Mycobacterium tuberculosis dihydrofolate reductase is a target for isoniazid

Isoniazid is a key drug used in the treatment of tuberculosis. Isoniazid is a pro-drug, which, after activation by the katG-encoded catalase peroxidase, reacts nonenzymatically with NAD(+) and NADP(+) to generate several isonicotinoyl adducts of these pyridine nucleotides. One of these, the acyclic 4S isomer of isoniazid-NAD, targets the inhA-encoded enoyl-ACP reductase, an enzyme essential for mycolic acid biosynthesis in Mycobacterium tuberculosis. Here we show that the acyclic 4R isomer of isoniazid-NADP inhibits the M. tuberculosis dihydrofolate reductase (DHFR), an enzyme essential for nucleic acid synthesis. This biologically relevant form of the isoniazid adduct is a subnanomolar bisubstrate inhibitor of M. tuberculosis DHFR. Expression of M. tuberculosis DHFR in Mycobacterium smegmatis mc(2)155 protects cells against growth inhibition by isoniazid by sequestering the drug. Thus, M. tuberculosis DHFR is the first new target for isoniazid identified in the last decade.     

Macrophage apoptosis in response to high intracellular burden of Mycobacterium tuberculosis is mediated by a novel caspase-independent pathway

We previously reported that macrophage exposure to attenuated strains of pathogenic mycobacteria at multiplicities of infection (MOI) < or = 10 triggers TNF-alpha-mediated apoptosis which reduces the viability of intracellular bacilli. Virulent strains were found to suppress macrophage apoptosis, and it was proposed that apoptosis is an innate defense against intracellular Mycobacterium tuberculosis analogous to apoptosis of virus-infected cells. The potential similarity of host cell responses to intracellular infection with mycobacteria and viruses suggests that M. tuberculosis might lyse infected macrophage when that niche is no longer needed. To investigate this question, we challenged murine macrophages with high intracellular bacillary loads. A sharp increase in cytolysis within 24 h was observed at MOI > or = 25. The primary death mode was apoptosis, based on nuclear morphology and phosphatidyl serine exposure, although the apoptotic cells progressed rapidly to necrosis. Apoptosis at high MOI differs markedly from low MOI apoptosis: it is potently induced by virulent M. tuberculosis, it is TNF-alpha-independent, and it does not reduce mycobacterial viability. Caspase inhibitors failed to prevent high MOI apoptosis, and macrophages deficient in caspase-3, MyD88, or TLR4 were equally susceptible as wild type. Apoptosis was reduced in the presence of cathepsin inhibitors, suggesting the involvement of lysosomal proteases in this novel death response. We conclude that the presence of high numbers of intracellular M. tuberculosis bacilli triggers a macrophage cell death pathway that could promote extracellular spread of infection and contribute to the formation of necrotic lesions in tuberculosis.     

Human mesenchymal stem cell based intracellular dormancy model of Mycobacterium tuberculosis

Understanding the biology of the tuberculosis pathogen during dormant asymptomatic infection, called latent tuberculosis is crucial to decipher a resilient therapeutic strategy for the disease. Recent discoveries exhibiting presence of pathogen’s DNA and bacilli in mesenchymal stem cells (MSCs) of human and mouse despite completion of antitubercular therapy, indicates that these specific cells could be one of the niches for dormant Mycobacterium tuberculosis in humans. To determine if in vitro infection of human MSCs could recapitulate the in vivo characteristics of dormant M. tuberculosis, we examined survival, phenotype, and drug susceptibility of the pathogen in MSCs. When a very low multiplicity of infection (1:1) was used, M. tuberculosis could survive in human bone marrow derived MSCs for more than 22 days without any growth. At this low level of infection, the pathogen did not cause any noticeable host cell death. During the later phase of infection, MSC-residing M. tuberculosis exhibited increased expression of HspX (a 16-kDa alpha-crystallin homolog) with a concurrent increase in tolerance to the frontline antitubercular drugs Rifampin and isoniazid. These results present a human MSC-based intracelllular model of M. tuberculosis infection to dissect the mechanisms through which the pathogen acquires and maintains dormancy in the host.     

Escape of Mycobacterium tuberculosis from oxidative killing by neutrophils

Neutrophils enter sites of infection, where they can eliminate pathogenic bacteria in an oxidative manner. Despite their predominance in active tuberculosis lesions, the function of neutrophils in this important human infection is still highly controversial. We observed that virulent Mycobacterium tuberculosis survived inside human neutrophils despite prompt activation of these defence cells' microbicidal effectors. Survival of M. tuberculosis was accompanied by necrotic cell death of infected neutrophils. Necrotic cell death entirely depended on radical oxygen species production since chronic granulomatous disease neutrophils were protected from M. tuberculosis-triggered necrosis. More, importantly, the M. tuberculosis ΔRD1 mutant failed to induce neutrophil necrosis rendering this strain susceptible to radical oxygen species-mediated killing. We conclude that this virulence function is instrumental for M. tuberculosis to escape killing by neutrophils and contributes to pathogenesis in tuberculosis.     

CD43 controls the intracellular growth of Mycobacterium tuberculosis through the induction of TNF-alpha-mediated apoptosis

Establishment of Tuberculosis infection begins with the successful entry and survival of the pathogen within macrophages. We previously showed that macrophage CD43 is required for optimal uptake and growth inhibition of Mycobacterium tuberculosis both in vitro and in vivo. Here, we explore the mechanisms by which CD43 restricts mycobacterial growth in murine macrophages. We found that although M. tuberculosis grows more readily in resting CD43-/- macrophages, priming of cells with IFN-gamma returns the bacterial growth rate to that seen in CD43+/+ cells. To discern the mechanisms by which M. tuberculosis exhibits enhanced growth within resting CD43-/- macrophages, we assessed the induction of inflammatory mediators in response to infection. We found that absence of CD43 resulted in reduced production of TNF-alpha, IL-12 and IL-6 by M. tuberculosis-infected macrophages. We also found that infected resting, but not activated CD43-/- macrophages, showed decreased apoptosis and increased necrosis. Exogenous addition of the pro-inflammatory cytokine TNF-alpha restored control of M. tuberculosis growth and induction of apoptosis to CD43+/+ levels. We propose that CD43 is involved in the inflammatory response to M. tuberculosis and, through the induction of pro-inflammatory mediators, can regulate apoptosis to control intracellular growth of the bacterium.     

Novel drug target strategies against Mycobacterium tuberculosis

The resurgence of drug resistant tuberculosis (TB) is a significant global healthcare challenge. Mycobacterium tuberculosis (MTB), TB's causative agent, evades the host immune system and drug regimes by entering prolonged periods of non-proliferation or dormancy. In infected individuals, the immune system sequesters MTB into structures called granulomas where the bacterium survives by shifting into a non-replicative state. Although still not well understood, progress has been made in characterizing the genetic program of MTB, activated by DosR (DevR) signal transduction that allows adaptation to the hypoxic, nutrient limiting granuloma microenvironment. Recent work, especially the identification genes involved in regulatory networks and the Enduring Hypoxic Response (EHR), hold promise for developing new drugs targeting dormancy phase MTB.     

Bim is a crucial regulator of apoptosis induced by Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis, induces apoptosis in infected macrophages in vitro and in vivo. However, the molecular mechanism controlling this process is not known. In order to study the involvement of the mitochondrial apoptotic pathway in M. tuberculosis-induced apoptosis, we analysed cell death in M. tuberculosis-infected embryonic fibroblasts (MEFs) derived from different knockout mice for genes involved in this route. We found that apoptosis induced by M. tuberculosis is abrogated in the absence of Bak and Bax, caspase 9 or the executioner caspases 3 and 7. Notably, we show that MEF deficient in the BH3-only BCL-2-interacting mediator of cell death (Bim) protein were also resistant to this process. The relevance of these results has been confirmed in the mouse macrophage cell line J774, where cell transfection with siRNA targeting Bim impaired apoptosis induced by virulent mycobacteria. Notably, only infection with a virulent strain, but not with attenuated ESX-1-defective strains, such as Bacillus Calmette-Guerin and live-attenuated M. tuberculosis vaccine strain MTBVAC, induced Bim upregulation and apoptosis, probably implicating virulence factor early secreted antigenic target 6-kDa protein in this process. Our results suggest that Bim upregulation and apoptosis is mediated by the p38MAPK-dependent pathway. Our findings show that Bim is a master regulator of apoptosis induced by M. tuberculosis.Mycobacterium tuberculosis, the causative agent of tuberculosis, is primarily an intracellular pathogen that has successfully developed strategies to colonise host alveolar macrophages and overcome their bactericidal defence mechanisms.¹Apoptosis is a physiological type of cell death characterised by the preservation of the plasma membrane integrity, which prevents local inflammatory reactions and tissue damage. Intracellular pathogens have co-evolved with the host to develop strategies for modulation of host cell apoptosis to favour infection.² During M. tuberculosis infection, presence of apoptotic cells has been detected in lungs from both infected humans and mice.^{3, 4, 5} ESX-1 secretion system, which regulates early secreted antigenic target 6-kDa protein (ESAT-6) secretion, seems to play a crucial role in apoptosis induction and virulence during mycobacterial infection.^{3, 6} It has been shown that attenuated strains, like Bacillus Calmette-Guerin (BCG) and the live-attenuated M. tuberculosis vaccine Mycobacterium tuberculosis vaccine strain (MTBVAC),⁷ which lack a functional ESX-1 secretion system, have lost their ability to induce apoptosis and cell death.^{3, 8} Altogether, these results suggest that the ability to induce apoptotic cell death is a feature characteristic of virulent strains. Indeed, similarly to other authors, we have shown that apoptosis triggered by virulent mycobacteria is required for bacterial spread.^{3, 9}The activation of the mitochondrial cell death pathway is regulated by the Bcl-2 family of proteins consisting of pro-apoptotic (Bak, Bax, Bim, Bid and so on) and anti-apoptotic (Bcl-2, Bcl-X_L, Mcl-1 and so on) members, whose activity is reciprocally modulated.¹⁰ BH3-only pro-apoptotic proteins (i.e., Bid, BCL-2-interacting mediator of cell death (Bim), Puma and Noxa) interfere with anti-apoptotic proteins Bcl-2, Bcl-X_L or Mcl-1, and induce Bak and Bax activation by conformational change, leading to mitochondrial permeabilization.¹¹ Pore formation on mitochondrial membrane leads to the release of pro-apoptotic factors to cytosol. One of these molecules, cytochrome c, is necessary to activate caspase 9,¹² which activates the effector caspases 3 and 7 by cleavage. These are ultimately responsible for the appearance of the apoptotic phenotype.The intracellular mediators of apoptosis induced by M. tuberculosis are poorly understood. Previous works have shown that virulent M. tuberculosis strains are able to activate the mitochondrial cell death pathway including cytochrome c release and caspase activation.^{4, 13} However, the molecular mechanism including the involvement of the Bcl-2 family in this process remains unknown. In this work, we conducted an in-depth analysis of the implication of different pro-apoptotic members of the Bcl-2 family during apoptosis induced by the clinical isolate MT103 in different cell lines. We have identified the BH3-only protein Bim as a key modulator of apoptosis induction and bacterial spread.     

Mitochondria are a primary target of hypericin phototoxicity: synergy of intracellular calcium mobilisation in cell killing

Hypericin, a naturally occurring anthraquinone synthesised by hypericum, upon light activation exhibits photodynamic cytotoxicity attributed mainly to the production of reactive oxygen species. This study aimed to elucidate the primary subcellular targets and mechanistic aspects of hypericin photosensitization in human prostate carcinoma cells. Depletion of intracellular glutathione (>85%) via inhibition of gamma-glutamyl-cysteine synthase had no effect on hypericin (5 microM) phototoxicity, thus precluding any direct oxidative involvement of H2O2. There was no change in intracellular SOD activity immediately after hypericin irradiation (1.5-5 J cm(-2)). Evaluation of the lysosomal enzyme hexosaminidase activity showed: (a) 60% cell loss 22 h following irradiation (1.5 J cm(-2)) and (b) a steady rate of lysosomal leakage to the cytosol (25%), at the same time and irradiation. However, lysosomal damage appears to be a slower process compared to the rapid loss of mitochondrial function, as reflected from parallel tetrazolium to formazan assays. The activity of cytosolic and mitochondrial aconitase, an enzyme exquisitely sensitive to oxidation, revealed a dose correlated loss of activity in the mitochondria immediately following hypericin photoactivation. The use of ionomycin, which modulates both internal Ca2+ stores and external Ca2+ transport during hypericin photosensitization, profoundly enhanced photocytotoxicity. Our data supports a direct mitochondrial hypericin phototoxicity that does not involve glutathione/H2O2 homeostasis. Further a potential synergistic treatment combining mitochondrial targeting of photosensitisers and Ca2+ mobilisation was identified.     

The susceptibility of strains of Mycobacterium tuberculosis to catalase-mediated peroxidative killing

At low pH and with continuous low concentrations of hydrogen peroxide generated in situ, catalase was able to replace peroxidase in the peroxidase/hydrogen peroxide/iodide microbicidal system. The system was effective against Escherichia coli and Mycobacterium tuberculosis. Iodide could not be replaced by chloride. The system was effective in lactate buffer, but not in citrate/phosphate buffer. Strains of M. tuberculosis with high and low virulence were equally susceptible. The observations are discussed in the context of an involvement of host-cell catalase in a possible intracellular killing mechanism against M. tuberculosis.     

Virulence of Mycobacterium tuberculosis and susceptibility to peroxidative killing systems.

At sub-bactericidal concentrations of hydrogen peroxide, Mycobacterium tuberculosis was killed by hydrogen peroxide/peroxidase/halide microbicidal systems. The halide cofactor could be either iodide or, with much lower efficiency, chloride. Omission of any one of the reactants eliminated the tuberculocidal effect. Differences in susceptibility between different strains of M. tuberculosis did not correlate with virulence differences. The observations are discussed in the context of host defence mechanisms against tuberculosis.     

Mycobacterium tuberculosis nuoG is a virulence gene that inhibits apoptosis of infected host cells

The survival and persistence of Mycobacterium tuberculosis depends on its capacity to manipulate multiple host defense pathways, including the ability to actively inhibit the death by apoptosis of infected host cells. The genetic basis for this anti-apoptotic activity and its implication for mycobacterial virulence have not been demonstrated or elucidated. Using a novel gain-of-function genetic screen, we demonstrated that inhibition of infection-induced apoptosis of macrophages is controlled by multiple genetic loci in M. tuberculosis. Characterization of one of these loci in detail revealed that the anti-apoptosis activity was attributable to the type I NADH-dehydrogenase of M. tuberculosis, and was mainly due to the subunit of this multicomponent complex encoded by the nuoG gene. Expression of M. tuberculosis nuoG in nonpathogenic mycobacteria endowed them with the ability to inhibit apoptosis of infected human or mouse macrophages, and increased their virulence in a SCID mouse model. Conversely, deletion of nuoG in M. tuberculosis ablated its ability to inhibit macrophage apoptosis and significantly reduced its virulence in mice. These results identify a key component of the genetic basis for an important virulence trait of M. tuberculosis and support a direct causal relationship between virulence of pathogenic mycobacteria and their ability to inhibit macrophage apoptosis.     

Ceramide-induced apoptosis in fas-resistant Hodgkin's disease cell lines is caspase independent

We investigated whether cell-permeable, synthetic ceramide (C6 ceramide) could induce apoptosis in Fas-resistant Hodgkin's disease (HD)-derived cell lines. Despite strongly expressing the Fas-receptor, two of three HD-derived cell lines were resistant to Fas-mediated apoptosis. This resistance to Fas could not be attributed to differential Fas isoform generation patterns between the Fas-resistant and the Fas-sensitive cell lines. The Fas-resistant cell lines did not demonstrate the presence of Fas exon 8 deletion. Bcl-2 and BclxL levels were comparable between the Fas-resistant and the Fas-sensitive cell lines. C6 ceramide could induce apoptosis in both Fas-resistant cell lines and this was associated with a decrease in BclxL level. Caspase-1, caspase-3, or pan-caspase inhibitors could not prevent ceramide-induced apoptosis. Furthur, ceramide treatment did not lead to cleavage of caspase 3 or poly(ADP-ribose) polymerase, but caused a loss in mitochondrial transmembrane potential which could not be prevented by caspase inhibitors. Thus, we conclude that ceramide-induced apoptosis in Fas-resistant HD cell lines is caspase independent.     

Analysis of intracellular expressed proteins of Mycobacterium tuberculosis clinical isolates

Background

Tuberculosis (TB) is the most threatening infectious disease globally. Although progress has been made to reduce global incidence of TB, emergence of multidrug resistant (MDR) TB threatens to undermine these advances. To combat the disease, novel intervention strategies effective against drug resistant and sensitive subpopulations of M. tuberculosis are urgently required as adducts in the present treatment regimen. Using THP-1 cells we have analyzed and compared the global protein expression profile of broth-cultured and intraphagosomally grown drug resistant and sensitive M.tuberculosis clinical isolates.

Results

On comparing the two dimensional (2-DE) gels, many proteins were found to be upregulated/expressed during intracellular state which were identified by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Four proteins (adenosylhomocysteinase, aspartate carbomyltransferase, putatitive thiosulfate sulfurtransferase and universal stress protein) were present in both intracellular MDR and sensitive isolates and three of these belonged to intermediary metabolism and respiration category. Two proteins (alanine dehydrogenase and adenosine kinase) of intracellular MDR isolate and two (glucose-6-phosphate isomerase and ATP synthase epsilon chain) of intracellular sensitive isolate belonged to intermediary metabolism and respiration category. One protein (Peroxidase/Catalase) of intracellular MDR and three (HSPX, 14 kDa antigen and 10 kDa chaperonin) of sensitive isolate belonged to virulence, detoxification and adaptation category. ESAT-6 of intracellular MDR belonged to cell wall and cell processes category. Two proteins (Antigen 85-C and Antigen 85-A) of intracellular sensitive isolate were involved in lipid metabolism while probable peptidyl-prolyl cis-trans isomerase A was involved in information pathways. Four (Rv0635, Rv1827, Rv0036c and Rv2032) of intracellular MDR and two proteins (Rv2896c and Rv2558c) of sensitive isolate were hypothetical proteins which were functionally characterized using bioinformatic tools. Bioinformatic findings revealed that the proteins encoded by Rv0036, Rv2032c, Rv0635, Rv1827 and Rv2896c genes are involved in cellular metabolism and help in intracellular survival.

Conclusions

Mass spectrometry and bioinformatic analysis of both MDR and sensitive isolates of M. tuberculosis during intraphagosomal growth showed that majority of commonly upregulated/expressed proteins belonged to the cellular metabolism and respiration category. Inhibitors of the metabolic enzymes/intermediate can therefore serve as suitable drug targets against drug-resistant and sensitive subpopulations of M. tuberculosis.     

Constrained intracellular survival of Mycobacterium tuberculosis in human dendritic cells

Dendritic cells (DCs) are likely to play a key role in immunity against Mycobacterium tuberculosis, but the fate of the bacterium in these cells is still unknown. Here we report that, unlike macrophages (Mphis), human monocyte-derived DCs are not permissive for the growth of virulent M. tuberculosis H37Rv. Mycobacterial vacuoles are neither acidic nor fused with host cell lysosomes in DCs, in a mode similar to that seen in mycobacterial infection of Mphis. However, uptake of the fluid phase marker dextran, and of transferrin, as well as accumulation of the recycling endosome-specific small GTPase Rab11 onto the mycobacterial phagosome, are almost abolished in infected DCs, but not in Mphis. Moreover, communication between mycobacterial phagosomes and the host-cell biosynthetic pathway is impaired, given that <10% of M. tuberculosis vacuoles in DCs stained for the endoplasmic reticulum-specific proteins Grp78/BiP and calnexin. This correlates with the absence of the fusion factor N-ethylmaleimide-sensitive factor onto the vacuolar membrane in this cell type. Trafficking between the vacuoles and the host cell recycling and biosynthetic pathways is strikingly reduced in DCs, which is likely to impair access of intracellular mycobacteria to essential nutrients and may thus explain the absence of mycobacterial growth in this cell type. This unique location of M. tuberculosis in DCs is compatible with their T lymphocyte-stimulating functions, because M. tuberculosis-infected DCs have the ability to specifically induce cytokine production by autologous T lymphocytes from presensitized individuals. DCs have evolved unique subcellular trafficking mechanisms to achieve their Ag-presenting functions when infected by intracellular mycobacteria.     

Siderocalin inhibits the intracellular replication of Mycobacterium tuberculosis in macrophages

Siderocalin is a secreted protein that binds to siderophores to prevent bacterial iron acquisition. While it has been shown to inhibit the growth of Mycobacterium tuberculosis ( M.tb ) in extracellular cultures, its effect on this pathogen within macrophages is not clear. Here, we show that siderocalin expression is upregulated following M.tb infection of mouse macrophage cell lines and primary murine alveolar macrophages. Furthermore, siderocalin added exogenously as a recombinant protein or overexpressed in the RAW264.7 macrophage cell line inhibited the intracellular growth of the pathogen. A variant form of siderocalin, which is expressed only in the macrophage cytosol, inhibited intracellular M.tb growth as effectively as the normal, secreted form, an observation that provides mechanistic insight into how siderocalin might influence iron acquisition by the bacteria in the phagosome. Our findings are consistent with an important role for siderocalin in protection against M.tb infection and suggest that exogenously administered siderocalin may have therapeutic applications in tuberculosis.     

Mycobacterium tuberculosis KatG is a peroxynitritase

Mycobacterium tuberculosis resides within the highly oxidative environment of the human macrophage and previous reports have indicated that these mycobacteria are susceptible to reactive nitrogen intermediates including peroxynitrite. This work provides evidence that the Mycobacterium tuberculosis hemoprotein KatG acts as an efficient peroxynitritase exhibiting a kapp of 1.4 x 10(5) M-1s-1 for peroxynitrite decomposition at pH 7.4 and 37 degrees C. The ability of KatG to act as a peroxynitritase adds to its growing list of enzymatic activities and may in part explain the ability of Mycobacterium tuberculosis to persist in macrophages.     

Camptothecin-induced apoptosis in non-small cell lung cancer is independent of cyclooxygenase expression

Recent observations show a positive correlation between the expression of cyclooxygenase (COX), especially COX-2), and cancer development. Here we tested the hypothesis that expression of COX-2 could influence apoptosis in lung cancer cell lines. To address this question, we determined the effects of camptothecin-induced apoptosis on three lung cancer cell lines which over express COX-1 (CORL23), COX-2 (MOR-P) and neither isoform (H-460), and determine if these effects were prostaglandin mediated. We also compared the effects of non-selective and isoenzyme selective COX-2 inhibitors on camptothecin-induced apoptosis in these three cell lines. Camptothecin induced apoptosis in all three cell lines independently of COX-1 or COX-2 expression. Indomethacin, a non-selective COX inhibitor and NS398, a selective COX-2 inhibitor had no effect on camptothecin-induced apoptosis at concentrations that abolished prostaglandin production. In conclusion, these finding suggest that the COX pathway is not involved in camptothecin-induced apoptosis of non-small cell lung cancer cell lines.