关键词： Mycobacteria   Lipids   mel2   PDIM   TAG   Bioluminescence   Redox stress  

摘要： Using a high throughput genetic strategy, designated Random Inducible Controlled Expression (RICE), we identified the six gene mel2 locus in Mtb and M. marinum. Interestingly, three of the genes present in mel2 have similarities to bioluminescence genes. Similar to other bacterial bioluminescence systems, mel2 facilitates detoxification of reactive oxygen species (ROS). Through the use of thin layer chromatography (TLC) we demonstrate enhanced production of the cell wall virulence lipid, pthiocerol dimycoserosate (PDIM), in a Mtb mel2 mutant relative to the wild type strain in the presence of both H2O2 and diamide oxidative stresses. Furthermore, propionate toxicity assays revealed increased accumulation of triacylglycerol (TAG) in the mel2 mutant relative to wild type. These observations provide the first evidence that mel2 plays a critical role in Mtb lipid biosynthesis. (C) 2013 Elsevier Ltd. All rights reserved.

关键词： 分枝杆菌   PDIM   脂质   致病机制  

摘要： 结核分枝杆菌(MycobacteriumTuberculosis,MTB)是导致人和动物结核病的病原体。MTB细胞壁成分复杂,功能多样,不但帮助分枝杆菌抵御外界不良的生活环境,同时在细菌逃逸宿主免疫攻击和致病过程中也发挥重要作用。脂质占细胞壁干重的60%以上,其高含量与细菌毒力密切相关。MTB不含内毒素,也不产生外毒素和侵袭性酶类,其致病性主要与占细胞壁干重60%以上的脂质成分密切相关。PDIM(phthioceroldimycocerosate)作为细胞壁组分中独特而重要的结构,在维持细菌正常结构上发挥重要作用。由于PDIM在细胞壁分布的广泛性,研究发现PDIM在病原体-宿主的相互作用过程中也扮演重要角色,尤其是在细菌的早期侵染及进入巨噬细胞环节中发挥积极作用。对PDIM重要致病和毒力损伤机制研究,不仅加深对病原体-宿主互作网络中的理解,还有望为结核病的治疗带来新的希望与突破。

关键词： Mycobacterium tuberculosis   Colony phenotype   Human macrophages infection   Capsule   PDIM   Virulence   TIME QUANTITATIVE PCR   SIGMA-FACTORS   IN-VITRO   H37RV   TRIACYLGLYCEROLS   REGULATORS   BACTERIA   CULTURE   STRESS   GENOME  

摘要： Mycobacterium tuberculosis (MTB) colony morphology was associated to the pathogen's virulence. We isolated a new MTB H37Rv smooth colony, which only appeared following human macrophages (MDM) infection. The new phenotype was Alcohol-Acid resistant, but devoid of a covering capsule and biofilm defective. We ascertained that there were no deletions in the Rv0096-Rv0101 PDIM Operon, but that its expression was repressed as compared to MTB wild type (wt). Its lipid composition displayed lower PDIM components and higher TAG as compared to wt. In MDM it induced the sigma factors sigB, sigI and sigL expression vs. synthetic medium culture, while it repressed other six sigma factors. It also induced, significantly more than wt, mprA, a mycobacterial persistence regulator. It was phagocytosed more than wt by MDM, where it grew significantly less, but persisted therein till 14 days infection. It induced significantly less IFN-gamma, IL-12 and IL-27 transcription than wt in infected MDM, while it increased the transcription of inducible NOS. It resided in mature, LAMP-3 positive phagolysosomes, where it never formed cords. This apparently "weaker" colony might represent an adaptive intracellular phenotype, whose infection may be less productive, but probably better equipped for a long lasting persistence in mildly activated host cells. (C) 2012 Elsevier Ltd. All rights reserved.

关键词： Mycobacterium   Virulence   Vaccines   PDIM   PGL   Lipopeptide   PHTHIOCEROL DIMYCOCEROSATE PDIM   GENE-CLUSTER   MOLECULAR DISSECTION   GENOME SEQUENCE   TUBERCULOSIS   BIOSYNTHESIS   IDENTIFICATION   COMPLEX   BCG   GLYCOPEPTIDOLIPIDS  

摘要： Mycobacterium bovis is an important pathogen of both domesticated and wild animals in many countries, and improved vaccines have great potential to assist in its control and eventual eradication. One of the hallmarks of members of the Mycobacterium tuberculosis complex, which includes both M. bovis and M. tuberculosis, is their ability to synthesise an impressive array of unique and complex lipids, many of which act as defensive, offensive or adaptive effectors of virulence.. For example, studies focussed on the development of rationally attenuated strains of both M. bovis and M. tuberculosis with efficacy as animal or human vaccines have shown that the phthiocerol dimycocerosates (PDIMs) and glycosylphenol-PDIM (phenolic glycolipid, PGL) are key virulence factors. The availability of the genome sequences for M. bovis and M. tuberculosis, together with mutants of these organisms carrying defects in lipid biosynthesis, and biochemical and molecular tools to dissect lipid biosynthesis pathways, has enabled developments in our understanding of the biosynthesis of PDIMs and PGL, as well as the possible roles played by PDIMs and PGL in virulence. In this article we review some of these developments, and also propose a cryptic lipid biosynthesis pathway in M. bovis and M. tuberculosis that may be involved in the production of an unrecognised, virulence-associated lipopeptide. (C) 2011 Elsevier B.V. All rights reserved.

关键词： MYCOBACTERIUM tuberculosis   VIRULENCE (Microbiology)   LIPIDS   LUNG diseases   CELL culture  

摘要： The article presents a study which determines the implications of the loss of phthiocerol dimycocerosate (PDIM) from Mycobacterium tuberculosis. The study conducted lipid and microarray analysis and competition experiments. The study suggests that all strains that were repeatedly subcultured might contain heterogeneous population of PDIM-positive and -negative cell types and that future virulence studies must confirm the presence of PDIM in recombinant clones and the parental strains.

关键词： ACYL CARRIER PROTEIN   MYCOBACTERIUM-TUBERCULOSIS   ENOYL REDUCTASE   MYCOLIC ACIDS   GENE-CLUSTER   SYSTEM   INHA   RESISTANCE   PURIFICATION   ADAPTATION  

摘要： The fatty acid biosynthesis (FAS-II) pathway in Mycobacterium tuberculosis generates long chain fatty acids that serve as the precursors to mycolic acids, essential components of the mycobacterial cell wall. Enzymes in the FAS-II pathway are thought to form one or more noncovalent multi-enzyme complexes within the cell, and a bacterial two-hybrid screen was used to search for missing components of the pathway and to furnish additional data on interactions involving these enzymes in vivo. Using the FAS-II beta-ketoacyl synthase, KasA, as bait, an extensive bacterial two-hybrid screen of a M. tuberculosis genome fragment library unexpectedly revealed a novel interaction between KasA and PpsB as well as PpsD, two polyketide modules involved in the biosynthesis of the virulence lipid phthiocerol dimycocerosate (PDIM). Sequence analysis revealed that KasA interacts with PpsB and PpsD in the region of the acyl carrier domain of each protein, raising the possibility that lipids could be transferred between the FAS-II and PDIM biosynthetic pathways. Subsequent studies utilizing purified proteins and radiolabeled lipids revealed that fatty acids loaded onto PpsB were transferred to KasA and also incorporated into long chain fatty acids synthesized using a Mycobacterium smegmatis lysate. These data suggest that in addition to producing PDIMs, the growing phthiocerol product can also be shuttled into the FAS-II pathway via KasA as an entry point for further elongation. Interactions between these biosynthetic pathways may exist as a simple means to increase mycobacterial lipid diversity, enhancing functionality and the overall complexity of the cell wall.

关键词： other bioactive products   lipids   carboxylic acid esters (acyclic compounds)  

摘要： ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

关键词： conjugate addition   alkyne addition   asymmetric synthesis   hydrosilylation   mycobacterium tuberculosis   PHTHIOCEROL DIMYCOCEROSATE LOCUS   CONJUGATE ADDITION   ASSIGNMENT   EFFICIENT   LIGANDS   COMPLEX   SYSTEM  

摘要： The first asymmetric synthesis of phthiocerol was achieved in 15 simple steps and 5.6% overall yield by applying three efficient catalytic transformations. The construction of building block 4 started with the copper/phosphoramidite-catalyzed asymmetric conjugate addition of Me 2Zn to cycloheptenone, followed by in situ ethylation. Baeyer Villiger oxidation using excess m-chloroperoxybenzoic acid (mCPBA) followed by treatment of the resulting lactone 7 with K2C03 in MeOH led to the formation of the linear product. Treatment with TBAF followed by a Fleming-Tamao oxidation using KHCO3 and H2O2, resulted in the formation of the corresponding hydroxy ketones, which could be separated by column chromatography affording 16 as a pure isomers.

关键词： lipid virulence factor   metabolic flux   pathogenesis   PDIM   sulfolipid-1   MYCOCEROSIC ACID SYNTHASE   GENE-CLUSTER   FATTY-ACIDS   ACYL-COA   MACROPHAGES   BIOSYNTHESIS   EXPRESSION   GROWTH   IDENTIFICATION   MMPL8  

摘要： Mycobacterium tuberculosis synthesizes specific polyketide lipids that interact with the host and are required for virulence. Using a mass spectrometric approach to simultaneously monitor hundreds of lipids, we discovered that the size and abundance of two lipid virulence factors, phthiocerol climycocerosate (PDIM) and sulfolipid-1 (SL-11), are controlled by the availability of a common precursor, methyl malonyl CoA (MMCoA). Consistent with this view, increased levels of MMCoA led to increased abundance and mass of both PDIM and SL-1. Furthermore, perturbation of MMCoA metabolism attenuated pathogen replication in mice. Importantly, we detected increased PDIM synthesis in bacteria growing within host tissues and in bacteria grown in culture on odd-chain fatty acids. Because M. tuberculosis catabolizes host lipids to grow during infection, we propose that growth of M. tuberculosis on fatty acids in vivo leads to increased flux of MMCoA through lipid biosynthetic pathways, resulting in increased virulence lipid synthesis. Our results suggest that the shift to host lipid catabolism during infection allows for increased virulence lipid anabolism by the bacterium.

关键词： TUBERCULOSIS COMPLEX   GENE-CLUSTER   MUTANTS   GLYCOPEPTIDOLIPIDS   PHENOLGLYCOLIPIDS   IDENTIFICATION   DISSECTION   VIRULENCE   SEQUENCE  

摘要： The unusual and complex cell wall of pathogenic mycobacteria plays a major role in pathogenesis, with specific complex lipids acting as defensive, offensive, or adaptive effectors of virulence. The phthiocerol and phthiodiolone dimycocerosate esters (PDIMs) comprise one such category of virulence-enhancing lipids. Recent work in several laboratories has established that the Mycobacterium tuberculosis fadD26-mmpL7 (Rv2930-Rv2942) locus plays a major role in PDIM biosynthesis and secretion and that PDIM is required for virulence. Here we describe two independent transposon mutants (WAg533 and WAg537) of Mycobacterium bovis, both of which carry an insertion in Mb0100 (= M. tuberculosis Rv0097) to reveal a new locus involved in PDIM biosynthesis. The mutations have a polar effect on expression of the downstream genes Mb0101, Mb0102 (fadD10), Mb0103, and Mb0104 (nrp), and Mb0100 is shown to be in an operon comprising these genes and Mb0099. Reverse transcription-PCR analysis shows elevated transcription of genes in the operon upstream from the transposon insertion sites in both mutants. Both mutants have altered colony morphology and do not synthesize PDIMs or glycosylphenol-PDIM. Both mutants are avirulent in a guinea pig model of tuberculosis, and when tested as a vaccine, WAg533 conferred protective immunity against M. bovis infection at least equal to that afforded by M. bovis bacillus Calmette-Guerin.