Macrophage response to Mycobacterium tuberculosis during HIV infection: relationships between macrophage activation and apoptosis

Human macrophages represent the first line of defense for the containment of Mycobacterium tuberculosis infection. After phagocytosis, macrophages express activation surface markers and produce proinflammatory cytokines and chemokines whose main role is to control pathogen spreading by recruiting peripheral lymphocytes and monocytes at the site of inflammation. However, in the case of a concomitant human immunodeficiency virus (HIV) infection, these signals strongly enhance the susceptibility to viral infection both at the viral entry and replication levels. Under these conditions, viral expansion extends beyond tissue macrophages to T cells and vice-versa, according to the emerging viral phenotype. In absence of an efficient immune response, Mycobacterium tuberculosis can replicate in macrophages in an uncontrolled fashion culminating in macrophage death by apoptosis. As a consequence, a more severe form of immunedepression, involving both innate and specific immune responses, could be responsible for both ematogenous mycobacterial dissemination and extrapulmonary form of tuberculosis in HIV-infected patients.     

CTL-mediated killing of intracellular Mycobacterium tuberculosis is independent of target cell nuclear apoptosis

Two subsets of human CTL have been defined based upon phenotype and function: CD4(-) CD8(-) double-negative (DN) CTL lyse susceptible targets via Fas-Fas ligand interaction and CD8(+) CTL via the granule exocytosis pathway. CD8(+) CTL, but not DN CTL, can mediate an antimicrobial activity against Mycobacterium tuberculosis-infected target cells that is dependent on cytotoxic granules that contain granulysin. We investigated the role of nuclear apoptosis for the antimicrobial effector function of CD1-restricted CTL using the caspase inhibitor N:-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. We found that DN CTL-induced target cell lysis was completely dependent on caspase activation, whereas the cytolytic activity of CD8(+) CTL was caspase independent. However, both DN and CD8(+) CTL-induced nuclear apoptosis required caspase activation. More important, the antimicrobial effector function of CD8(+) CTL was not diminished by inhibition of caspase activity. These data indicate that target cell nuclear apoptosis is not a requirement for CTL-mediated killing of intracellular M. tuberculosis.     

HIV impairs TNF-alpha mediated macrophage apoptotic response to Mycobacterium tuberculosis

The factors that contribute to the exceptionally high incidence of Mycobacterium tuberculosis (MTb) disease in HIV(+) persons are poorly understood. Macrophage apoptosis represents a critical innate host cell response to control MTb infection and limit disease. In the current study, virulent live or irradiated MTb (iMTbRv) induced apoptosis of differentiated human U937 macrophages in vitro, in part dependent on TNF-alpha. In contrast, apoptosis of differentiated HIV(+) human U1 macrophages (HIV(+) U937 subclone) was markedly reduced in response to iMTbRv and associated with significantly reduced TNF-alpha release, whereas apoptosis and TNF-alpha release were intact to TLR-independent stimuli. Furthermore, reduced macrophage apoptosis and TNF-alpha release were independent of MTb phagocytosis. Whereas surface expression of macrophage TLR2 and TLR4 was preserved, IL-1 receptor associated kinase-1 phosphorylation and NF-kappaB nuclear translocation were reduced in HIV(+) U1 macrophages in response to iMTbRv. These findings were confirmed using clinically relevant human alveolar macrophages (AM) from healthy persons and asymptomatic HIV(+) persons at clinical risk for MTb infection. Furthermore, in vitro HIV infection of AM from healthy persons reduced both TNF-alpha release and AM apoptosis in response to iMTbRv. These data identify an intrinsic specific defect in a critical macrophage cellular response to MTb that may contribute to disease pathogenesis in HIV(+) persons.     

Fibroblast apoptosis induced by Porphyromonas gingivalis is stimulated by a gingipain and caspase-independent pathway that involves apoptosis-inducing factor

Porphyromonas gingivalis is an oral bacterium that causes pathology in a number of dental infections that are associated with increased fibroblast cell death. Studies presented here demonstrated that P. gingivalis stimulates cell death by apoptosis rather than necrosis. Unlike previous studies apoptosis was induced independent of proteolytic activity and was also independent of caspase activity because a pancaspase inhibitor, Z-VAD-fmk, had little effect. Moreover, P. gingivalis downregulated caspase-3 mRNA levels and caspase-3 activity. The consequence of this downregulation was a significant reduction in tumour necrosis factor-alpha-induced apoptosis, which is caspase-3-dependent. Immunofluorescence and immunoblot analysis revealed P. gingivalis-induced translocation of apoptosis-inducing factor (AIF) from the cytoplasm to the nucleus. siRNA studies were undertaken and demonstrated that P. gingivalis stimulated cell death was significantly reduced when AIF was silenced (P < 0.05). Treatment of human gingival fibroblasts with H-89, a protein kinase A inhibitor that blocks AIF activation also reduced P. gingivalis-induced apoptosis (P < 0.05). These results indicate that P. gingivalis causes fibroblast apoptosis through a pathway that involves protein kinase A and AIF, is not dependent upon bacterial proteolytic activity and is also independent of the classic apoptotic pathways involving caspase-3.     

CsoR is a novel Mycobacterium tuberculosis copper-sensing transcriptional regulator

Copper is an essential element that becomes highly cytotoxic when concentrations exceed the capacity of cells to sequester the ion. Here, we identify a new copper-specific repressor (CsoR) of a copper-sensitive operon (cso) in Mycobacterium tuberculosis (Mtb) that is representative of a large, previously uncharacterized family of proteins (DUF156). Electronic and X-ray absorption spectroscopies reveal that CsoR binds a single-monomer mole equivalent of Cu(I) to form a trigonally coordinated (S(2)N) Cu(I) complex. The 2.6-A crystal structure of copper-loaded CsoR shows a homodimeric antiparallel four-helix bundle architecture that represents a novel DNA-binding fold. The Cu(I) is coordinated by Cys36, Cys65' and His61' in a subunit bridging site. Cu(I) binding negatively regulates the binding of CsoR to a DNA fragment encompassing the operator-promoter region of the Mtb cso operon; this results in derepression of the operon in Mtb and the heterologous host Mycobacterium smegmatis. Substitution of Cys36 or His61 with alanine abolishes Cu(I)- and CsoR-dependent regulation in vivo and in vitro. Potential roles of CsoR in Mtb pathogenesis are discussed.     

Long-term control of Mycobacterium tuberculosis infection is mediated by dynamic immune responses

The primary goal of this study was to determine how chronic exposure to Ag influences the functionality of Mycobacterium tuberculosis-specific T cell responses. The frequency of IFN-gamma-producing effector CD4(+) and CD8(+) T cells dynamically changed during persistent M. tuberculosis infection. CD8(+) T cells used differential effector functions during acute and chronic phases of the immune response, where CD8(+) T cells produced negligible amounts of IFN-gamma early in infection, but switched to cytokine production during the chronic stage of infection. Using limiting dilution analysis, CD8(+) T cells isolated during the initial phase of infection demonstrated lytic potential, but this waned in the chronic stage. The apparent loss of cytotoxic activity was not associated with the lack of perforin. Ag dose could potentially govern the functional program of CD8(+) T cells. Collectively, these results depict a host immune response mounted against M. tuberculosis of a significantly more dynamic nature than previously recognized.     

Execution of macrophage apoptosis by PE_PGRS33 of Mycobacterium tuberculosis is mediated by Toll-like receptor 2-dependent release of tumor necrosis factor-alpha

Combating tuberculosis requires a detailed understanding of how mycobacterial effectors modulate the host immune response. The role of the multigene PE family of proteins unique to mycobacteria in the pathogenesis of tuberculosis is still poorly understood, although certain PE_PGRS genes have been linked to virulence. Tumor necrosis factor-alpha (TNF-alpha) is essential for successfully combating tuberculosis. In this study we provide evidence that PE_PGRS33, a surface exposed protein, elicits TNF-alpha release from macrophages in a TLR2 (Toll-like receptor 2)-dependent manner. ASK1 (apoptosis signal-regulating kinase 1) is activated downstream of TLR2. ASK1 activates the MAPKs p38 and JNK. PE_PGRS33-induced signaling leads to enhanced expression of TNF-alpha and TNF receptor I (TNFRI) genes. Mycobacterium smegmatis expressing PE_ PGRS33 elicits the same effects as purified PE_PGRS33. TNF-alpha release occurs even when internalization of the bacteria is blocked by cytochalasin D, suggesting that interaction of PE_ PGRS33 with TLR2 is sufficient to trigger the effects described. Release of TNF-alpha plays the determining role in triggering apoptosis in macrophages challenged with PE_PGRS33. The death receptor-dependent signals are amplified through classical caspase 8-dependent mitochondrial release of cytochrome c, leading to the activation of caspases 9 and 3. An important aspect of our findings is that deletions within the PGRS domain (simulating those occurring in clinical strains) attenuate the TNF-alpha-inducing ability of PE_PGRS33. These results provide the first evidence that variations in the polymorphic repeats of the PGRS domain modulate the innate immune response.     

烟曲霉感染时内在化Dectin-1介导巨噬细胞自噬活化

目的探究在烟曲霉感染时Dectin-1是否内在化表达并介导了巨噬细胞的自噬活化,初步明确其作用机制。方法采用Western blot法和免疫荧光技术,观察经β-1,3-D-葡聚糖酶消化前后的烟曲霉孢子刺激下,RAW264.7细胞内Dectin-1与LC3Ⅱ的表达与定位,通过DCFH-DA探针检测消化β-葡聚糖对活性氧(ROS)生成的影响。结果烟曲霉孢子刺激后RAW264.7细胞的Dectin-1与LC3Ⅱ表达水平显著升高,同时二者呈斑点状聚集并共定位于烟曲霉孢子表面;消化β-葡聚糖后Dectin-1与LC3Ⅱ表达量降低,荧光斑点消失,并且ROS的生成受到抑制。结论烟曲霉感染时Dectin-1提高自身内在化表达并诱导了巨噬细胞自噬功能的活化。     

The OtsAB pathway is essential for trehalose biosynthesis in Mycobacterium tuberculosis

The disaccharide trehalose is the major free sugar in the cytoplasm of mycobacteria; it is a constituent of cell wall glycolipids, and it plays a role in mycolic acid transport during cell wall biogenesis. The pleiotropic role of trehalose in the biology of Mycobacterium tuberculosis and its absence from mammalian cells suggests that its biosynthesis may provide a useful target for novel drugs. However, there are three potential pathways for trehalose biosynthesis in M. tuberculosis, and the aim of the present study was to introduce mutations into each of the pathways to determine whether or not they are functionally redundant. The results show that the OtsAB pathway, which generates trehalose from glucose and glucose-6-phosphate, is the dominant pathway required for M. tuberculosis growth in laboratory culture and for virulence in a mouse model. Of the two otsB homologues annotated in the genome sequence of M. tuberculosis, only OtsB2 (Rv3372) has a functional role in the pathway. OtsB2, trehalose-6-phosphate phosphatase, is strictly essential for growth and provides a tractable target for high throughput screening. Inactivation of the TreYZ pathway, which can generate trehalose from alpha-1,4-linked glucose polymers, had no effect on the growth of M. tuberculosis in vitro or in mice. Deletion of the treS gene altered the late stages of pathogenesis of M. tuberculosis in mice, significantly increasing the time to death in a chronic infection model. Because the TreS enzyme catalyzes the interconversion of trehalose and maltose, the mouse phenotype could reflect either a requirement for synthesis of additional trehalose or, conversely, a requirement for breakdown of stored trehalose to liberate free glucose.     

Mediating of caspase-independent apoptosis by cadmium through the mitochondria-ROS pathway in MRC-5 fibroblasts

Cadmium (Cd) is an environmental pollutant of global concern with a 10-30-year biological half-life in humans. Accumulating evidence suggests that the lung is one of the major target organs of inhaled Cd compounds. Our previous report demonstrated that 100 microM Cd induces MRC-5 cells, normal human lung fibroblasts, to undergo caspase-independent apoptosis mediated by mitochondrial membrane depolarization and translocation of apoptosis-inducing factor (AIF) from mitochondria into the nucleus. Here, using benzyloxycarbonyl-Val-Ala-Asp-(ome) fluoromethyl ketone (Z-VAD.fmk) as a tool, we further demonstrated that Cd could induce caspase-independent apoptosis at concentrations varied from 25 to 150 microM, which was modulated by reactive oxygen species (ROS) scavengers, such as N-acetylcysteine (NAC), mannitol, and tiron, indicating that ROS play a crucial role in the apoptogenic activity of Cd. Consistent with this notion, the intracellular hydrogen peroxide (H2O2) was 2.9-fold elevated after 3 h of Cd treatment and diminished rapidly within 1 h as detected by flow cytometry with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Using inhibitors of the mitochondrial electron transport chain (ETC) (oligomycin A and rotenone for complex I and V, respectively) and mitochondrial permeability transition pore (MPTP) (cyclosporin A and aristolochic acid), we coincidently found the ROS production, mitochondrial membrane depolarization, and apoptotic content were almost completely or partially abolished. As revealed by confocal microscopy staining with chloromethyl-X-rosamine (CMXRos) and an anti-AIF antibody, the collapse of mitochondrial membrane potential induced by Cd (3 h-treatment) was a prelude to the translocation of caspase-independent pro-apoptotic factor, AIF, into the nucleus (after 4 h of Cd treatment). In summary, this study demonstrated that, in MRC-5 fibroblasts, Cd induced caspase-independent apoptosis through a mitochondria-ROS pathway. More importantly, we provide several lines of evidence supporting a role of mitochondrial ETC and MPTP in the regulation of caspase-independent cell death triggered by Cd.     

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.     

The forest behind the tree: phylogenetic exploration of a dominant Mycobacterium tuberculosis strain lineage from a high tuberculosis burden country

Background

Genotyping of Mycobacterium tuberculosis isolates is a powerful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. Standardized PCR-based typing, based on 15 to 24 mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) loci combined with spoligotyping, has been shown to have adequate resolution power for tracing TB transmission and to be useful for predicting diverse strain lineages in European settings. Its informative value needs to be tested in high TB-burden countries, where the use of genotyping is often complicated by dominance of geographically specific, genetically homogeneous strain lineages.

Methodology/Principal Findings

We tested this genotyping system for molecular epidemiological analysis of 369 M. tuberculosis isolates from 3 regions of Brazil, a high TB-burden country. Deligotyping, targeting 43 large sequence polymorphisms (LSPs), and the MIRU-VNTRplus identification database were used to assess phylogenetic predictions. High congruence between the different typing results consistently revealed the countrywide supremacy of the Latin-American-Mediterranean (LAM) lineage, comprised of three main branches. In addition to an already known RDRio branch, at least one other branch characterized by a phylogenetically informative LAM3 spoligo-signature seems to be globally distributed beyond Brazil. Nevertheless, by distinguishing 321 genotypes in this strain population, combined MIRU-VNTR typing and spoligotyping demonstrated the presence of multiple distinct clones. The use of 15 to 24 loci discriminated 21 to 25% more strains within the LAM lineage, compared to a restricted lineage-specific locus set suggested to be used after SNP analysis. Noteworthy, 23 of the 28 molecular clusters identified were exclusively composed of patient isolates from a same region, consistent with expected patterns of mostly local TB transmission.

Conclusions/Significance

Standard MIRU-VNTR typing combined with spoligotyping can reveal epidemiologically meaningful clonal diversity behind a dominant M. tuberculosis strain lineage in a high TB-burden country and is useful to explore international phylogenetical ramifications.     

Somatostatin receptor-3 mediated intracellular signaling and apoptosis is regulated by its cytoplasmic terminal

In the present study, we describe the role of cytoplasmic terminal (C-tail) domain in regulating coupling to adenylyl cyclase, signaling, and apoptosis in human embryonic kidney (HEK-293) cells transfected with wild type (wt)-hSSTR3 and C-tail deleted mutants. Cells transfected with wt-hSSTR3 and C-tail mutants show comparable membrane expression; however, display decreased expression in presence of agonist. wt-hSSTR3 exists as preformed homodimer at cell surface in basal conditions and decreases in response to agonist. Cells expressing C-tail mutants also show evidence of homodimerization with the same intensity as wt-hSSTR3. The agonist-dependent inhibition of cyclic adenosine monophosphate (cAMP) was lost in cells expressing C-tail mutants. Agonist treatment in cells expressing wt-hSSTR3 resulted in inhibition of cell proliferation, increased expression of PARP-1, and TUNEL positivity in proliferating cell nuclear antigen (PCNA)-positive cells. The agonist mediated increase in membrane expression of protein tyrosine phosphatase (PTP) seen with wt-hSSTR3 was diminished in C-tail mutants, which was accompanied with the loss of receptor's ability to induce apoptosis. Taken together, our data provide new insights into C-tail-dependent regulation of cell signaling and apoptosis by hSSTR3.     

Mycobacterium tuberculosis virulence is mediated by PtpA dephosphorylation of human vacuolar protein sorting 33B

Entry into host macrophages and evasion of intracellular destruction mechanisms, including phagosome-lysosome fusion, are critical elements of Mycobacterium tuberculosis (Mtb) pathogenesis. To achieve this, the Mtb genome encodes several proteins that modify host signaling pathways. PtpA, a low-molecular weight tyrosine phosphatase, is a secreted Mtb protein of unknown function. The lack of tyrosine kinases in the Mtb genome suggests that PtpA may modulate host tyrosine phosphorylated protein(s). We report that a genetic deletion of ptpA attenuates Mtb growth in human macrophages, and expression of PtpA-neutralizing antibodies simulated this effect. We identify VPS33B, a regulator of membrane fusion, as a PtpA substrate. VPS33B and PtpA colocalize in Mtb-infected human macrophages. PtpA secretion combined with active-phosphorylated VPS33B inhibited phagosome-lysosome fusion, a process arrested in Mtb infections. These results demonstrate that PtpA is essential for Mtb intracellular persistence and identify a key host regulatory pathway that is inactivated by Mtb.     

A novel nucleoid-associated protein of Mycobacterium tuberculosis is a sequence homolog of GroEL

The Mycobacterium tuberculosis genome sequence reveals remarkable absence of many nucleoid-associated proteins (NAPs), such as HNS, Hfq or DPS. In order to characterize the nucleoids of M. tuberculosis, we have attempted to identify NAPs, and report an interesting finding that a chaperonin-homolog, GroEL1, is nucleoid associated. We report that M. tuberculosis GroEL1 binds DNA with low specificity but high affinity, suggesting that it might have naturally evolved to bind DNA. We are able to demonstrate that GroEL1 can effectively function as a DNA-protecting agent against DNase I or hydroxyl-radicals. Moreover, Atomic Force Microscopic studies reveal that GroEL1 can condense a large DNA into a compact structure. We also provide in vivo evidences that include presence of GroEL1 in purified nucleoids, in vivo crosslinking followed by Southern hybridizations and immunofluorescence imaging in M. tuberculosis confirming that GroEL1: DNA interactions occur in natural biological settings. These findings therefore reveal that M. tuberculosis GroEL1 has evolved to be associated with nucleoids.     

Downregulation of VRK1 by p53 in response to DNA damage is mediated by the autophagic pathway

Human VRK1 induces a stabilization and accumulation of p53 by specific phosphorylation in Thr18. This p53 accumulation is reversed by its downregulation mediated by Hdm2, requiring a dephosphorylated p53 and therefore also needs the removal of VRK1 as stabilizer. This process requires export of VRK1 to the cytosol and is inhibited by leptomycin B. We have identified that downregulation of VRK1 protein levels requires DRAM expression, a p53-induced gene. DRAM is located in the endosomal-lysosomal compartment. Induction of DNA damage by UV, IR, etoposide and doxorubicin stabilizes p53 and induces DRAM expression, followed by VRK1 downregulation and a reduction in p53 Thr18 phosphorylation. DRAM expression is induced by wild-type p53, but not by common human p53 mutants, R175H, R248W and R273H. Overexpression of DRAM induces VRK1 downregulation and the opposite effect was observed by its knockdown. LC3 and p62 were also downregulated, like VRK1, in response to UV-induced DNA damage. The implication of the autophagic pathway was confirmed by its requirement for Beclin1. We propose a model with a double regulatory loop in response to DNA damage, the accumulated p53 is removed by induction of Hdm2 and degradation in the proteasome, and the p53-stabilizer VRK1 is eliminated by the induction of DRAM that leads to its lysosomal degradation in the autophagic pathway, and thus permitting p53 degradation by Hdm2. This VRK1 downregulation is necessary to modulate the block in cell cycle progression induced by p53 as part of its DNA damage response.     

Exit of intracellular Porphyromonas gingivalis from gingival epithelial cells is mediated by endocytic recycling pathway

Gingival epithelial cells function as an innate host defence system to prevent intrusion by periodontal bacteria. Nevertheless, Porphyromonas gingivalis, the most well‐known periodontal pathogen, can enter gingival epithelial cells and pass through the epithelial barrier into deeper tissues. However, it is poorly understood how this pathogen exits from infected cells for further transcellular spreading. The present study was performed to elucidate the cellular machinery exploited by P. gingivalis to exit from immortalized human gingival epithelial cells. P. gingivalis was shown to be internalized with early endosomes positive for the FYVE domain of EEA1 and transferrin receptor, and about half of the intracellular bacteria were then sorted to lytic compartments, including autolysosomes and late endosomes/lysosomes, while a considerable number of the remaining organisms were sorted to Rab11‐ and RalA‐positive recycling endosomes. Inhibition experiments revealed that bacterial exit was dependent on actin polymerization, lipid rafts and microtubule assembly. Dominant negative forms and RNAi knockdown of Rab11, RalA and exocyst complex subunits (Sec5, Sec6 and Exo84) significantly disturbed the exit of P. gingivalis. These results strongly suggest that the recycling pathway is exploited by intracellular P. gingivalis to exit from infected cells to neighbouring cells as a mechanism of cell‐to‐cell spreading.     

Artesunate induces apoptosis via a Bak-mediated caspase-independent intrinsic pathway in human lung adenocarcinoma cells

This report is designed to explore the exact molecular mechanism by which artesunate (ART), a semisynthetic derivative of the herbal antimalaria drug artemisinin, induces apoptosis in human lung adenocarcinoma (ASTC‐a‐1 and A549) cell lines. ART treatment induced ROS‐mediated apoptosis in a concentration‐ and time‐dependent fashion accompanying the loss of mitochondrial potential and subsequent release of Smac and AIF indicative of intrinsic apoptosis pathway. Blockage of casapse‐8 and ‐9 did not show any inhibitory effect on the ART‐induced apoptosis, but which was remarkably prevented by silencing AIF. Of the utmost importance, ART treatment induced the activation of Bak but not Bax, and silencing Bak but not Bax remarkably inhibited ART‐induced apoptosis and AIF release. Furthermore, although ART treatment did not induced a significant down‐regulation of voltage‐dependent anion channel 2 (VDAC2) expression and up‐regulation of Bim expression, silencing VDAC2 potently enhanced the ART‐induced Bak activation and apoptosis which were significantly prevented by silencing Bim. Collectively, our data firstly demonstrate that ART induces Bak‐mediated caspase‐independent intrinsic apoptosis in which Bim and VDAC2 as well as AIF play important roles in both ASTC‐a‐1 and A549 cell lines, indicating a potential therapeutic effect of ART for lung cancer. J. Cell. Physiol. 227: 3778–3786, 2012. © 2012 Wiley Periodicals, Inc.     

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.