Mycobacterium tuberculosis eis regulates autophagy, inflammation, and cell death through redox-dependent signaling

The "enhanced intracellular survival" (eis) gene of Mycobacterium tuberculosis (Mtb) is involved in the intracellular survival of M. smegmatis. However, its exact effects on host cell function remain elusive. We herein report that Mtb Eis plays essential roles in modulating macrophage autophagy, inflammatory responses, and cell death via a reactive oxygen species (ROS)-dependent pathway. Macrophages infected with an Mtb eis-deletion mutant H37Rv (Mtb-Δeis) displayed markedly increased accumulation of massive autophagic vacuoles and formation of autophagosomes in vitro and in vivo. Infection of macrophages with Mtb-Δeis increased the production of tumor necrosis factor-α and interleukin-6 over the levels produced by infection with wild-type or complemented strains. Elevated ROS generation in macrophages infected with Mtb-Δeis (for which NADPH oxidase and mitochondria were largely responsible) rendered the cells highly sensitive to autophagy activation and cytokine production. Despite considerable activation of autophagy and proinflammatory responses, macrophages infected with Mtb-Δeis underwent caspase-independent cell death. This cell death was significantly inhibited by blockade of autophagy and c-Jun N-terminal kinase-ROS signaling, suggesting that excessive autophagy and oxidative stress are detrimental to cell survival. Finally, artificial over-expression of Eis or pretreatment with recombinant Eis abrogated production of both ROS and proinflammatory cytokines, which depends on the N-acetyltransferase domain of the Eis protein. Collectively, these data indicate that Mtb Eis suppresses host innate immune defenses by modulating autophagy, inflammation, and cell death in a redox-dependent manner.     

Mycobacterium tuberculosis H37Rv induces monocytic release of interleukin-6 via activation of mitogen-activated protein kinases: inhibition by N-acetyl-L-cysteine

The release of proinflammatory cytokines after mycobacterial infection is a host immune response that may be propitious or deleterious to the host. Elevated levels of interleukin (IL)-6 are present in plasma of patients with active tuberculosis infection. The aim of this study was to investigate the role of mitogen-activated protein kinases in the secretion of interleukin-6 in THP-1 cells and human primary monocytes that were infected with Mycobacterium tuberculosis H37Rv, and its regulation by N-acetyl-L-cysteine, a potential antimycobacterial agent. Exposure of THP-1 human monocytes to M. tuberculosis H37Rv induced rapidly, in a time-dependent manner, the phosphorylation of mitogen-activated protein kinase kinase 3/6 and p38 mitogen-activated protein kinase, accompanied by an upregulation of interleukin-6. Using highly specific inhibitors of mitogen-activated protein kinase kinase-1, p38 mitogen-activated protein kinase and nuclear factor-kappaB, we found that extracellular-signal regulated kinase 1/2, p38 mitogen-activated protein kinase and nuclear factor-kappaB were essential for M. tuberculosis H37Rv-induced interleukin-6 production in human primary monocytes. Pretreatment with N-acetyl-L-cysteine reduced, in a dose-dependent manner, M. tuberculosis H37Rv-induced activation of mitogen-activated protein kinase kinase 3/6 and interleukin-6 production in THP-1 cells.     

Thermostable hexameric form of Eis (Rv2416c) protein of M. tuberculosis plays an important role for enhanced intracellular survival within macrophages

Eis protein is reported to enhance the intracellular survival of Mycobacterium tuberculosis in human macrophages. Eis protein is not only known to skew away the immunity by disturbing the protective T(H)1 response, but aminoglycoside acetyltransferase activity of Eis is reported to regulate autophagy, inflammation and cell death. Here we have gained insight into the structure-function properties of Eis. Eis protein is a hexameric αβ protein. Although urea and guanidinium hydrochloride (GdmCl) was found to induce one-step unfolding of Eis but size exclusion chromatography showed that GdmCl treated Eis maintained its hexameric form. SDS-PAGE assay confirmed that hexameric form of Eis is partially stable to SDS and converts into trimers and monomers. Out of these three forms, aminoglycoside acetyltransferase activity is found to be associated only with hexamers. The Tm of Eis was found to be ～75°C. Aminoglycoside acetyltransferase Eis demonstrated remarkable heat stability retaining >80% of their activity at 70°C which falls down to ～50% at 75°C and is completely inactive at 80°C. Further, intracellular survival assay with heated samples of M. smegmatis harboring eis gene of M. tuberculosis H37Rv demonstrated a possible role for the thermostability associated with Eis protein in the enhanced intracellular survival within macrophages. In sum, these data reveal that only hexameric form of Eis has a thermostable aminoglycoside acetyltransferase activity. This is the first report showing the thermostability associated with aminoglycoside acetyltransferase activity of Eis protein being one of the essential features for the execution of its biological role.     

Th2-type immune response observed in healthy individuals to sonicate antigen prepared from the most prevalent Mycobacterium tuberculosis strain with single copy of IS6110

Different Mycobacterium tuberculosis strains operate different immune evasion strategies for their survival in the host. This mainly depends on the virulence of the strain and the host immune responses. The most virulent strains are actively involved in the transmission, widely spread in the community and induce differential immune responses. We evaluated the immune response of a sonicate antigen prepared from one predominant strain (S7) from M. tuberculosis harbouring a single copy of IS6110. Significant lymphoproliferative response against purified protein derivative from tubercle bacillus (PPD) and H37Rv antigens was observed in PPD positive normal individuals and tuberculosis patients. Interferon-gamma (IFN-gamma) levels against these antigens were significantly increased in normal individuals but not in tuberculosis patients. The antigen S7 showed marginal T-cell proliferation but did not induce IFN-gamma secretion in both groups. Conversely, it induced significantly high levels of cytokine interleukin 4 (IL-4) in normal individuals. The macrophage cytokines, IL-12 and tumour necrosis factor alpha (TNF-alpha), did not show S7 antigen specific stimulation. The intracellular cytokine further confirmed an increase in IL-4(+)/CD4+ T-cells and a decrease in IFN-gamma(+)/CD4+ T-cells upon stimulation. The antibody response showed an increase in IgG and IgA levels against this antigen in normal individuals. These observations suggest that antigen S7 modulates the immune response towards T helper cell type 2 by suppressing T helper cell type 1 protective immune response in PPD positive normal individuals. We speculate that some components of this sonicate antigen are associated with immunosuppressive response.     

Programmed death (PD)-1:PD-ligand 1/PD-ligand 2 pathway inhibits T cell effector functions during human tuberculosis

Protective immunity against Mycobacterium tuberculosis requires the generation of cell-mediated immunity. We investigated the expression and role of programmed death 1 (PD-1) and its ligands, molecules known to modulate T cell activation, in the regulation of IFN-gamma production and lytic degranulation during human tuberculosis. We demonstrated that specific Ag-stimulation increased CD3+PD-1+ lymphocytes in peripheral blood and pleural fluid from tuberculosis patients in direct correlation with IFN-gamma production from these individuals. Moreover, M. tuberculosis-induced IFN-gamma participated in the up-regulation of PD-1 expression. Blockage of PD-1 or PD-1 and its ligands (PD-Ls: PD-L1, PD-L2) enhanced the specific degranulation of CD8+ T cells and the percentage of specific IFN-gamma-producing lymphocytes against the pathogen, demonstrating that the PD-1:PD-Ls pathway inhibits T cell effector functions during active M. tuberculosis infection. Furthermore, the simultaneous blockage of the inhibitory receptor PD-1 together with the activation of the costimulatory protein signaling lymphocytic activation molecule led to the promotion of protective IFN-gamma responses to M. tuberculosis, even in patients with weak cell-mediated immunity against the bacteria. Together, we demonstrated that PD-1 interferes with T cell effector functions against M. tuberculosis, suggesting that PD-1 has a key regulatory role during the immune response of the host to the pathogen.     

Mycobacterium tuberculosis lipoarabinomannan-mediated IRAK-M induction negatively regulates Toll-like receptor-dependent interleukin-12 p40 production in macrophages

Mannose-capped lipoarabinomannans (Man-LAMs) are members of the repertoire of Mycobacterium tuberculosis modulins that the bacillus uses to subvert the host innate immune response. Interleukin-12 (IL-12) production is critical for mounting an effective immune response by the host against M. tuberculosis. We demonstrate that Man-LAM inhibits IL-12 p40 production mediated by subsequent challenge with lipopolysaccharide (LPS). Man-LAM inhibits LPS-induced IL-12 p40 expression in an IL-10-independent manner. It attenuates LPS-induced NF-kappaB-driven luciferase gene expression, suggesting that its effects are likely directly related to inhibition of NF-kappaB. This is probably because of dampening of the Toll-like receptor signaling. Man-LAM inhibits IL-1 receptor-associated kinase (IRAK)-TRAF6 interaction as well as IkappaB-alpha phosphorylation. It directly attenuates nuclear translocation and DNA binding of c-Rel and p50. Man-LAM exerts these effects by inducing the expression of Irak-M, a negative regulator of TLR signaling. Knockdown of Irak-M expression by RNA interference reinstates LPS-induced IL-12 production in Man-LAM-pretreated cells. The fact that Irak-M expression could be elicited by yeast mannan suggested that ligation of the mannose receptor by the mannooligosaccharide caps of LAM was the probable trigger for IRAK-M induction.     

Organelle membrane proteomics reveals differential influence of mycobacterial lipoglycans on macrophage phagosome maturation and autophagosome accumulation

The mycobacterial cell wall component lipoarabinomannan (LAM) has been described as one of the key virulence factors of Mycobacterium tuberculosis. Modification of the terminal arabinan residues of this lipoglycan with mannose caps in M. tuberculosis or with phosphoinositol caps in Mycobacterium smegmatis results in distinct host immune responses. Given that M. tuberculosis typically persists in the phagosomal vacuole after being phagocytosed by macrophages, we performed a proteomic analysis of that organelle after treatment of macrophages with LAMs purified from the two mycobacterial species. The quantitative changes in phagosomal proteins suggested a distinct role for mannose-capped LAM in modulating protein trafficking pathways that contribute to the arrest of phagosome maturation. Enlightened by our proteomic data, we performed further experiments to show that only the LAM from M. tuberculosis inhibits accumulation of autophagic vacuoles in the macrophage, suggesting a new function for this virulence-associated lipid.     

Ins and outs of Mycobacterium tuberculosis PPE family in pathogenesis and implications for novel measures against tuberculosis

Mycobacterium tuberculosis is the most successful pathogen with multiple mechanisms to subvert host immune response, resulting in insidious disease. A unique Mycobacterium antigen family termed PPE (Pro-Pro-Glu) has long been widely speculated as "molecular mantra" to escape host immunity. Members of this family are characterized by a conserved N terminal and a variable C terminal. This family associated closely with ESAT-6(ESX) secretion system and largely located in cell wall or cell membrane. The expression of PPE protein is temporally regulated, and highly expressed during M. tuberculosis persistence. Importantly, the distribution of PPE family is so far limited to Mycobacterium genus, prevalent among pathogenic Mycobacterium species. It is tempting to explore this family due to its potential in the latency and reactivation of M. tuberculosis. The evolution, structure, and functions of most PPE proteins remain elusive. The understanding of these questions will deepen our appreciation of the pathogenesis of M. tuberculosis and accelerate novel anti-TB measures discovery.     

Aminoglycoside multiacetylating activity of the enhanced intracellular survival protein from Mycobacterium smegmatis and its inhibition

The enhanced intracellular survival (Eis) protein improves the survival of Mycobacterium smegmatis (Msm) in macrophages and functions as the acetyltransferase responsible for kanamycin A resistance, a hallmark of extensively drug-resistant (XDR) tuberculosis, in a large number of Mycobacterium tuberculosis (Mtb) clinical isolates. We recently demonstrated that Eis from Mtb (Eis_Mtb) efficiently multiacetylates a variety of aminoglycoside (AG) antibiotics. Here, to gain insight into the origin of substrate selectivity of AG multiacetylation by Eis, we analyzed AG acetylation by Eis_Msm, investigated its inhibition, and compared these functions to those of Eis_Mtb. Even though for several AGs the multiacetylation properties of Eis_Msm and Eis_Mtb are similar, there are three major differences. (i) Eis_Msm diacetylates apramycin, a conformationally constrained AG, which Eis_Mtb cannot modify. (ii) Eis_Msm triacetylates paromomycin, which can be only diacetylated by Eis_Mtb. (iii) Eis_Msm only monoacetylates hygromycin, a structurally unique AG that is diacetylated by Eis_Mtb. Several nonconserved amino acid residues lining the AG-binding pocket of Eis are likely responsible for these differences between the two Eis homologues. Specifically, we propose that because the AG-binding pocket of Eis_Msm is more open than that of Eis_Mtb, it accommodates apramycin for acetylation in Eis_Msm, but not in Eis_Mtb. We also demonstrate that inhibitors of Eis_Mtb that we recently discovered can inhibit Eis_Msm activity. These observations help define the structural origins of substrate preference among Eis homologues and suggest that Eis_Mtb inhibitors may be applied against all pathogenic mycobacteria to overcome AG resistance caused by Eis upregulation.     

The Us3 Protein of Herpes Simplex Virus 1 Inhibits T Cell Signaling by Confining Linker for Activation of T Cells (LAT) Activation via TRAF6 Protein

Herpes simplex virus 1 (HSV-1) is the most prevalent human virus and causes global morbidity because the virus is able to infect multiple cell types. Remarkably, HSV infection switches between lytic and latent cycles, where T cells play a critical role. However, the precise way of virus-host interactions is incompletely understood. Here we report that HSV-1 productively infected Jurkat T-cells and inhibited antigen-induced T cell receptor activation. We discovered that HSV-1-encoded Us3 protein interrupted TCR signaling and interleukin-2 production by inactivation of the linker for activation of T cells. This study unveils a mechanism by which HSV-1 intrudes into early events of TCR-mediated cell signaling and may provide novel insights into HSV infection, during which the virus escapes from host immune surveillance.     

果蝇免疫研究进展及其感染分枝杆菌的免疫特征

耐药性、持续感染以及与HIV病毒的共感染等诸多因素导致一度得到控制的结核病死灰复燃, 有效控制日益严峻的结核病迫切需要深入认识其致病菌——结核分枝杆菌Mycobacterium tuberculosis的基础生物学特性, 以及宿主相应的免疫控制机理。目前尚无一个动物模型能够同时回答这些关键问题, 而利用多种动物模型有望从不同角度回答上述问题, 普遍认为果蝇Drosophila 是比较理想的研究结核病天然免疫的简易模式动物之一。本文综述了果蝇免疫研究的最新进展, 包括免疫途径及其新成员与负调控子, 重点总结了用海分枝菌杆菌M. marinum、偶发分枝杆菌M. fortuitum和耻垢分枝杆菌M. smegmatis等分枝杆菌感染果蝇的新发现, 其中包括感染期间不诱导抗菌肽表达, 多个宿主因子(如CD36家族成员和ESCRT)参与了应答, 鉴定出具有杀灭分支杆菌作用的β-己糖酰胺酶, 感染期间能量代谢相关基因差异表达等。这些工作为利用果蝇模型快速筛选治疗结核病的新药物靶标和药物先导物提供了思路。     

Activation of an NLRP3 inflammasome restricts Mycobacterium kansasii infection

Mycobacterium kansasii has emerged as an important nontuberculous mycobacterium pathogen, whose incidence and prevalence have been increasing in the last decade. M. kansasii can cause pulmonary tuberculosis clinically and radiographically indistinguishable from that caused by Mycobacterium tuberculosis infection. Unlike the widely-studied M. tuberculosis, little is known about the innate immune response against M. kansasii infection. Although inflammasome activation plays an important role in host defense against bacterial infection, its role against atypical mycobacteria remains poorly understood. In this report, the role of inflammasome activity in THP-1 macrophages against M. kansasii infection was studied. Results indicated that viable, but not heat-killed, M. kansasii induced caspase-1-dependent IL-1β secretion in macrophages. The underlying mechanism was found to be through activation of an inflammasome containing the NLR (Nod-like receptor) family member NLRP3 and the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD). Further, potassium efflux, lysosomal acidification, ROS production and cathepsin B release played a role in M. kansasii-induced inflammasome activation. Finally, the secreted IL-1β derived from caspase-1 activation was shown to restrict intracellular M. kansasii. These findings demonstrate a biological role for the NLRP3 inflammasome in host defense against M. kansasii.     

结核分枝杆菌感染对人巨噬细胞离子通道及其调控元件转录表达的影响

为研究人巨噬细胞的离子通道及其调控元件是否参与了抗结核分枝杆菌感染免疫,利用表达谱芯片技术研究细菌感染后主巨噬细胞基因的表达情况,在全局表达谱分析的基础上,重点分析了离子通道及其调控元件的表达,并比较无毒株和临床分离有毒株在诱导离子通道及其调控元件表达方面的差异。结果表明,细菌感染影响离子通道及其调控元件基因的表达,涉及的离子通道包括钾通道、钠通道、氯通道、钙通道,差异表达的调控元件包括G蛋白、G蛋白偶联受体、蛋白质激酶和磷酸化酶;临床株感染影响的离子通道及其调控元件较无毒株广泛和丰富。这些观察结果提示,离子通道及其调控元件参与了宿主细胞对感染细菌的免疫应答,有关离子通道及其调控元件在抗结核免疫中的作用有待进一步研究。芯片研究的结果为将来的研究提供了线索。     

Flotillin and RacH modulate the intracellular immunity of Dictyostelium to Mycobacterium marinum infection

Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, provides a useful model to study the pathogenesis of tuberculosis in genetically tractable model organisms. Using the amoeba Dictyostelium discoideum as a host, we show that expression of the M. marinum protein MAG24-1 is crucial to interfere with phagosome maturation. We find that two host proteins - the flotillin homologue vacuolin and p80, a predicted copper transporter - accumulate at the vacuole during pathogen replication until it finally ruptures and the bacteria are released into the host cytosol. Flotillin-1 accumulation at the replication niche and its rupture were also observed in human peripheral blood monocytes. By infecting various Dictyostelium mutants, we show that the absence of one of the two Dictyostelium vacuolin isoforms renders the host more immune to M. marinum. Conversely, the absence of the small GTPase RacH renders the host more susceptible to M. marinum proliferation but inhibits its cell-to-cell spreading.     

Structural analysis of the N‐acetyltransferase Eis1 from Mycobacterium abscessus reveals the molecular determinants of its incapacity to modify aminoglycosides

Enhanced intracellular survival (Eis) proteins belonging to the superfamily of the GCN5‐related N‐acetyltransferases play important functions in mycobacterial pathogenesis. In Mycobacterium tuberculosis, Eis enhances the intracellular survival of the bacilli in macrophages by modulating the host immune response and is capable to chemically modify and inactivate aminoglycosides. In nontuberculous mycobacteria (NTM), Eis shares similar functions. However, Mycobacterium abscessus, a multidrug resistant NTM, possesses two functionally distinct Eis homologues, Eis1_Mab and Eis2_Mab. While Eis2_Mab participates in virulence and aminoglycosides resistance, this is not the case for Eis1_Mab, whose exact biological function remains to be determined. Herein, we show that overexpression of Eis1_Mab in M. abscessus fails to induce resistance to aminoglycosides. To clarify why Eis1_Mab is unable to modify this class of antibiotics, we solved its crystal structure bound to its cofactor, acetyl‐CoA. The structure revealed that Eis1_Mab has a typical homohexameric Eis‐like organization. The structural analysis supported by biochemical approaches demonstrated that while Eis1_Mab can acetylate small substrates, its active site is too narrow to accommodate aminoglycosides. Comparison with other Eis structures showed that an extended loop between strands 9 and 10 is blocking the access of large substrates to the active site and movement of helices 4 and 5 reduces the volume of the substrate‐binding pocket to these compounds in Eis1_Mab. Overall, this study underscores the molecular determinants explaining functional differences between Eis1_Mab and Eis2_Mab, especially those inherent to their capacity to modify aminoglycosides.     

斑马鱼-海分枝杆菌模型研究对结核病致病机理的启示

全世界约三分之一的人口感染过结核分枝杆菌,其导致的结核病仍然是全球公共卫生的严重威胁。结核菌是典型的胞内致病菌。结核菌的致病性与其成功逃避和利用宿主免疫应答等密切相关。控制结核病需要深入了解致病菌和宿主之间的相互作用。不同的动物模型是揭示致病菌-宿主相互作用的关键。海分枝杆菌-斑马鱼模型是最近才得以发展并获得了不少新见解的研究系统之一。本文总结了该模型揭示的海分枝杆菌毒力因子Erp、Esx-1、pmiA、Mel1和Mel2、KasB等,以及该模型的优缺点。这些结果为大动物模型研究和深入了解结核分枝杆菌感染人体的致病机理提供了线索。     

Protein kinase E of Mycobacterium tuberculosis has a role in the nitric oxide stress response and apoptosis in a human macrophage model of infection

Mycobacterium tuberculosis , an intracellular pathogen, inhibits macrophage apoptosis to support survival and replication inside the host cell. We provide evidence that the functional serine/threonine kinase, PknE, is important for survival of M. tuberculosis that enhances macrophage viability by inhibiting apoptosis. A promoter of PknE identified in this study was shown to respond to nitric oxide stress. Deletion of pknE in virulent M. tuberculosis , H37Rv, resulted in a strain that has increased resistance to nitric oxide donors and increased sensitivity to reducing agents. The deletion mutant created by specialized transduction induced enhanced apoptosis while inhibiting necrosis. The pknE mutant also modifies the innate immune response as shown by the marked decline in the pro-inflammatory cytokines in a macrophage model of infection. These findings suggest a novel mechanism, by which PknE senses nitric oxide stress and prevents apoptosis by interfering with host signalling pathways.