cytochrome-c-t and Tuberculosis

Host metabolism reprogramming is a key feature of Mycobacterium tuberculosis (

Topics: Cytochromes c; Energy Metabolism; Host-Pathogen Interactions; Humans; Mitochondria; Mycobacterium tuberculosis; Tuberculosis

The mycobacterial cytochrome

Topics: Cryoelectron Microscopy; Cytochromes c; Humans; Mycobacterium tuberculosis; Oxygen; Protons; Tuberculosis

Apoptosis inhibition is a critical strategy of mycobacteria facilitating its survival in macrophages, but the underlying mechanism is not completely understood. In this study, we found that Rv3033, a secreted virulence factor of mycobacteria, played an important role in bacillary survival within macrophages. Forced over-expressed of Rv3033 in macrophages could efficiently resist mycobacteria-induced early and late apoptosis, accompanied with the obvious increased cellular bacterial burden. By exploring the underlying mechanism, we found that Rv3033 efficiently repressed the intrinsic (caspase-9 meditated), but not the extrinsic (caspase-8 mediated) apoptotic pathway in mycobacteria-infected macrophages. And this repression relied on the orchestrating blockade of both mitochondrial cytochrome c release and endoplasmic reticulum (ER) stress PERK branch activation. Our study uncovered a novel function of mycobacterial virulence factor Rv3033 as an anti-apoptotic protein, which may provide a new target for tuberculosis (TB) treatment.

Topics: Animals; Apoptosis; Bacterial Proteins; Caspase 9; Cytochromes c; eIF-2 Kinase; Endoplasmic Reticulum Stress; HEK293 Cells; Humans; Inhibitor of Apoptosis Proteins; Macrophages; Mice; Mycobacterium tuberculosis; RAW 264.7 Cells; Tuberculosis; Virulence Factors

The emergence of drug resistant strains of Mycobacterium tuberculosis (M. tuberculosis) together with reports of co-infections with the human immunodeficiency virus (HIV) has renewed interest to better understand the intricate mechanisms prevalent during co-infections. In this study we report a synergistic effect of M. tuberculosis and HIV-1, and their antigens Rv3416 and Nef, respectively, in inhibiting apoptosis of macrophages. This inhibition involves the TLR2 pathway and second messengers that play complementing and contrasting roles in regulating apoptosis. Interestingly, the route of calcium influx into cells differentially regulates apoptosis during antigenic co-stimulation. While calcium released from intracellular stores was anti-apoptotic, calcium influx from the external milieu was pro-apoptotic. Further, molecular sensors of intracellular calcium release aid in antigen mediated inhibition of apoptosis. A cross-regulation between oxidative burst and differential routing of calcium influx governed apoptosis. Interestingly, the HIV-1 Nef supported anti-apoptotic responses in macrophages whereas Vpu had no significant effect. These results point to a synergistic liaison between M. tuberculosis and HIV-1 in regulating macrophage apoptosis.

Topics: Antigens; Apoptosis; Bacterial Proteins; Calcium; Cells, Cultured; Coinfection; Cytochromes c; Gene Expression Regulation; HEK293 Cells; HIV Infections; HIV-1; Homeostasis; Humans; Leukocytes, Mononuclear; Macrophages; Membrane Potential, Mitochondrial; Mycobacterium tuberculosis; nef Gene Products, Human Immunodeficiency Virus; Respiratory Burst; RNA, Small Interfering; Signal Transduction; Toll-Like Receptor 2; Tuberculosis

Mycobacterium tuberculosis depends on aerobic respiration for growth and utilizes an aa3-type cytochrome c oxidase for terminal electron transfer. Cytochrome c maturation in bacteria requires covalent attachment of heme to apocytochrome c, which occurs outside the cytoplasmic membrane. We demonstrate that in M. tuberculosis the thioredoxin-like protein Rv3673c, which we named CcsX, is required for heme insertion in cytochrome c. Inactivation of CcsX resulted in loss of c-type heme absorbance, impaired growth and virulence of M. tuberculosis, and induced cytochrome bd oxidase. This suggests that the bioenergetically less efficient bd oxidase can compensate for deficient cytochrome c oxidase activity, highlighting the flexibility of the M. tuberculosis respiratory chain. A spontaneous mutation in the active site of vitamin K epoxide reductase (VKOR) suppressed phenotypes of the CcsX mutant and abrogated the activity of the disulfide bond-dependent alkaline phosphatase, which shows that VKOR is the major disulfide bond catalyzing protein in the periplasm of M. tuberculosis.. Mycobacterium tuberculosis requires oxygen for growth; however, the biogenesis of respiratory chain components in mycobacteria has not been explored. Here, we identified a periplasmic thioredoxin, CcsX, necessary for heme insertion into cytochrome c. We investigated the consequences of disrupting cytochrome c maturation (CCM) for growth and survival of M. tuberculosis in vitro and for its pathogenesis. Appearance of a second-site suppressor mutation in the periplasmic disulfide bond catalyzing protein VKOR indicates the strong selective pressure for a functional cytochrome c oxidase. The observation that M. tuberculosis is able to partially compensate for defective CCM by upregulation of the cytochrome bd oxidase exposes a functional role of this alternative terminal oxidase under normal aerobic conditions and during pathogenesis. This suggests that targeting both oxidases simultaneously might be required to effectively disrupt respiration in M. tuberculosis.

Topics: Animals; Bacterial Proteins; Cytochromes c; Electron Transport; Electron Transport Complex IV; Female; Heme; Humans; Male; Mice, Inbred C57BL; Mycobacterium tuberculosis; Oxygen; Thioredoxins; Tuberculosis

Topics: Animals; Bacterial Proteins; Catalase; Cytochromes; Cytochromes c; Electron Transport Chain Complex Proteins; Escherichia coli; Escherichia coli Proteins; Female; Humans; Male; Mycobacterium tuberculosis; Oxidative Stress; Oxidoreductases; Tuberculosis

Experimental models have shown that lipoproteins from Mycobacterium tuberculosis induce apoptosis via Toll-like receptor 2 (TLR2) in the THP-1 cell line and in monocyte-derived macrophages from healthy volunteers. We found an increased percentage of circulating monocytes in patients with tuberculosis (TB) in comparison to healthy controls. Patients with TB showed a higher TLR2 and TLR4 expression density on monocytes, and a higher proportion of TLR2(+) monocytes, as well as increased serum tumour necrosis factor-α level. In culture, monocytes from TB patients were more susceptible to death than monocytes from healthy controls. Moreover, death-susceptible monocytes were positive to both TLR2 and TLR4 at the start of culture. Freshly obtained monocytes from TB patients exhibited cleaved caspase 9 and denaturalized cytochrome c. For levels of caspase 8, apoptosis-regulating signal kinase 1, and phospho-p38 mitogen-activated protein kinase there was no difference between samples from TB patients and from healthy controls. The culture filtrate antigen extract from M. tuberculosis H37Rv strain induced the death of monocytes from patient with TB after a 4-hr incubation, which was abrogated by neutralizing antibodies for TLR2 but not TLR4. Similarly, Pam3CSK4, a synthetic agonist triacylated ligand to TLR2, also induced the death of monocytes, although it did not increase levels of cleaved caspase 9. Our findings suggest that monocytes from TB patients are more susceptible to death, probably through mitochondrial damage, and that cell death increases in the presence of mycobacterial antigen by a TLR2-dependent pathway.

Topics: Adult; Antigens, Bacterial; Apoptosis; Caspase 9; Cytochromes c; Female; Humans; Male; Middle Aged; Monocytes; Mycobacterium tuberculosis; Toll-Like Receptor 2; Toll-Like Receptor 4; Tuberculosis; Young Adult

The present study was conducted to see the role of NF-kappaB in virulent (Mycobacterium tuberculosis H37Rv) and avirulent (M. tuberculosis H37Ra) mycobacterial infection in THP-1 cells. To inactivate NF-kappaB, pCMV-IkappaBalphaM dn containing THP-1 cell line was generated which showed marked increase in apoptosis with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Infected THP-1-IkappaBalphaM dn cells showed decrease in mitochondrial membrane potential, cytochrome c release, activation of caspase-3 and enhanced TNF-alpha production. Increase in apoptosis of infected THP-1-IkappaBalphaM dn cells resulted in inhibition of intracellular mycobacterial growth. Differential NF-kappaB activation potential was observed with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Both the strains activated NF-kappaB after 4 h in THP-1 cells however after 48 h only M. tuberculosis H37Rv activated NF-kappaB which lead to up-regulation of bcl-2 family anti-apoptotic member, bfl-1/A1. Our results indicated that NF-kappaB activation may be a determinant factor for the success of virulent mycobacteria within macrophages.

Topics: Apoptosis; Caspase 3; Cell Line; Cytochromes c; Enzyme Activation; Humans; I-kappa B Proteins; Macrophages; Membrane Potential, Mitochondrial; Microbial Viability; Minor Histocompatibility Antigens; Mitochondria; Mutation; Mycobacterium tuberculosis; NF-kappa B; NF-KappaB Inhibitor alpha; Protein Transport; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Time Factors; Transcription Factor RelA; Transfection; Tuberculosis; Tumor Necrosis Factor-alpha; Up-Regulation; Virulence

CD4(+) T cells are central in mediating granuloma formation and limiting growth and dissemination of mycobacterial infections. To determine whether T cells responding to influenza infection can interact with T cells responding to Mycobacterium bovis bacille Calmette-Guérin (BCG) infection and disrupt granuloma formation, we infected mice containing two monoclonal T cell populations specific for the model Ags pigeon cytochrome c (PCC) and hen egg lysozyme (HEL). These mice were chronically infected with PCC epitope-tagged BCG (PCC-BCG) and acutely infected with HEL epitope-tagged influenza virus (HEL-flu). In these mice, PCC-BCG infection is much more abundant in the liver than the lung, whereas HEL-flu infection is localized to the lung. We observe that both T cells have access to both inflammatory sites, but that PCC-specific T cells dominate the PCC-BCG inflammatory site in the liver, whereas HEL-specific T cells dominate the HEL-flu inflammatory site in the lung. Influenza infection, in the absence of an influenza-specific T cell response, is able to increase the activation state and IFN-gamma secretion of PCC-BCG-specific T cells in the granuloma. Activation of HEL-specific T cells allows them to secrete IFN-gamma and contribute to protection in the granuloma. Ultimately, infection with influenza has little effect on bacterial load, and bacteria do not disseminate. In summary, these data illustrate complex interactions between T cell responses to infectious agents that can affect effector responses to pathogens.

Topics: Animals; Antigens; Cell Communication; Chickens; Clone Cells; Columbidae; Cytochromes c; Granuloma; Humans; Immunity; Influenza, Human; Mice; Mice, Knockout; Muramidase; Mycobacterium bovis; T-Lymphocytes; Tuberculosis