关键词： PDIM   Gal-3   炎症反应   NF-κB   肉芽肿  

摘要： 目的:研究鉴定分枝杆菌细胞壁脂质PDIM诱导Gal-3表达的TLR信号通路,明确Gal-3对PDIM调控炎症介质释放的影响及其机制;并进一步研究Gal-3对PDIM介导肉芽肿形成的意义与机制。方法:将Raw264.7细胞分为正常对照组(Mock)、WT野生株和PDIM缺失突变株(命名为△PDIM)感染组。(1)Western blot检测各感染组0.5-4 h内Gal-3蛋白表达,qRT-PCR检测Gal-3 mRNA表达,细胞免疫荧光检测各组感染4 h时Gal-3蛋白表达强度,并统计比较各感染组不同时间点胞内菌数量,明确PDIM诱导Gal-3蛋白表达与侵入胞内的细菌数量无关。(2)Western blot和qRT-PCR分别检测各感染组0.5-4 h内TLR2和TLR4蛋白与mRNA表达变化;并分别使用TLR2受体特异性抑制剂(C29)和NF-κB特异性转录活性抑制剂(JSH-23)预处理细胞,western blot和qRT-PCR分别比较各感染组0.5-4 h Gal-3蛋白和mRNA表达,初步明确PDIM诱导Gal-3表达的TLR信号通路。(3)各组感染4 h时,qRT-PCR检测TNF-α、IL-6、iNOS、IL-10和TGF-β表达,western blot检测各感染组0.5-4 h内ERK、NF-κBp65和IKK总蛋白及其磷酸化水平,confocal观察Gal-3与NF-κBp65的共定位情况,免疫共沉淀(Co-IP)检测Gal-3与NF-κBp65之间的相互作用。(4)转染Gal-3的siRNA干扰质粒到Raw264.7细胞,再感染WT和△PDIM菌株4 h,qRT-PCR检测各感染组TNF-α、IL-6、iNOS、IL-10和TGF-β表达,confocal检测各感染组中Gal-3与NF-κBp65的胞内表达及定位情况。(5)构建Gal-3过表达质粒并转染Raw264.7细胞,再感染△PDIM菌株4 h,qRT-PCR检测TNF-α、iNOS和TGF-β表达,confocal检测Gal-3表达以及NF-κBp65入核情况,明确PDIM诱导Gal-3对NF-κB活化及下游炎症相关因子表达变化影响。(6)建立海分枝杆菌小鼠尾静脉感染模型,然后再注射Gal-3特异性抑制剂TD139,比较大体病变和计算病变面积,组织匀浆铺板计数载菌量,HE染色和抗酸染色观察小鼠尾部组织病理变化及细菌分布,免疫组化染色和图像分析技术检测小鼠组织中Gal-3、TNF-α和MMP-9蛋白表达与分布,明确TD139对小鼠组织损伤的干预效应。结果:(1)海分枝杆菌感染小鼠Raw264.7巨噬细胞后,WT感染组中Gal-3蛋白和mRNA均呈上升趋势;细菌侵染实验发现,含有PDIM的WT菌株诱导Gal-3表达与侵入Raw264.7细胞内的细菌数无关。(2)相比于△PDIM感染组,WT感染组中TLR2蛋白和mRNA表达逐渐升高,而两组中TLR4蛋白和mRNA表达均降低;使用TLR2受体特异性抑制剂(C29)和NF-κB特异性转录活性抑制剂(JSH-23)预处理细胞后,WT感染组中Gal-3蛋白和mRNA表达明显降低,提示PDIM可能通过TLR2/NF-κB通路诱导Gal-3表达。(3)感染4 h后,相比于WT感染组,△PDIM感染组中促炎因子TNF-α、IL-6和iNOS表达显著升高,而抑炎因子IL-10和TGF-β表达降低,且差异显著。Western blot检测发现WT感染组中ERK、NF-κBp65和IKK蛋白的磷酸化水平较低,而△PDIM感染组中ERK、NF-κBp65和IKK蛋白则是持续磷酸化状态,表明PDIM可抑制宿主细胞早期免疫反应。Confocal观察发现Gal-3和NF-κBp65存在明显共定位,并且Co-IP证实两者之间存在较强的相互作用,说明PDIM诱导Gal-3可抑制NF-κB活化及下游炎症相关因子表达。(4)转染Gal-3的siRNA干扰质粒到Raw264.7细胞后,能完全逆转WT感染组中促炎因子和抑炎因子的表达趋势,并且显著增加NF-κBp65入核活化率。(5)转染Gal-3过表达质粒到Raw264.7细胞后,也能逆转△PDIM感染组中炎症介质表达,并且显著降低NF-κBp65入核活化率(6)小鼠尾静脉感染模型中,WT感染组小鼠尾部出现明显肿胀、溃烂,组织病理学观察发现有典型肉芽肿病变,并检测到大量细菌,TNF-α和MMP-9表达呈强阳性;给予WT感染组小鼠注射TD139干预后,小鼠尾部病变得到明显改善,TNF-α和MMP-9炎性介质表达减少;而△PDIM感染组小鼠尾部有轻微肿胀,仅检测到少许细菌。结论:分枝杆菌细胞壁脂质PDIM可能通过TLR2/NF-κB通路诱导Gal-3表达,Gal-3负反馈于NF-κB信号通路

关键词： virulence   Cyclipostins   Cyclophostin   PDIM   PGL  

摘要： With the high number of patients infected by tuberculosis and the sharp increase of drug-resistant tuberculosis cases, developing new drugs to fight this disease has become increasingly urgent. In this context, analogs of the naturally occurring enolphosphates Cyclipostins and Cyclophostin (CyC analogs) offer new therapeutic opportunities. The CyC analogs display potent activity both in vitro and in infected macrophages against several pathogenic mycobacteria including Mycobacterium tuberculosis and Mycobacterium abscessus. Interestingly, these CyC inhibitors target several enzymes with active-site serine or cysteine residues that play key roles in mycobacterial lipid and cell wall metabolism. Among them, TesA, a putative thioesterase involved in the synthesis of phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs), has been identified. These two lipids (PDIM and PGL) are non-covalently bound to the outer cell wall in several human pathogenic mycobacteria and are important virulence factors. Herein, we used biochemical and structural approaches to validate TesA as an effective pharmacological target of the CyC analogs. We confirmed both thioesterase and esterase activities of TesA, and showed that the most active inhibitor CyC17, binds covalently to the catalytic Ser104 residue leading to a total loss of enzyme activity. These data were supported by the X-ray structure, obtained at a 2.6-angstrom resolution, of a complex in which CyC17 is bound to TesA. Our study provides evidence that CyC17 inhibits the activity of TesA, thus paving the way to a new strategy for impairing the PDIM and PGL biosynthesis, potentially decreasing the virulence of associated mycobacterial species. (C) 2018 Elsevier Ltd. All rights reserved.

关键词： Mycobacterium tuberculosis   Aloe emodin   PDIM   Vancomycin   MDR  

摘要： Treatment of tuberculosis still represent a major public health issue. The emergence of multi-and extensively-drug resistant (MDR and XDR) Mycobacterium tuberculosis clinical strains further pinpoint the urgent need for new anti-tuberculous drugs. We previously showed that vancomycin can target mycobacteria lacking cell wall integrity, especially those lacking related phthiocerol and phthiodolone dimycocerosates, PDIM A and PDIM B, respectively. As aloe emodin was previously hypothesized to be able to target the synthesis of mycobacterial cell wall lipids, we tested its ability to potentiate glycopeptides antimycobacterial activity. The aloe emodin with the vancomycin induced a combination effect beyond simple addition, close to synergism, at a concentration lower to reported IC50 cytotoxic value, on M. bovis BCG and on H37Rv M. tuberculosis. Interestingly, out of six MDR and pre-XDR clinical strains, one showed a strong synergic susceptibility to the drug combination. Mycobacterial cell wall lipid analyses highlighted a selective reduction of PDIM B by aloe emodin.

关键词： Methylcitrate cycle   Vitamin B-12   Methylmalonyl pathway   mce4A   Cholesterol   String  

摘要： Lipid metabolism forms the heart and soul of Mycobacterium tuberculosis life cycle. Starting from macrophage invasion at cholesterol rich micro-domains to a sustainable survival for infection by utilizing cholesterol, Mycobacterium displays the nexus of metabolic pathways around host derived lipids. mce4 operon acts as cholesterol import system in M. tuberculosis and here we demonstrate role of mce4A gene of this operon in cholesterol catabolism. Here M. tuberculosis H37Rv overexpressing Rv3499c (mce4A) recombinant was used as a model to decipher the metabolic flux during intake and utilization of host lipids by mycobacteria. We analysed the impact of mce4A expression on carbon shift initiated during cholesterol utilization necessary for long term survival of mycobacterium. Through transcriptional analysis, upregulation in methylcitrate cycle (MCC) and methylmalonyl pathway (MMP) genes was observed in Rv3499c overexpressing recombinants of M. tuberculosis H37Rv. Up-regulation of methylmalonyl pathway associated enzyme encoding genes increased accumulation of virulence associated mycobacterial lipids phthiocerol dimycocerates (PDIM) and sulfolipid (SL1). We demonstrate that MCC and MMP associated enzyme encoding genes are upregulated upon mce4A overexpression and lead to enhanced accumulation of PDIM and SL1 which are responsible for pathogenicity of M. tuberculosis.

关键词： Mycobacterium tuberculosis   Tuberculosis   Phthiocerol dimycocerosates   PDIM   Lymphatic endothelial cells   hLEC   Macrophages   Phagosome damage   Cytosol   Xenophagy  

摘要： Background: Phthiocerol dimycocerosates (PDIM), glycolipids found on the outer surface of virulent members of the Mycobacterium tuberculosis (Mtb) complex, are a major contributing factor to the pathogenesis of Mtb. Myelocytic cells, such as macrophages and dendritic cells, are the primary hosts for Mtb after infection and previous studies have shown multiple roles for PDIM in supporting Mtb in these cells. However, Mtb can infect other cell types. We previously showed that Mtb efficiently replicates in human lymphatic endothelial cells (hLECs) and that the hLEC cytosol acts as a reservoir for Mtb in humans. Here, we examined the role of PDIM in Mtb translocation to the cytosol in hLECs. Results: Analysis of a Mtb mutant unable to produce PDIM showed less co-localisation of bacteria with the membrane damage marker Galectin-8 (Gal8), indicating that PDIM strongly contribute to phagosomal membrane damage. Lack of this Mtb lipid also leads to a reduction in the proportion of Mtb co-localising with markers of macroautophagic removal of intracellular bacteria (xenophagy) such as ubiquitin, p62 and NDP52. hLEC imaging with transmission electron microscopy shows that Mtb mutants lacking PDIM are much less frequently localised in the cytosol, leading to a lower intracellular burden. Conclusions: PDIM is needed for the disruption of the phagosome membrane in hLEC, helping Mtb avoid the hydrolytic phagolysosomal milieu. It facilitates the translocation of Mtb into the cytosol, and the decreased intracellular burden of Mtb lacking PDIM indicates that the cytosol is the preferred replicative niche for Mtb in these cells. We hypothesise that pharmacological targeting of PDIM synthesis in Mtb would reduce the formation of a lymphatic reservoir of Mtb in humans.

关键词： Mycobacterium tuberculosis   I3-Ag85   TDM   PDIM  

摘要： The multiplicity of drug resistant Mycobacterium tuberculosis (Mtb) strains is a growing health issue. New therapies are needed, acting on new targets. The I3-Ag85 was already reported to reduce the amount of trehalose dimycolate lipid of the mycobacterial cell wall. This inhibitor of Ag85C increased the mycobacterial wall permeability. We previously showed that M. tuberculosis strains, even multi-drug resistant and extensively-drug resistant strains, can be susceptible to vancomycin when concomitantly treated with a drug altering the cell envelope integrity. We investigated the effect of the I3-Ag85 on vancomycin susceptibility of M. tuberculosis. Although no synergy was observed, a new target of this drug was discovered: the production of phthiodiolone dimycocerosate (PDIM B).

关键词： 5-HETE   5-hydroxyicosatetraenoic acid   12-HETE   12-hydroxyicosatetraenoic acid   15-HETE   15-hydroxyicosatetraenoic acid   aa   diacyl   ae   acyl-alkyl   ACs   acylcarnitines   ACOG   The American College of Obstetricians and Gynecologists   AD   Alzheimer’s disease   Aβ   amyloid-beta   aSAH   aneurysmal subarachnoid hemorrhage   AA   arachidonic acid   CL   cardiolipins   C1P   ceramide-1-phosphate   CERK   ceramide kinase   CSF   cerebrospinal fluid   CEH   cholesterol ester hydrolase   COX-1   cyclooxygenase 1   COX-2   cyclooxygenase 2   cPLA 2α   group IVA cytosolic phospholipase A 2   P450   cytochrome P450   DHA   docosahexaenoic acid   EPA   eicosapentaenoic acid   ESI   electrospray ionization   ER   endoplasmic reticulum   ELISA   enzyme-linked immunosorbent assay   FT-ICR MS   Fourier transform ion cyclotron resonance mass spectrometry   Gal-G5   galactose-functionalized polyamidoamine dendrimer generation 5   HDL   high-density lipoprotein   HPLC   high pressure liquid chromatography   HTS   high-throughput screening   HMG-CoA   hydroxymethylglutaryl-CoA   HMGCS   hydroxymethylglutaryl-CoA synthase   ICU   intensive care unit   LTB 4   leukotriene B 4   LXA 4   lipoxin A 4   LOX   lipoxygenase   LC/MS-MS   liquid chromatography mass spectrometry   LDL   low-density lipoprotein   LPA   lysophosphatidic acid   LPC   lysophosphatidylcholine   m/z   mass/charge ratio   mApoE-PA-LIP   modified ApoE-derived peptide   MRM   multiple reaction monitoring   PLS-DA   partial least squares discriminant analysis   PLA 2   phospholipase A 2   PA   phosphatidic acid   PC   phosphatidylcholine   PE   phosphatidylethanolamine   PG   phosphatidylglycerols   PI   phosphatidylinositol   PIP 2   phosphatidylinositol 4,5-bisphosphate   PS   phosphatidylserine   PDIM   phthiocerol dimycocerosate   PRP   platelet-rich plasma   PAMAM   polyamidoamine   PUFA   polyunsaturated fatty acid   PIH   pregnancy-induced hypertension   PGE 1   prostaglandin E 1   PGE 2   prostaglandin E 2   PGF 1a   prostaglandin F1 alpha   PGF 2a   prostaglandin F2 alpha   PGJ 2   prostaglandin J 2   SPMs   pro-resolving lipid mediators   PD 1   protection D 1   QTOF   quadrupole time-of-flight   RNS   reactive nitrogen species   ROS   reactive oxygen species   RvD 1   resolvin D 1   RvD 2   resolvin D 2   RvD 5   res  

摘要： Lipidomics is a rapidly developing field of study that focuses on the identification and quantitation of various lipid species in the lipidome. Lipidomics has now emerged in the forefront of scientific research due to the importance of lipids in metabolism, cancer, and disease. Using both targeted and untargeted mass spectrometry as a tool for analysis, progress in the field has rapidly progressed in the last decade. Having the ability to assess these small molecules in vivo has led to better understanding of several lipid-driven mechanisms and the identification of lipid-based biomarkers in neurodegenerative disease, cancer, sepsis, wound healing, and pre-eclampsia. Biomarker identification and mechanistic understanding of specific lipid pathways linked to a disease's pathologies can form the foundation in the development of novel therapeutics in hopes of curing human disease.

摘要： The cell wall of Mycobacterium tuberculosis is composed of unique lipids that are important for pathogenesis. Indeed, the first-ever genetic screen in M. tuberculosis identified genes involved in the biosynthesis and transport of the cell wall lipid PDIM (phthiocerol dimycocerosates) as crucial for the survival of M. tuberculosis in mice. Here we show evidence for a novel molecular mechanism of the PDIM-mediated virulence in M. tuberculosis. We characterized the DNA interaction and the regulon of Rv3167c, a transcriptional repressor that is involved in virulence regulation of M. tuberculosis, and discovered that it controls the PDIM operon. A loss-of-function genetic approach showed that PDIM levels directly correlate with the capacity of M. tuberculosis to escape the phagosome and induce host cell necrosis and macroautophagy. In conclusion, our study attributes a novel role of the cell wall lipid PDIM in intracellular host cell modulation, which is important for host cell exit and dissemination of M. tuberculosis. IMPORTANCE Mycobacterium tuberculosis is a major human pathogen that has co-evolved with its host for thousands of years. The complex and unique cell wall of M. tuberculosis contains the lipid PDIM (phthiocerol dimycocerosates), which is crucial for virulence of the bacterium, but its function is not well understood. Here we show that PDIM expression by M. tuberculosis is negatively regulated by a novel transcriptional repressor, Rv3167c. In addition, we discovered that the escape of M. tuberculosis from its intracellular vacuole was greatly augmented by the presence of PDIM. The increased release of M. tuberculosis into the cytosol led to increased host cell necrosis. The discovery of a link between the cell wall lipid PDIM and a major pathogenesis pathway of M. tuberculosis provides important insights into the molecular mechanisms of host cell manipulation by M. tuberculosis.

关键词： Esx-1   PDIM   complementation   mycobacteria   nonsense suppression   spontaneous mutation  

摘要： Mycobacterial pathogens use the ESAT-6 system 1 (Esx-1) exporter to promote virulence. Previously, we used gene disruption and complementation to conclude that the MMAR_0039 gene in Mycobacterium marinum is required to promote Esx-1 export. Here we applied molecular genetics, proteomics, and whole-genome sequencing to demonstrate that the MMAR_0039 gene is not required for Esx-1 secretion or virulence. These findings suggest that we initially observed an indirect mechanism of genetic complementation. We identified a spontaneous nonsense mutation in a known Esx-1-associated gene which causes a loss of Esx-1 activity. We show that the Esx-1 function was restored by nonsense suppression. Moreover, we identified a polar mutation in the ppsC gene which reduced cellular impermeability but did not impact cytotoxicity in macrophages. Our studies reveal insight into Esx-1 export, nonsense suppression, and cell envelope lipid biogenesis.

关键词： Phthiocerol dimycocerosate A (PDIM A)   Tuberculosis   Total synthesis  

摘要： Total synthesis of phthiocerol dimycocerosate A (PDIM A), a virulent factor of Mycobacterium tuberculosis, has been achieved. Phthiocerol, a component of PDIM A, has been synthesized by subsequent epoxide-opening allcylation reactions with arabinose-derived diepoxide. This route is concise and efficient in supplying PDIM A for biological studies.