摘要： Mycobacterium tuberculosis(Mtb) infects several lung macrophage populations, which have distinct abilities to restrict Mtb. What enables Mtb survival in certain macrophage populations is not well understood. Here we used transposon sequencing analysis of Mtb in wild-type and autophagy-deficient mouse macrophages lacking ATG5 or ATG7, and found that Mtb genes involved in phthiocerol dimycocerosate (PDIM) virulence lipid synthesis confer resistance to autophagy. UsingppsDmutant Mtb, we found that PDIM inhibits LC3-associated phagocytosis (LAP) by inhibiting phagosome recruitment of NADPH oxidase. In mice, PDIM protected Mtb from LAP and classical autophagy. During acute infection, PDIM was dispensable for Mtb survival in alveolar macrophages but required for survival in non-alveolar macrophages in an autophagy-dependent manner. During chronic infection, autophagy-deficient mice succumbed to infection with PDIM-deficient Mtb, with impairments in B-cell accumulation in lymphoid follicles. These findings demonstrate that PDIM contributes to Mtb virulence and immune evasion, revealing a contributory role for autophagy in B-cell responses.

关键词： PHTHIOCEROL DIMYCOCEROSATE   METHYLCITRATE CYCLE   METABOLISM   IDENTIFICATION   GENOME   SUSCEPTIBILITY   RESISTANCE   TRANSPOSON   CATABOLISM   REQUIRES  

摘要： Phthiocerol dimycocerosate (PDIM) is an essential virulence lipid of Mycobacterium tuberculosis. In vitro culturing rapidly selects for spontaneous PDIM-negative mutants that have attenuated virulence and increased cell wall permeability, thus impacting the relevance of experimental findings. PDIM loss can also reduce the efficacy of the BCG Pasteur vaccine. Here we show that vancomycin susceptibility can rapidly screen for M. tuberculosis PDIM production. We find that metabolic deficiency of methylmalonyl-CoA impedes the growth of PDIM-producing bacilli, selecting for PDIM-negative variants. Supplementation with odd-chain fatty acids, cholesterol or vitamin B12 restores PDIM-positive bacterial growth. Specifically, we show that propionate supplementation enhances PDIM-producing bacterial growth and selects against PDIM-negative mutants, analogous to in vivo conditions. Our study provides a simple approach to screen for and maintain PDIM production, and reveals how discrepancies between the host and in vitro nutrient environments can attenuate bacterial pathogenicity.

关键词： PDIM   Mycobacterium tuberculosis   Tn-seq   antibiotic resistance   drug tolerance   membrane permeability   nutrient limitation   PROPIONATE METABOLISM   GROWTH   PERSISTENCE   STRESS   SUSCEPTIBILITY   IDENTIFICATION   EXPRESSION   EVOLUTION   PATHWAY   TOXIN  

摘要： Tolerance of Mycobacterium tuberculosis to antibiotics contributes to the long duration of tuberculosis (TB) treatment and the emergence of drug-resistant strains. M. tuberculosis drug tolerance is induced by nutrient restriction, but the genetic determinants that promote antibiotic tolerance triggered by nutrient limitation have not been comprehensively identified. Here, we show that M. tuberculosis requires production of the outer membrane lipid phthiocerol dimycocerosate (PDIM) to tolerate antibiotics under nutrient-limited conditions. We developed an arrayed transposon (Tn) mutant library in M. tuberculosis Erdman and used orthogonal pooling and transposon sequencing (Tn-seq) to map the locations of individual mutants in the library. We screened a subset of the library (similar to 1,000 mutants) by Tn-seq and identified 32 and 102 Tn mutants with altered tolerance to antibiotics under stationary-phase and phosphate-starved conditions, respectively. Two mutants recovered from the arrayed library, ppgK::Tn and clpS::Tn, showed increased susceptibility to two different drug combinations under both nutrient-limited conditions, but their phenotypes were not complemented by the Tn-disrupted gene. Whole-genome sequencing revealed single nucleotide polymorphisms in both the ppgK::Tn and clpS::Tn mutants that prevented PDIM production. Complementation of the clpS::Tn ppsD Q291* mutant with ppsD restored PDIM production and antibiotic tolerance, demonstrating that loss of PDIM sensitized M. tuberculosis to antibiotics. Our data suggest that drugs targeting production of PDIM, a critical M. tuberculosis virulence determinant, have the potential to enhance the efficacy of existing antibiotics, thereby shortening TB treatment and limiting development of drug *** Mycobacterium tuberculosis causes 10 million cases of active TB disease and over 1 million deaths worldwide each year. TB treatment is complex, requiring at least 6 months of therapy with a combinat

关键词： NATURAL-PRODUCTS   REQUIREMENTS   MUTAGENESIS   MECHANISM   PROTEINS   SURVIVAL   GROWTH   GENES   PDIM  

摘要： Isonitrile lipopeptides (INLPs) are known to be related to the virulence of pathogenic mycobacteria by mediating metal transport, but their biosynthesis remains obscure. In this work, we use in vitro biochemical assays, site-directed mutagenesis, chemical synthesis, and spectroscopy techniques to scrutinize the activity of core enzymes required for INLP biosynthesis in mycobacteria. Compared to environmental Streptomyces, pathogenic Mycobacterium employ a similar chemical logic and enzymatic machinery in INLP biosynthesis, differing mainly in the fatty-acyl chain length, which is controlled by multiple enzymes in the pathway. Our in-depth study on the non-heme iron(II) and alpha- ketoglutarate-dependent dioxygenase for isonitrile generation, including Rv0097 from Mycobacterium tuberculosis (Mtb), demonstrates that it recognizes a free-standing small molecule substrate, different from the recent hypothesis that a carrier protein is required for Rv0097 in Mtb. A key residue in Rv0097 is further identified to dictate the varied fatty-acyl chain length specificity between Streptomyces and Mycobacterium.

关键词： Tuberculosis   Rifampicin   rpoB   Fitness   Virulence   PDIM  

摘要： Multidrug-resistant tuberculosis (isoniazid/rifampin[RIF]-resistant TB) ravages developing countries. Fitness is critical in clinical outcomes. Previous studies on RIF-resistant TB (RR-TB) showed competitive fitness gains and losses, with rpoB -S450L as the most isolated/fit mutation. This study measured virulence/resistance genes, phthiocerol dimycocerosate (PDIM) levels and their relationship with rpoB S450L ATCC25618 RR-TB strain fitness. After obtaining 10 different RR-TB GenoType MTBDRplus 2.0-genotyped isolates (with nontyped, S441, H445 and S450 positions), only one S450L isolate (R9, rpoB -S450L ATCC 25618, RR 1 μg/mL) was observed, with H445Y being the most common. A competitive fitness in vitro assay with wild-type (wt) ATCC 25618: R9 1:1 in 50 mL Middlebrook 7H9/OADC was performed, and generation time (G) in vitro and relative fitness were obtained. mRNA and PDIM were extracted on log and stationary phases. Fitness decreased in rpoB S450L and H445Y strains, with heterogeneous fitness cues in three biological replicas of rpoB -S450L: one high and two low fitness replicas. S450L strain had significant pknG increase. Compared with S450L, wt- rpoB showed increased polyketide synthase ppsA expression and high PDIM peak measured by HPLC-MS in log phase compared to S450L. This contrasts with previously increased PDIM in other RR-TB isolates.

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： 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： Lipid transport   MmpL   Mycobacterium tuberculosis   PDIM   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. © 2021 The Author(s)

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。