关键词： Mycobacterium tuberculosis   MmpL   PDIM   Lipid transport   RND transporters  

摘要： Mycobacterial membrane protein Large (MmpL7) is a Resistance-Nodulation-Division (RND) family transporter required for the export of the virulence lipid, phthiocerol dimycocerosate (PDIM), in Mycobacterium tuberculosis . Using a null mutant of the related, vaccine strain Mycobacterium bovis BCG, we show that MmpL7 is also involved in the transport of the structurally related phenolic glycolipid (PGL), which is also produced by the hypervirulent M. tuberculosis strain HN878, but absent in M. tuberculosis H37Rv. Furthermore, we generated an in silico model of M. tuberculosis MmpL7 that revealed MmpL7 as a functional outlier within the MmpL-family, missing a canonical proton-relay signature sequence, suggesting that it employs a yet-unidentified mechanism for energy coupling for transport. In addition, our analysis demonstrates that the periplasmic porter domain 2 insert (PD2-insert), which doesn't share any recognisable homology, is highly alpha-helical in nature, suggesting an organisation similar to that seen in the hopanoid PD3/4 domains. Using the M. bovis BCG mmpL7 mutant for functional complementation with mutated alleles of mmpL7 , we were able to identify residues present in the transmembrane domains TM4 and TM10, and the PD2 domain insert that play a crucial role in PDIM transport, and in certain cases, biosynthesis of PDIM.

关键词： Mycobacterium leprae   cholesterol   cholestenone   PGL-I   PDIM   3 beta-HSD   reductive power   oxidation  

摘要： Upon infection, Mycobacterium leprae, an obligate intracellular bacillus, induces accumulation of cholesterol-enriched lipid droplets (LDs) in Schwann cells (SCs). LDs are promptly recruited to M. leprae-containing phagosomes, and inhibition of this process decreases bacterial survival, suggesting that LD recruitment constitutes a mechanism by which host-derived lipids are delivered to intracellular M. leprae. We previously demonstrated that M. leprae has preserved only the capacity to oxidize cholesterol to cholestenone, the first step of the normal cholesterol catabolic pathway. In this study we investigated the biochemical relevance of cholesterol oxidation on bacterial pathogenesis in SCs. Firstly, we showed that M. leprae increases the uptake of LDL-cholesterol by infected SCs. Moreover, fluorescence microscopy analysis revealed a close association between M. leprae and the internalized LDL-cholesterol within the host cell. By using Mycobacterium smegmatis mutant strains complemented with M. leprae genes, we demonstrated that ml1942 coding for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), but not ml0389 originally annotated as cholesterol oxidase (ChoD), was responsible for the cholesterol oxidation activity detected in M. leprae. The 3 beta-HSD activity generates the electron donors NADH and NADPH that, respectively, fuel the M. leprae respiratory chain and provide reductive power for the biosynthesis of the dominant bacterial cell wall lipids phthiocerol dimycocerosate (PDIM) and phenolic glycolipid (PGL)-I. Inhibition of M. leprae 3 beta-HSD activity with the 17 beta-[N-(2,5-di-t-butylphenyl)carbamoyl]-6-azaandrost-4-en-3one (compound 1), decreased bacterial intracellular survival in SCs. In conclusion, our findings confirm the accumulation of cholesterol in infected SCs and its potential delivery to the intracellular bacterium. Furthermore, we provide strong evidence that cholesterol oxidation is an essential catabolic pathway for M. leprae pathogenicit

关键词： Mycobacterium tuberculosis   mycobacteria outer membrane   PDIM   ESX-1  

摘要： The success of Mycobacterium tuberculosis as a pathogen is well established: tuberculosis is the leading cause of death by a single infectious agent worldwide. The threat of multi- and extensively drug- resistant bacteria has renewed global concerns about this pathogen and understanding its virulence strategies will be essential in the fight against tuberculosis. The current review will focus on phthiocerol dimycocerosates (PDIMs), a long- known and well- studied group of complex lipids found in the M. tuberculosis cell envelope. Numerous studies show a role for PDIMs in several key steps of M. tuberculosis pathogenesis, with recent studies highlighting its involvement in bacterial virulence, in association with the ESX-1 secretion system. Yet, the mechanisms by which PDIMs help M. tuberculosis to control macrophage phagocytosis, inhibit phagosome acidification and modulate host innate immunity, remain to be fully elucidated.

关键词： Mycobacterium bovis   PhoP   Acidic stress   PDIM  

摘要： PhoP is part of the two-component PhoPR system that regulates the expression of virulence genes of Mycobacteria. The goal of this work was to elucidate the role of PhoP in the mechanism that Mycobacterium bovis, the causative agent of bovine tuberculosis, displays upon stress. An analysis of gene expression and acidic growth curves indicated that M. bovis neutralized the external acidic environment by inducing and secreting ammonia. We found that PhoP is essential for ammonia production/secretion and its role in this process seems to be the induction of asparaginase and urease expression. We also demonstrated that the lack of PhoP negatively affected the synthesis of phthiocerol dimycocerosates. This finding is consistent with the role of the lipid anabolism in maintaining the redox environment upon stress in mycobacteria. Altogether the results of this study indicate that PhoP plays an important role in the response mechanisms to stress of M. bovis.

关键词： HIV-1   TB   Mycobacterium tuberculosis   in vitro   DC-SIGN   liposomes   trans-infection   SL1   TDM   PDIM   H37Rv   HN878   CDC1551   EU127   BCG   M   smegmatis  

摘要： Tuberculosis (TB) is the leading cause of death among HIV-1-infected individuals and Mycobacterium tuberculosis (Mtb) co-infection is an early precipitate to AIDS. We aimed to determine whether Mtb strains differentially modulate cellular susceptibility to HIV-1 infection (cis- and trans-infection), via surface receptor interaction by their cell envelope lipids. Total lipids from pathogenic (lineage 4 Mtb H37Rv, CDC1551 and lineage 2 Mtb HN878, EU127) and non-pathogenic (Mycobacterium bovis BCG and Mycobacterium smegmatis) Mycobacterium strains were integrated into liposomes mimicking the lipid distribution and antigen accessibility of the mycobacterial cell wall. The resulting liposomes were tested for modulating in vitro HIV-1 cis- and trans-infection of TZM-bl cells using single-cycle infectious virus particles. Mtb glycolipids did not affect HIV-1 direct infection however, trans-infection of both R5 and X4 tropic HIV-1 strains were impaired in the presence of glycolipids from M. bovis, Mtb H37Rv and Mtb EU127 strains when using Raji-DC-SIGN cells or immature and mature dendritic cells (DCs) to capture virus. SL1, PDIM and TDM lipids were identified to be involved in DC-SIGN recognition and impairment of HIV-1 trans-infection. These findings indicate that variant strains of Mtb have differential effect on HIV-1 trans-infection with the potential to influence HIV-1 disease course in co-infected individuals.

关键词： 分枝杆菌   PDIM   IQGAP1   VEGF  

摘要： 研究背景:众所周知,结核病是一种危害严重的传染性,引起结核病的是结核分枝杆菌,是一种细胞内的病原菌。结核分枝杆菌既不产生内毒素,也不分泌外毒素和侵袭性酶,它的致病性主要与细菌细胞壁上高含量的脂质成分有关。目的:探讨骨架蛋白IQGAP1(IQ motif-containing GTPase-activating protein 1)在分枝杆菌细胞壁脂质PDIM介导感染和肉芽肿形成中的作用,揭示IQGAP1调控p38MAPK和VEGF表达对分枝杆菌感染与致病的机制;并进一步研究骨架蛋白IQGAP1的小分子抑制剂对分枝杆菌PDIM介导致病的影响,为寻找和鉴定新的结核病的治疗靶点提供实验依据。方法:研究工作主要通过结合体外Raw264.7巨噬细胞感染实验和小鼠体内感染实验完成。1.揭示IQGAP1对分枝杆菌感染早期炎症因子表达的影响及p38MAPK信号在该过程中的作用。(1)Raw264.7巨噬细胞培养传代后,随机分为3组:无菌PBS处理作为对照(Mock)、野生型海分枝杆菌感染组(wide-type,WT)、海分枝杆菌细胞壁毒力成分PDIM缺失突变株(ΔPDIM)感染组。western blot检测感染后0.5 h、1 h、2 h、4 h时IQGAP1的蛋白表达。(2)感染4 h时,q RT-PCR检测促炎因子(TNF-α、IL-6)和抑炎因子(TGF-β、IL-10)的表达;western blot检测IQGAP1、TNF-α、TGF-β及p38MAPK和NF-κBp65的磷酸化水平。(3)设计合成针对IQGAP1的si RNA序列和过表达质粒(GV219＿Iqgap1)并分别转染Raw264.7巨噬细胞,再分别感染WT或?PDIM菌株,q RT-PCR检测上述促炎或抑炎因子的表达;western blot检测IQGAP1、TNF-α、TGF-β及p38MAPK和NF-κBp65的磷酸化水平。Confocal检测IQGAP1的si RNA干扰或过表达对细菌感染过程NF-κBp65入核的影响。(4)Raw264.7细胞分别感染WT和?PDIM菌株,q RT-PCR检测p38MAPK磷酸化级联反应的关键激酶如MKK3和MKK6及失活激酶的磷酸化酶如MKP1和MKP5的表达;Raw264.7细胞分别转染IQGAP1的si RNA和过表达质粒后再检测上述分子的表达;在上述两步实验结果基础上,western blot检测WT和?PDIM菌株感染后MKK3磷酸化水平及过表达或抑制IQGAP1后MKK3磷酸化水平的变化。(5)ΔPDIM菌感染Raw264.7细胞,再用p38MAPK抑制剂SB203580处理细胞,western blot检测不同时间点NF-κBp65磷酸化表达;用ΔPDIM菌和Gossypetin(MKK3/MKK6的特异抑制剂)共孵育Raw264.7细胞,western blot检测MKK3磷酸化水平。(6)构建海分枝杆菌的小鼠尾静脉感染模型,然后第7天时小鼠腹腔注射骨架蛋白抑制剂——Nocodazole(每2天一次),连续注射七次后处死小鼠,比较小鼠尾巴大体病变,收集小鼠尾组织研磨涂板计算菌载量,HE染色观察比较组织病理学变化;同时western blot检测IQGAP1等蛋白的表达变化。2.探讨分枝杆菌感染过程中诱导IQGAP1对VEGF表达的意义与机制。(1)建立上述相同的Raw264.7感染模型,感染4 h时,western blot和ELISA分别检测细胞及上清液中VEGF的蛋白表达。(2)将IQGAP1的si RNA序列和GV219＿Iqgap1质粒分别转染Raw264.7巨噬细胞,再分别感染WT或?PDIM菌株,western blot检测VEGF的表达。(3)用VEGF-receptor的特异性抑制剂阿西替尼(Axitinib)处理Raw264.7细胞,然后western blot检测VEGF及IQGAP1的表达。(4)收取小鼠尾静脉感染模型中的尾组织,western blot检测VEGF的表达;免疫组化染色观察IQGAP1和VEGF的表达和分布。(5)结核分枝杆菌感染的小鼠、兔子和猴子的肺组织及临床结核病人样本中IQGAP1和VEGF的表达与分布。结果:***1通过调控p38MAPK信号通路影响分枝杆菌感染早期炎症因子的表达。(1)Raw264.7巨噬细胞感染模型上,WT菌株感染组IQGAP1蛋白水平明显上调(**P<0.01),而在ΔPDIM组表达微弱。(2)q RT-PCR检测发现WT菌株感染组中抑炎因子(TGF-β、IL-10)的表达显著高于ΔPDIM组(**P<0.01),而促炎因子(TNF-α、IL-6)显著低于ΔPDIM组(**P<0.01)。Western blot检测发现ΔPDIM组中

关键词： Mycobacteria   PDIM   biochemistry   bioorthogonal chemistry   chemical biology   infectious disease   microbiology   zebrafish  

摘要： Several virulence lipids populate the outer cell wall of pathogenic mycobacteria. Phthiocerol dimycocerosate (PDIM), one of the most abundant outer membrane lipids, plays important roles in both defending against host antimicrobial programs and in evading these programs altogether. Immediately following infection, mycobacteria rely on PDIM to evade Myd88-dependent recruitment of microbicidal monocytes which can clear infection. To circumvent the limitations in using genetics to understand virulence lipids, we developed a chemical approach to track PDIM during Mycobacterium marinum infection of zebrafish. We found that PDIM's methyl-branched lipid tails enabled it to spread into host epithelial membranes to prevent immune activation. Additionally, PDIM's affinity for cholesterol promoted this phenotype;treatment of zebrafish with statins, cholesterol synthesis inhibitors, decreased spreading and provided protection from infection. This work establishes that interactions between host and pathogen lipids influence mycobacterial infectivity and suggests the use of statins as tuberculosis preventive therapy by inhibiting PDIM spread.

关键词： PDIM   HpoR   转录调控   胞内生存   IL-6   细胞凋亡  

摘要： 结核分枝杆菌具有独特的细胞壁结构,与病原菌致病性息息相关。其中,最具代表性的成分是结核菌醇蜡分支酸酯(PDIM),它位于细菌细胞壁的表面,对维持细胞壁的完整性十分重要。同时,PDIM也是结核分枝杆菌重要的毒力因子之一,它的生物合成和转运对细菌感染宿主以及感染后在宿主胞内的存活发挥了不可或缺的重要作用。但是,目前我们对于PDIM代谢基因的表达调控了解得非常少,直接相关的转录调控因子尚未鉴定。本研究利用*** BCG为模式菌株,首次发现和鉴定了转录因子HpoR可以直接调控PDIM合成基因簇的表达,具体结果如下:(1)利用凝胶阻滞实验(EMSA)以及染色质免疫沉淀实验(ChIP)证明了HpoR在*** BCG细菌体内以及体外均能与PDIM操纵子的启动子bcg950p结合,而且这种结合具有明显的序列特异性;(2)通过设计、制备涵盖不同长度的bcg950p DNA片段,EMSA实验发现HpoR特异性结合在bcg950p的P1区段,进一步测序分析发现其中含有一个保守的反向回文结构;(3)β-半乳糖苷酶活性实验及实时荧光定量PCR(qRT-PCR)证明了HpoR是一个转录抑制子,阻遏靶基因的表达;超表达HpoR时PDIM基因簇的表达下调,HpoR敲除则导致PDIM基因簇表达上调;(4)利用不同HpoR重组菌株感染巨噬细胞后发现,敲除HpoR会增强BCG菌株在巨噬细胞内的存活,相反HpoR超表达则导致细菌在巨噬细胞内存活率下降;(5)通过比较不同重组菌株感染巨噬细胞后细胞因子分泌水平,发现HpoR敲除菌株导致促炎细胞因子IL-6分泌明显上升;(6)检测感染后细胞表型,发现HpoR超表达BCG菌株感染的巨噬细胞凋亡率上升,ROS水平也相应上升。因此,本论文成功鉴定了一个直接参与调控PDIM基因簇表达的新转录因子HpoR,并初步发现了其在分枝杆菌感染宿主细胞过程中的重要作用,这些工作为后续进一步研究结核分枝杆菌的基因调控与致病机理提供了重要线索。