ATP stimulates human macrophages to kill intracellular virulent Mycobacterium tuberculosis via calcium-dependent phagosome-lysosome fusion.

Advances in therapy for tuberculosis will require greater understanding of the molecular mechanisms of pathogenesis and the human immune response in this disease. Exposure of Mycobacterium tuberculosis-infected human macrophages to extracellular ATP (ATP(e)) results in bacterial killing, but the molecular mechanisms remain incompletely characterized. In this study, we demonstrate that ATP(e)-induced bactericidal activity toward virulent M. tuberculosis requires an increase in cytosolic Ca(2+) in infected macrophages. Based on our previous work with primary infection of human macrophages, we hypothesized that the Ca(2+) dependence of ATP-induced killing of intracellular M. tuberculosis was linked to promotion of phagosome-lysosome fusion. Using confocal laser-scanning microscopy, we demonstrate that ATP(e) induces fusion of the M. tuberculosis-containing phagosome with lysosomes, defined by accumulation of three lysosomal proteins and an acidophilic dye. Stimulation of phagosome-lysosome fusion by ATP(e) exhibited distinct requirements for both Ca(2+) and phospholipase D and was highly correlated with killing of intracellular bacilli. Thus, key signal transduction pathways are conserved between two distinct models of human macrophage antituberculous activity: primary infection of naive macrophages and physiologic stimulation of macrophages stably infected with M. tuberculosis.     

Induction of TNF in human alveolar macrophages as a potential evasion mechanism of virulent Mycobacterium tuberculosis.

The ability of macrophages to release cytokines is crucial to the host response to intracellular infection. In particular, macrophage-derived TNF plays an important role in the host response to infection with the intracellular pathogen Mycobacterium tuberculosis. In mice, TNF is indispensable for the formation of tuberculous granulomas, which serve to demarcate the virulent bacterium. TNF is also implicated in many of the immunopathological features of tuberculosis. To investigate the role of TNF in the local immune response, we infected human alveolar macrophages with virulent and attenuated mycobacteria. Infection with virulent strains induced the secretion of significantly higher levels of bioactive TNF than attenuated strains correlating with their ability to multiply intracellularly. Treatment of infected macrophages with neutralizing anti-TNF Abs reduced the growth rate of intracellular bacteria, whereas bacterial replication was augmented by addition of exogenous TNF. Infected and uninfected macrophages contributed to cytokine production as determined by double-staining of M. tuberculosis and intracellular TNF. The induction of TNF by human alveolar macrophages at the site of infection permits the multiplication of intracellular bacteria and may therefore present an evasion mechanism of human pathogens.     

Evidence for the cytotoxic effects of Mycobacterium tuberculosis phospholipase C towards macrophages

Phospholipase Cs (PLCs) contribute importantly to the virulence and pathogenicity of several bacteria. It has been reported in previous studies that mutations in the four predicted plc genes of Mycobacterium tuberculosis inhibit the growth of these bacteria during the late phase of infection in mice. These enzymes have not yet been fully characterised, mainly because they are not easy to produce in large quantities. With a view to elucidating the role of all Mycobacterium tuberculosis phospholipase Cs (PLC-A, PLC-B, PLC-C and PLC-D), a large amount of active, soluble recombinant PLCs, were expressed and purified using Mycobacterium smegmatis as expression system. These enzymes showed different pH activity profiles. PLC-C was found to be the most active of the four recombinant PLCs under acidic conditions. All the enzymes tested induced cytotoxic effects on mouse macrophage RAW 264.7 cell lines, via direct or indirect enzymatic hydrolysis of cell membrane phospholipids. These results open new prospects for characterising biochemical and structural features of Mycobacterium tuberculosis PLCs, which might lead to the identification of novel anti-tuberculosis drug targets. All mycobacterial phospholipase Cs can now be studied in order to determine their role in the virulence and pathogenicity of bacteria of this kind.     

Identification of two Mycobacterium tuberculosis H37Rv ORFs involved in resistance to killing by human macrophages

Background  

The ability of Mycobacterium tuberculosis to survive and replicate in macrophages is crucial for the mycobacterium's ability to infect the host and cause tuberculosis. To identify Mycobacterium tuberculosis genes involved in survival in macrophages, a library of non-pathogenic Mycobacterium smegmatis bacteria, each carrying an individual integrated cosmid containing M. tuberculosis H37Rv genomic DNA, was passed through THP-1 human macrophages three times.     

Mycobacterium tuberculosis activates the DNA-dependent cytosolic surveillance pathway within macrophages

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Highlights? M. tuberculosis permeabilizes phagosomes and activates cytosolic signaling pathways ? Host cytoplasmic DNA receptors sense M. tuberculosis extracellular DNA ? DNA recognition activates the STING/TBK1/IRF3 pathway ? Cytosolic sensing promotes M. tuberculosis infection     

cAMP levels within Mycobacterium tuberculosis and Mycobacterium bovis BCG increase upon infection of macrophages

Adenosine 3',5'-cyclic monophosphate (cAMP)-mediated signal transduction is common in both prokaryotes and eukaryotes, and several bacterial pathogens modulate cAMP signaling pathways of their mammalian hosts during infection. In this study, cAMP levels associated with Mycobacterium tuberculosis and Mycobacterium bovis BCG were measured during macrophage infection. cAMP levels within both bacteria increased c . 50-fold during infection of J774.16 macrophages, relative to the cAMP levels within bacteria incubated in tissue culture media alone. cAMP levels also increased within the macrophage cytoplasm upon uptake of live, but not dead, mycobacteria. The presence of albumin in the absence of oleic acid significantly decreased cAMP secretion and production by both M. tuberculosis and M. bovis BCG. These results suggest that cAMP signaling plays a role in the interaction of tuberculosis-complex mycobacteria with macrophages during infection, and that albumin may be a physiological indicator differentiating host environments during infection.     

Interactions of attenuated Mycobacterium tuberculosis phoP mutant with human macrophages

Background

Mycobacterium tuberculosis phoP mutant SO2 derived from a clinical isolate was shown to be attenuated in mouse bone marrow-derived macrophages and in vivo mouse infection model and has demonstrated a high potential as attenuated vaccine candidate against tuberculosis.

Methodology/Principal Findings

In this study, we analyze the adhesion and the intracellular growth and trafficking of SO2 in human macrophages. Our results indicate an enhanced adhesion to phagocitic cells and impaired intracellular replication of SO2 in both monocyte-derived macrophages and human cell line THP-1 in comparison with the wild type strain, consistent with murine model. Intracellular trafficking analysis in human THP-1 cells suggest that attenuation of SO2 within macrophages could be due to an impaired ability to block phagosome-lysosome fusion compared with the parental M. tuberculosis strain. No differences were found between SO2 and the wild-type strains in the release and mycobacterial susceptibility to nitric oxide (NO) produced by infected macrophages.

Conclusions/Significance

SO2 has enhanced ability to bind human macrophages and differs in intracellular trafficking as to wild-type M. tuberculosis. The altered lipid profile expression of the phoP mutant SO2 and its inability to secrete ESAT-6 is discussed.     

TNF-alpha and IL-10 modulate the induction of apoptosis by virulent Mycobacterium tuberculosis in murine macrophages

The Bcg/Nramp1 gene controls early resistance and susceptibility of macrophages to mycobacterial infections. We previously reported that Mycobacterium tuberculosis-infected (Mtb) B10R (Bcgr) and B10S (Bcgs) macrophages differentially produce nitric oxide (NO-), leading to macrophage apoptosis. Since TNF-alpha and IL-10 have opposite effects on many macrophage functions, we determined the number of cells producing TNF-alpha and IL-10 in Mtb-infected or purified protein derivative-stimulated B10R and B10S macrophages lines, and Nramp1+/+ and Nramp1-/- peritoneal macrophages and correlated them with Mtb-mediated apoptosis. Mtb infection and purified protein derivative treatment induced more TNF-alpha+Nramp1+/+ and B10R, and more IL-10+Nramp1-/- and B10S cells. Treatment with mannosylated lipoarabinomannan, which rescues macrophages from Mtb-induced apoptosis, augmented the number of IL-10 B10R+ cells. Anti-TNF-alpha inhibited apoptosis, diminished NO- production, p53, and caspase 1 activation and increased Bcl-2 expression. In contrast, anti-IL-10 increased caspase 1 activation, p53 expression, and apoptosis, although there was no increment in NO- production. Murine rTNF-alpha induced apoptosis in noninfected B10R and B10S macrophages that was reversed by murine rIL-10 in a dose-dependent manner with concomitant inhibition of NO- production and caspase 1 activation. NO- and caspase 1 seem to be independently activated in that aminoguanidine did not affect caspase 1 activation and the inhibitor of caspase 1, Tyr-Val-Ala-Asp-acylooxymethylketone, did not block NO- production; however, both treatments inhibited apoptosis. These results show that Mtb activates TNF-alpha- and IL-10-dependent opposite signals in the induction of macrophage apoptosis and suggest that the TNF-alpha-IL-10 ratio is controlled by the Nramp1 background of resistance/susceptibility and may account for the balance between apoptosis and macrophage survival.     

Sulfolipid-1 biosynthesis restricts Mycobacterium tuberculosis growth in human macrophages

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a highly evolved human pathogen characterized by its formidable cell wall. Many unique lipids and glycolipids from the Mtb cell wall are thought to be virulence factors that mediate host-pathogen interactions. An intriguing example is Sulfolipid-1 (SL-1), a sulfated glycolipid that has been implicated in Mtb pathogenesis, although no direct role for SL-1 in virulence has been established. Previously, we described the biochemical activity of the sulfotransferase Stf0 that initiates SL-1 biosynthesis. Here we show that a stf0-deletion mutant exhibits augmented survival in human but not murine macrophages, suggesting that SL-1 negatively regulates the intracellular growth of Mtb in a species-specific manner. Furthermore, we demonstrate that SL-1 plays a role in mediating the susceptibility of Mtb to a human cationic antimicrobial peptide in vitro, despite being dispensable for maintaining overall cell envelope integrity. Thus, we hypothesize that the species-specific phenotype of the stf0 mutant is reflective of differences in antimycobacterial effector mechanisms of macrophages.     

Isolation of two subpopulations of Mycobacterium avium within human macrophages.

Mycobacterium avium is an intracellular pathogen that is associated with disseminated infection in acquired immunodeficiency syndrome (AIDS) patients. Human monocyte-derived macrophages were infected with M. avium strain 101 and a quinolone (Bay y 3118) was used at 8 micrograms ml-1, a concentration that kills growing bacteria but fails to eliminate static organisms. Infected monolayers were treated with Bay y 3118 for 4 days and viable bacteria obtained from the lysis of macrophages were used to infect other macrophages without passage in media. The procedure was repeated five times, after which seven different subpopulations that failed to grow within macrophages were identified. While the DNA fingerprinting confirmed that all came from the same strain, three protein profiles were observed. Static subpopulations were not killed by cytokine-stimulated macrophages, in contrast to the replicating subpopulation. Three of the static subpopulation strains were shown to be auxotrophic for glutamic acid or methionine. All seven non-duplicating subpopulation strains grew well in complete 7H10 agar. The importance of a static subpopulation of M. avium within macrophages is presently unknown. It is possible, however, that the non-growing bacteria would persist within macrophages.     

Mycobacterium tuberculosis gene expression in macrophages

This review provides a discussion on the current information about the response of Mycobacterium tuberculosis to the environment encountered in the macrophage. We focus on the types of genes shown to be upregulated when the pathogen grows in macrophages and discuss the possible roles of these genes in adaptation to the conditions in the eukaryotic cell, in the context of enhancing the survival of the pathogen during infection.     

结核分枝杆菌与巨噬细胞相互作用的研究进展

结核分枝杆菌是一种胞内感染菌,巨噬细胞是其寄生场所。结核分枝杆菌通过阻止吞噬溶酶体的融合、减少巨噬细胞凋亡、降低巨噬细胞对刺激应答的敏感性等途径逃避巨噬细胞的免疫监视和攻击,并在细胞内存活、增殖;而巨噬细胞又是抗菌免疫的主要效应细胞,通过直接杀伤和分泌多种细胞因子,对结核分枝杆菌具有免疫调节、呈递抗原等作用。深入研究结核分枝杆菌对巨噬细胞的免疫逃逸机制及巨噬细胞抗结核免疫作用,对研究宿主抗结核免疫机制及设计新型结核病疫苗有重要意义。     

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.     

结核分枝杆菌对巨噬细胞抗原呈递功能的抑制作用

本文旨在通过观察结核分枝杆菌刺激后巨噬细胞抗原呈递功能的变化, 探讨结核分枝杆菌的免疫逃逸机制。在体外, 结核分枝杆菌刺激巨噬细胞24 h 后, 用流式细胞仪检测γ干扰素( IFN-γ) 诱导的主要组织相容性复合物( MHC) Ⅱ类分子、CD86 和CD80 的表达变化; 酶联免疫吸附试验( ELISA) 检测巨噬细胞的抗原呈递功能; 反转录-聚合酶链反应检测巨噬细胞CⅡTA 及其启动子PⅠ、PⅢ和PⅣ的mRNA 水平。结果发现, 结核分枝杆菌抑制IFN-γ诱导的巨噬细胞表面MHCⅡ 类分子和CD86 的表达, 且呈剂量依赖性, 但CD80的表达变化不明显; 抗原呈递功能明显降低; 结核分枝杆菌刺激后巨细胞CⅡTA 及其启动子PⅠ、PⅢ和PⅣ的mRNA 水平显著降低。提示结核分枝杆菌可能通过降低CⅡTA 及其启动子PⅠ、PⅢ和PⅣ 的mRNA 水平, 抑制IFN-γ诱导的巨噬细胞MHCⅡ类分子的表达; 结核分枝杆菌可降低巨噬细胞CD86 的表达, 抑制IFN-γ诱导的巨噬细胞抗原呈递功能。