according to the dosage stated for next 15 days. Five batches of six mice each (No treatment, Vehicle, INH, ATM-I or INH+ATM-I) at 30 day p.i were used for assessing Mtb CFUs in lungs and spleen.

Cell cycle analysis and apoptosis assay

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RAW cells infected with GFP expressing Rv at MOI of 1 for indicated time points. Cells were fixed with 70% ethanol for overnight at 4°C followed by PBS washes and staining with 10 µg/ml propidium iodide (PI) solution (PBS+0.05% Triton-X + 100 µg/ml RNase A). Cells were analyzed through flow cytometry (FACS Calibur, Becton Dickinson, USA) to assess the distribution of cells in different phases of the cell cycle. For the apoptosis assay, PΦ infected with Rv and treated with the indicated inhibitors were stained with FITC Annexin V-7AAD according to manufacturer’s protocol (BioLegend). Following staining, cells were fixed with 2% PFA before assessing the cell percentage undergoing apoptosis through flow cytometry.

Culture filtrate protein (CFP) preparation

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Mtb strains were cultured in modified sauton’s medium (2.9 mM KH2PO4, 4.2 mM MgSO4, 10.4 mM citric acid, 0.2 mM Ferric ammonium citrate, 6% Glycerol, 30.5 mM asparagine, pH 7.2) till mid-log phase. Culture supernatants were passed through 0.22 µM filter and concentrated to 1/100th of the original volume using Amicon concentrators with a 3 kDa cut off (Rosenkrands and Andersen, 2001). The proteins concentration in the obtained culture filtrates was quantitated.

Statistical analysis

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GraphPad Prism eight software and MS office Excel 2010 was used to execute statistical calculations. Unpaired student’s t test was used to determine the p-values. Significant p values of the data sets were considered significant *p<0.05, **p<0.005, ***p<0.0005 and not significant (ns) if p>0.05. Densitometric analysis of important western blots (Figure 7—figure supplement 2) was performed using Image J software (Schneider et al.).

Appendix 1

Supplementary methods

Cell viability assays

MTT Assay was performed in triplicates to evaluate the cell viability during the treatment of ATM-I, AKT-I and Chk-I. PΦ were pre-treated for 1 hr with different doses (1, 5, 10 or 20 µM) ATM-I or Chk-I or Akt-I prior to infection. Post-infection cells were washed with PBS, and supplemented with fresh media with or without inhibitors. 4, 24, 48 hr p.i soluble MTT substrate (3-(4,5- dimethylthiazol-2-y1)−2,5-diphenyl tetrazolium bromide) was added at a final concentration of 500 µg/ml and cells were incubated for 4 hr. 500 µl of DMSO was added to each well, mixed and the absorbance was measured at 595 nm. Cell viability of the untreated samples were considered 100%. Cell viability in the presence of the inhibitor/Cell viability in the presence DMSO for the samples were calculated with respect to the corresponding value for the untreated sample.

Average O.D. of sample = [Average O.D. of the sample- Average O.D. of blank]

Cell viability (%) = [average O.D. of treated sample/average O.D. of untreated sample] x 100

Minimum inhibitory concentration (MIC) determination

A stock of 73 mM of INHwas prepared in sterile water, which was serially diluted in 7H9 media to a final concentration of 73 µM. 10 mM stock of ATM-I in DMSO was used to make final concentration as 100 µM in complete 7H9 media. Actively growing Rv cultures at 0.6 OD were centrifuged and resuspended in fresh 7H9 complete media. 2-fold serial dilutions of INH and ATM-I in triplicates were prepared in sterile 96-well plates. The resuspended bacilli were then added in the 96-well plate such that each well receives ~10,000 bacilli. Wells with Rv alone was used as the control. The plates were sealed with parafilm and incubated at 37°C for 6 days followed by the addition of 20 µl resazurin (0.02% in sterile water). The plates were further incubated for 12–24 hr at 37°C till the development of color. While blue color of resazurin converts into pink for viable cells, the color remains blue in case of dead cells. MIC is defined as the lowest drug concentration that inhibits the growth bacilli. Results were assessed on the basis of color development.

Densitometric analysis

Densitometric analysis of major findings was performed with the help of Image J software. Briefly, scans of autoradiograms (300 dpi) were opened in Image J software (Schneider et al., 2012) and the intensities of bands in each lane was individually measured. The intensities of γH2AX/pATM/ pATR/pChk2/pChk1/pAkt/p53 in an image were normalized with respect to their corresponding loading control β-actin /β-tubulin/GAPDH. Once the values were obtained for all the lanes in an image, the fold change was calculated as follows:

Fold Change = [Normalized intensity of a band/highest normalized intensity in that image]

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Decision letter

  1. Wendy S Garrett
    Senior Editor; Harvard TH Chan School of Public Health, United States
  2. Bavesh D Kana
    Reviewing Editor; University of the Witwatersrand, South Africa
  3. Alasdair Lesley
    Reviewer

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This study provides novel insight suggesting that pathogenic mycobacteria, such as Mycobacterium tuberculosis, secrete a genotoxic agent through the SecA2 pathway with important implications for immune responses and pathogenesis. As such, inhibitors of the DNA damage response, include the ATP kinase described in your work, and DNA-PK inhibitors, could be used as host-directed therapy in combination with current antimicrobials to improve tuberculosis treatment outcomes. This work provides promising new avenues for further research and opens the possibility of testing existing agents, with good safety and toxicity profiles, in clinical trials as adjunctive therapy to standard TB treatment.

Decision letter after peer review:

Thank you for submitting your article "Mycobacterium tuberculosis exploits host ATM kinase for survival advantage through SecA2 secretome" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Wendy Garrett as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Alasdair Lesley (Reviewer #1).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

The submission by Nandicoori explores activation of the ATM kinase, which occurs during colonization of macrophages by Mycobacterium tuberculosis. The authors study this in human and mouse macrophage cell lines and in primary mouse macrophages. The activation of ATM kinase led to the phosphorylation of cognate substrates Chk2 and γH2AX, the latter being detected in the form of nuclear foci, an observation that supports the induction of DNA double-strand breaks in response to M. tuberculosis infection. In contrast, a non-virulent strain (H37Ra) led to activation of the human ATR kinase, detected by its phosphorylation and phosphorylation of its substrate, Chk1. DNA damage induction could be recapitulated using bacterial supernatants and was abolished when using heat-killed bacteria or a mutant of the SecA2 secretion system. These data support the SecA2-dependent secretion of a genotoxic agent/s by virulent mycobacteria. Inhibition of ATM led to a small reduction in bacterial load in infected mouse macrophages and when combined with the antibiotic isoniazid, a synergistic effect was demonstrated. Inhibition of ATM led to apoptosis in a large proportion of infected macrophages, supporting a model where ATM promotes M. tuberculosis survival in its host. Inhibition of the Chk2 kinase, acting classically downstream of ATM to block cell cycle progression, did not phenocopy ATM inhibition while inhibition of Akt did. Akt has been reported as being activated by ATM but also by DNA-PK (another kinase activated together with ATM upon DNA double-strand breaks). Consistent with this, the authors report here an ATM dependent activation of Akt upon infection. These findings are of potential interest as they support the hypothesis that virulent mycobacteria secrete a genotoxic agent, that still awaits characterization, through the SecA2 pathway and suggest that inhibitors of the human DNA damage response, including ATM inhibitors and probably DNA-PK inhibitors, could be used as host-directed therapy in combination with current antimicrobials. Several of these inhibitors have already undergone clinical trials to be used alone or in combination with DNA damaging agents to cure various human cancers and some of them have demonstrated safety and efficacy.

Essential revisions:

1) In Figure 2D, phosphoATM seems nucleolar on the micrographs. This is unexpected. This could be an important finding if confirmed. To confirm signal specificity, an immunofluorescence (IF) experiment would be needed using the ATM inhibitor KU55933 to show that the nucleolar staining disappears after treatment. To confirm the localization, co-staining of phosphoATM upon infection should be performed with a nucleolar marker (e.g. nucleolin).

2) Considering that the formation of γH2AX foci is the strongest evidence for DNA double-strand break induction presented in the manuscript, pictures acquired at higher resolution showing the focal pattern of the staining should be provided. In addition, IF should also be performed using an antibody against another protein forming foci in response to DNA double-strand breaks, for example 53BP1.

3) When considering (i) the major role of NHEJ in repairing DNA double-strand break in human cells (Ciccia and Elledge, 2010) and (ii) the role of the NHEJ complex DNA-PK in activating Akt (Lan et al. Oncotarget 2016; Stronach et al. Neoplasia 2011), the impact of the inhibition of DNA-PK kinase activity on M. tuberculosis infection should be investigated in this study in parallel to ATM inhibition. This could be tested using one of the specific DNA-PK inhibitors available, such as NU7441.

4) After treatment with classical DNA double-strand break inducing agent, production of γH2AX is mainly ATM dependent, but has also been reported in some circumstance to be DNA-PK and/or ATR dependent. In Figure 5B, treatment with the ATM inhibitor only reduces γH2AX production, which suggests that other kinases might be at work. The authors should test the implication of ATR and DNA-PK in this residual phosphorylation, since specific inhibitors against these kinases are commercially available.

5) The authors report ATR activation upon infection by the H37Ra attenuated strain. IF should be performed (after H37Ra and H37Rv infection) to monitor for the accumulation of the protein RPA (complex of RPA1, RPA2 and RPA3) in the form of foci, which would support the accumulation of single-strand DNA upon infection (RPA is recruited on single-strand DNA regions).

6) Previous work has implicated ROS in the production of DNA damage in infected host cells, the role of ROS should be investigated here. An experiment with N-acetyl-cysteine or membrane permeable reduced glutathione should be performed.

7) Figure 6A and B do not provide sufficient data to support conclusions. Rather, the authors should test the effect of the KU inhibitor in vivo.

8) Furthermore, the model suggests AKT signalling promotes survival by blocking M. tuberculosis induced apoptosis. Therefore, M. tuberculosis infection with AKT or ATM inhibition should lead to increased cell death by apoptosis. However, this is very hard to assess with the data presented as, in Figure 5H, the effect of inhibitor alone is not tested (so the increased apoptosis could just be due to the inhibitor – which, as from Figure 5—figure supplement 1, does cause cell death); and in Figure 5—figure supplement 1, there is no increased cell death when M. tuberculosis is added. The authors should assess the effect of the inhibitors (KU and AKT-I) alone on apoptosis without M. tuberculosis infection.

9) Western blot data should be quantified and statistically analyzed. Some blots are sliced/edited others not in the same panel (Figure 3C); a single membrane should be used.

10) The model that decreased apoptosis is associated with increased bacterial growth is also contrary to recently published live imaging studies suggesting that macrophage death is essential for rapid bacterial growth (Mahamed et al., 2017). The authors should discuss this.

https://doi.org/10.7554/eLife.51466.sa1

Author response

Essential revisions:

1) In Figure 2D, phosphoATM seems nucleolar on the micrographs. This is unexpected. This could be an important finding if confirmed. To confirm signal specificity, an immunofluorescence (IF) experiment would be needed using the ATM inhibitor KU55933 to show that the nucleolar staining disappears after treatment. To confirm the localization, co-staining of phosphoATM upon infection should be performed with a nucleolar marker (e.g. nucleolin).

We concur with the observation regarding localization of pATM. In order to address the question, we performed immunofluorescence of PФ infected with GFP-Rv at 24 h p.i. To obtain better resolution, we captured the images at 100X (in the previous version we did it at 63X) with an optical zoom of 2.9X. It is apparent from the newly added data (Figure 2E) that pATM foci are observed throughout the nuclei. We also observed distinct localization of pATM foci at what appears to be nucleolar. We confirmed the specificity of these signals by performing pATM immunofluorescence in the presence of ATM inhibitor KU55933 (Figure 5B). Treatment of cells with ATM inhibitor led to disappearance of pATM foci, suggesting that the foci indeed correspond to pATM.

Further we made several attempts to co-stain nucleoli with nucleolin (cat no. 396400; Thermo Fisher Scientific) and pATM antibody. Unfortunately, despite our best efforts and even when utilized two other nucleolin antibodies, we were unsuccessful in getting the staining for the nucleolin. Therefore, while we agree with the reviewers that pATM localization in the nucleolus seems to be interesting, we have no definitive experimental proof of this observation.

2) Considering that the formation of γH2AX foci is the strongest evidence for DNA double-strand break induction presented in the manuscript, pictures acquired at higher resolution showing the focal pattern of the staining should be provided. In addition, IF should also be performed using an antibody against another protein forming foci in response to DNA double-strand breaks, for example 53BP1.

Thank you for the comment. We agree with the reviewers that images at higher resolution would provide us a better staining pattern for one of the most important DNA damage marker, γH2AX. Presence of mediator proteins such as 53BP1, which accumulate at the DNA damage site would corroborate our findings. We therefore performed immunofluorescence of PФ infected with GFP-Rv at 24 h p.i. for γH2AX and 53BP1 (Figure 2F). To obtain higher resolution images, they were captured at 100X magnification with an optical zoom of 2.9X. Results show distinct focal pattern and colocalization of γH2AX and 53BP1 in Rv infected PФ (Figure 2F).

3) When considering (i) the major role of NHEJ in repairing DNA double-strand break in human cells (Ciccia and Elledge, 2010) and (ii) the role of the NHEJ complex DNA-PK in activating Akt (Lan et al. Oncotarget 2016; Stronach et al. Neoplasia 2011), the impact of the inhibition of DNA-PK kinase activity on M. tuberculosis infection should be investigated in this study in parallel to ATM inhibition. This could be tested using one of the specific DNA-PK inhibitors available, such as NU7441.

We thank reviewers for an insightful suggestion. Infection of PФ with Rv did indeed result in the activation of DNA-PK, albeit to much lower levels and at later time points compared with the pATM (Figure 6A). To assess the role of DNA-PK and ATR in the survival of pathogen in the host, cells treated with inhibitors individually were infected with Rv and CFUs were enumerated (Figure 6C). It is evident that the addition of NU7441 (inhibitor of DNA-PK) or VE-281 (inhibitor of ATR) did not alter the CFUs, suggesting that even though DNA-PK seems to be activated in the DDR pathway, it does not influence the survival of the pathogen (Figure 6C).

4) After treatment with classical DNA double-strand break inducing agent, production of γH2AX is mainly ATM dependent, but has also been reported in some circumstance to be DNA-PK and/or ATR dependent. In Figure 5B, treatment with the ATM inhibitor only reduces γH2AX production, which suggests that other kinases might be at work. The authors should test the implication of ATR and DNA-PK in this residual phosphorylation, since specific inhibitors against these kinases are commercially available.

Results in Figure 5A (Figure 5B in the previous version) showed presence of residual γH2AX despite KU treatment. While phosphorylation of H2AX is predominantly modulated by ATM; ATR and DNA-PKcs may also contribute either partially or entirely to its phosphorylation (Ciccia and Elledge, 2010). Thus, as suggested by reviewers we assessed the possible role of combinatorically inhibiting ATM and DNA-PK or ATR on the levels of γH2AX. PФ treated with KU or KU + NU or KU + VE were infected with Rv and the extent of H2AX phosphorylation was evaluated by western blot. While addition of VE+KU did not alter the residual levels of γH2AX, addition of NU +KU resulted in further decrease in the levels of γH2AX, suggesting that DNA-PKcs also contributes to H2AX phosphorylation. To assess the role of DNA-PK and ATR in the ex-vivo survival of pathogen, host cells treated individually with inhibitors were infected with Rv and CFUs were enumerated (Figure 6C). It is evident that addition of NU or VE did not alter the CFUs, suggesting that even though DNA-PK seems to be activated in the DDR pathway, it does not influence the survival of the pathogen (Figure 6C).

5) The authors report ATR activation upon infection by the H37Ra attenuated strain. IF should be performed (after H37Ra and H37Rv infection) to monitor for the accumulation of the protein RPA (complex of RPA1, RPA2 and RPA3) in the form of foci, which would support the accumulation of single-strand DNA upon infection (RPA is recruited on single-strand DNA regions).

As suggested by reviewers, we evaluated differential accumulation of RPA2 and pRPA2 in UI, Ra and Rv infected cells. Infection by Ra led to the formation of distinct nuclear pRPA2 (Figure 2C) and RPA2 (Figure 2—figure supplement 1) foci, which corroborate robust activation of ATR-Chk1 pathway (Figure 2B; right panel), suggesting that Ra mediates generation of ssDNA. On the other hand, infection by Rv showed far fewer foci for RPA2 or pRPA2.

6) Previous work has implicated ROS in the production of DNA damage in infected host cells, the role of ROS should be investigated here. An experiment with N-acetyl-cysteine or membrane permeable reduced glutathione should be performed.

We agree with the reviewers that ROS is known to generate DNA damage as exemplified in case of C. trachomatis infection (Chumduri et al., 2013). On the contrary, H. pylori, L. monocytogenes mediated DSBs are ROS-independent. SodAMtb, which is secreted through SecA2 pathway plays an important role in detoxification of ROS generated inside the host thus partially neutralizing the anti-microbial property of ROS. In RvΔSecA2 mutant, SodAMtb secretion is expected to be compromised and hence one would expect higher levels of host ROS upon infection. The fact that RvΔSecA2 mutant fails to inflict DSBs suggests that Mtb mediated damage is unlikely to be ROS-dependent. In agreement with this, addition of N-acetyl cysteine (NAC), a scavenger of ROS, to the infected cells did not alter the γH2AX, suggesting that DNA damage is not mediated through ROS (Figure 7—figure supplement 1B).

7) Figure 6A and B do not provide sufficient data to support conclusions. Rather, the authors should test the effect of the KU inhibitor in vivo.

We agree with the reviewer that in vivo validation of PФ infection data (Figure 7C in the revised manuscript) is necessary. We investigated the possibility of utilizing ATM inhibitor towards adjunct host directed therapy (HDT) for TB with the help of murine infection model. Mice were infected aerosolically with Rv and 15 days post infection mice were treated with vehicle or INH or KU or INH + KU for next 15 days and CFUs were enumerated in both lungs and spleen (Figure 7D). As anticipated, treatment with INH resulted in ~ 1 log fold reduction in the load of Mtb. While treatment with KU alone did not show any difference in the clearance of the pathogen, combination of KU + INH treatment resulted in ~ 1 log fold better clearance compared with INH treatment alone (Figure 7E). Notably, the impact of KU+INH treatment compared with INH treatment alone was more evident in the spleen (Figure 7F), suggesting that combined therapy compromises dissemination of the pathogen. Taken together, we propose that ATM inhibitor is a potential candidate for HDT.

8) Furthermore, the model suggests AKT signalling promotes survival by blocking M. tuberculosis induced apoptosis. Therefore, M. tuberculosis infection with AKT or ATM inhibition should lead to increased cell death by apoptosis. However, this is very hard to assess with the data presented as, in Figure 5H, the effect of inhibitor alone is not tested (so the increased apoptosis could just be due to the inhibitor – which, as from Figure 5—figure supplement 1, does cause cell death); and in Figure 5—figure supplement 1, there is no increased cell death when M. tuberculosis is added. The authors should assess the effect of the inhibitors (KU and AKT-I) alone on apoptosis without M. tuberculosis infection.

The MTT assay quantifies the relative quantity of viable and healthy cells by measuring the reduction of yellow tetrazolium dye, MTT by mitochondrial enzymes. We agree with the reviewers that inhibitors alone do impact the health of cell, which we could observe when we plotted the graphs at an inhibitor concentration of 10 µM. (Graphs in Author response image 1, same as Figure 6—figure supplement 1 (presently) but plotted UI and Rv in the same graph).

Author response image 1
Cell viability upon treatment with inhibitors.

However, we have not statistically quantitated the difference. To quantify the number of cells undergoing apoptosis we performed Annexin V-7AAD staining wherein Annexin V recognizes the phosphatidylserine (PS) of the inner membrane and 7AAD is a nuclear stain. Even though these two assays utilize different approaches and have different sensitivity levels, in principle we observed similar readout of compromised health of Rv infected cells undergoing inhibitor treatment.

To address the point raised by reviewers, we performed Annexin V-7AAD staining both in UI and Rv infected cells in the presence of DMSO or KU or Akt-I or KU +Akt-I. Previously we performed the staining at 72 h. Since in MTT assay, we followed only up to 48 h, this time round, we performed Annexin V-7AAD staining at 48 h. While the addition of inhibitors did not impact the percent live cells (Figure 6G; top panel) in UI cells, their presence decreased the percent live cells Rv infected cells (Figure 6G; top panel). We observed concomitant increase in the early and late apoptotic cells in the presence of KU or Akt-I or KU + Akt-I (Figure 6G; bottom panel).

9) Western blot data should be quantified and statistically analyzed. Some blots are sliced/edited others not in the same panel (Figure 3C); a single membrane should be used.

To address the concerns, we have quantified and statistically analyzed the blots with major findings. Briefly densitometric analysis of major findings was performed with the help of Image J software. Scans of autoradiograms (300 dpi) were opened in Image J software and the intensities of bands in each lane was individually measured. The intensities of γH2AX/pATM/pATR/pChk2/pChk1/pAkt/p53 were normalized with respect to their corresponding loading control β-actin/β-tubulin/GAPDH. Once the values were obtained for all the lanes in an image, the fold change was calculated as follows.

Fold Change = [Normalized intensity of a band / highest normalized intensity in that panel]

The figure numbers of the corresponding western blot, which is a representative blot of one of the replicates, is given above each graph. The quantitation is provided in the revised manuscript in Figure 6—figure supplement 2 and Figure 7—figure supplement 2.

Sliced blots in Figure 3C have been replaced with unsliced blots. However, the sliced blots in Figure 4B, C, G and H are originally from the same membrane (please find the raw data for Figure 4B, C, G, and H). We have sliced out Ra infected lanes from these blots.

Author response image 2
Raw data for Figure 4B and C.

Lanes corresponding to Ra (lanes 9-12 in 4B and lanes 4-6 in 4C) have been spliced out. The splice junctions are indicated in the figure.

Author response image 3
Raw data for Figure 4G.

Lanes corresponding to Ra (lanes 5 and 6) have been spliced out. The splice junctions are indicated in the figure.

Author response image 4
Raw data for Figure 4H.

Lanes corresponding to Ra (lanes 5 and 6) have been spliced out. The splice junctions are indicated in the figure.

10) The model that decreased apoptosis is associated with increased bacterial growth is also contrary to recently published live imaging studies suggesting that macrophage death is essential for rapid bacterial growth (Mahamed et al., 2017). The authors should discuss this.

In the recent paper published in eLife (Mahamed et al., 2017), it was reported that internalization of Mtb aggregates by the macrophage results in killing of the host cell and there was direct correlation between the size of aggregates and cell death. However, the death in such cases was through non-apoptotic pathway. In our experiments we kept MOI at 1 to circumvent aggregation and non-apoptotic cell death of the host. Mtb infection of the host cells can induce host cell death through both apoptotic and non-apoptotic pathways. Apoptotic death is considered to be advantageous for the host as it results in the elimination of pathogen. Induction of non-apoptotic death of the host cells is beneficial for the pathogen as it fails to eliminate the pathogen. The above aspects have been included in the Discussion with appropriate references. We observed significantly increased apoptosis upon the addition of inhibitors for either ATM or Akt or both (Figure 6). These results are in line with the previous findings, wherein bacterial pathogens such as Salmonella typhimurium and Coxiella burnetii have been shown to get survival advantage through Akt activation (Kuijl et al., 2007; Voth and Heinzen, 2009).

https://doi.org/10.7554/eLife.51466.sa2

Article and author information

Author details

  1. Savita Lochab

    1. National Institute of Immunology, New Delhi, India
    2. Department of Zoology, University of Delhi, New Delhi, India
    Contribution
    Conceptualization, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8153-7867
  2. Yogendra Singh

    Department of Zoology, University of Delhi, New Delhi, India
    Contribution
    Funding acquisition
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3902-4355
  3. Sagar Sengupta

    National Institute of Immunology, New Delhi, India
    Contribution
    Conceptualization, Formal analysis, Supervision
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6365-1770
  4. Vinay Kumar Nandicoori

    National Institute of Immunology, New Delhi, India
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Project administration, Writing - review and editing
    For correspondence
    vinaykn@nii.ac.in
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5682-4178

Funding

Department of Biotechnology, Ministry of Science and Technology (BT/PR13522/COE/34/27/2015)

  • Vinay Kumar Nandicoori

University Grants Commission (DS Kothari Postdoctoral Fellowship)

  • Savita Lochab

Department of Science and Technology, Ministry of Science and Technology (JCB/2018/000013)

  • Sagar Sengupta

National Institute of Immunology

  • Sagar Sengupta

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was supported by the funding provided by Department of Biotechnology, Government of India (BT/PR13522/COE/34/27/2015) and National Institute of Immunology (NII) core funding. SL is thankful to NII Young Investigator fellowship and UGC-DS Kothari postdoctoral fellowship. SS acknowledges National Institute of Immunology Core funding and JC Bose Fellowship (JCB/2018/000013) for financial assistance. We thank the bio-containment facility (BSL3), central FACS and confocal facilities at NII. We thank Dr. Miriam Braunstein and Dr. Dheeraj Kumar for their kind gifts of RvΔsecA2 and RvΔRD1 strains of Mtb, respectively. Mice infection experiments were carried out at DBT funded Tuberculosis Aerosol Challenge Facility at ICGEB, Delhi, India. We thank Dr. Lakhyaveer Singh and his team for their help during animal infection experiments at TACF, ICGEB. We thank Dr. Swati Saha for her critical reading and helpful suggestions.

Ethics

Animal experimentation: The experimental protocol for the animal experiments was approved by the Animal Ethics Committee of the National Institute of Immunology, New Delhi, India. The approval (IAEC#409/16 & IAEC#462/18) is as per the guidelines issued by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.

Senior Editor

  1. Wendy S Garrett, Harvard TH Chan School of Public Health, United States

Reviewing Editor

  1. Bavesh D Kana, University of the Witwatersrand, South Africa

Reviewer

  1. Alasdair Lesley

Publication history

  1. Received: August 29, 2019
  2. Accepted: March 27, 2020
  3. Accepted Manuscript published: March 30, 2020 (version 1)
  4. Version of Record published: April 16, 2020 (version 2)
  5. Version of Record updated: May 27, 2020 (version 3)

Copyright

© 2020, Lochab et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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