放线菌蛋白O-甘露糖基化的研究进展

蛋白质糖基化作为一种翻译后修饰方式不仅在真核生物中广泛存在,而且在原核生物中尤其是放线菌中也存在。本文针对放线菌中的分枝杆菌属、链霉菌属和棒状杆菌属存在的O-甘露糖基化蛋白的聚糖链结构、糖基化过程以及生物学意义加以论述。     

真核生物蛋白质O-甘露糖基化的研究进展

蛋白质O-甘露糖基化是真核生物中广泛存在的蛋白质翻译后修饰过程,即在甘露糖转移酶(PMT)作用下将长萜酰甘露糖上活化的甘露糖连接到肽链上Ser或者Thr羟基的过程。本文着重介绍在真核生物里真菌和哺乳动物中O-甘露糖基化蛋白的O-甘露聚糖结构、合成过程和生物学意义。     

肽聚糖脱乙酰酶Rv1096对分枝杆菌与宿主细胞相互作用的影响

目的探讨结核分枝杆菌(Mycobacterium tuberculosis, Mtb)肽聚糖脱乙酰酶Rv1096对分枝杆菌与宿主细胞相互作用的影响。方法利用过表达Rv1096基因的重组耻垢分枝杆菌(Mycobacterium smegmatis, MS)_Rv1096,通过差速离心及胰蛋白酶消化试验,确定Rv1096蛋白的亚细胞定位;通过氨基酸定点突变联合伴刀豆凝集素A(Concanavalin A, ConA)免疫印迹确定Rv1096的O-甘露糖基化位点;运用刃天青显色法检测MS_Rv1096对溶菌酶的抵抗力;通过巨噬细胞感染试验,分析了Rv1096对耻垢分枝杆菌细胞内存活能力和宿主细胞炎症应答的影响。结果确定了Rv1096在重组耻垢分枝杆菌MS_Rv1096中的亚细胞定位在细胞壁;发现Rv1096蛋白含有三个O-甘露糖基修饰位点(~(265)Thr,~(266)Ser,~(267)Ser);细胞外测试结果表明,Rv1096能增强耻垢分枝杆菌对溶菌酶的抵抗能力,最低抑菌质量浓度从1.5 mg/mL升至2.5 mg/mL,而细胞感染显示其并不能显著增强耻垢分枝杆菌在人单核细胞白血病细胞(THP-1细胞)内的存活能力;定量PCR检测结果显示,过表达Rv1096的耻垢分枝杆菌刺激THP-1细胞分泌炎症因子(TNF-α,IL-6和IL-1β)的能力显著下降。通过平行对比证明若删除O-甘露糖基化位点(~(265)Thr,~(266)Ser,~(267)Ser),对Rv1096基因功能无显著影响。结论 Rv1096是一个细胞壁相关蛋白,具有三个O-甘露糖基化位点。过表达Rv1096对耻垢杆菌在宿主细胞内的存活能力无显著影响,但能够降低宿主细胞对耻垢分枝杆菌的炎症因子应答,且上述功能不受O-甘露糖基化修饰影响。     

利用伴刀豆球蛋白检测O-甘露糖基化

目的:基于伴刀豆球蛋白A(ConA)特异性识别并结合甘露糖的特性,建立一种检测O-甘露糖基化的方法,为酵母等宿主表达蛋白的O-糖基化提供一种高效筛选和分析的方法。方法:利用糖苷酶F(PNGF)切除检测蛋白的N-糖链,排除N-糖基化的干扰;通过Q阴离子交换柱和ConA Sepharose 4B柱纯化Western印迹膜封闭蛋白牛血清白蛋白(BSA),除去BSA中甘露糖修饰的蛋白的干扰,优化膜封闭条件;利用辣根过氧化物酶标记的ConA检测具有低甘露糖型N-糖基化修饰能力的毕赤酵母GJK01-HL(Δoch1)表达的抗Her-2抗体是否存在O-甘露糖基化现象。结果:通过PNGF酶切处理,可以完全去除糖蛋白的N-糖链的干扰;BSA经过Q阴离子交换柱和ConASepharose 4B柱纯化后,除去了大部分甘露糖蛋白,可作为封闭蛋白;用建立的方法检测,发现毕赤酵母工程菌GJK01-HL(Δoch1)表达的抗Her-2抗体存在O-甘露糖基化现象。结论:本方法是研究糖蛋白是否发生O-甘露糖基化的有效检测手段,可用于酵母等表达蛋白的O-糖基化的高效筛选和分析。     

结核分枝杆菌ESX-1分泌蛋白EspB结构和功能分析

结核分枝杆菌作为肺结核病的病原菌,在人类中致死率远高于其他病原菌.结核分枝杆菌具有特殊的疏水性细胞壁结构,这种致密的细胞壁结构帮助结核分枝杆菌抵御外界环境压力和来自宿主细胞的毒素.同时,它利用特殊的分泌系统将体内的毒力蛋白输出体外,ESX-1分泌系统就是其中之一.结核分枝杆菌ESX-1系统在结核分枝杆菌进入宿主细胞吞噬小体、逃逸至细胞质以及杀死吞噬细胞这些过程中发挥重要作用.研究表明,在结核分枝杆菌内膜上存在一个由多亚基组成、旨在帮助结核分枝杆菌向外输送分泌蛋白的分泌装置.在这个分泌装置的帮助下,结核分枝杆菌重要的毒力蛋白ESAT-6跨内膜向外分泌,EspB也通过这个内膜上的分泌装置被转运至胞外.EspB存在于静置培养的结核分枝杆菌的胶囊层中,也可在振荡培养的结核分枝杆菌的培养液中被检测.通过X射线晶体衍射分析,我们解析了EspB的晶体结构,相比于其他同源结构,发现了EspB的不同构象,即EspB单体能够自组装成为七聚体的规则结构,联系其与毒力因子ESAT-6具有共分泌的特点,七聚体构象的发现为解释EspB在结核分枝杆菌向外分泌蛋白的过程中发挥的作用提供线索,即EspB具有锚定在结核分枝杆菌胶囊层中,作为运输ESAT-6的孔道而存在的可能.     

结核分枝杆菌ESX分泌系统研究进展

在结核分枝杆菌中存在能够使某些蛋白通过其高度疏水和通透性极差的细胞壁的分泌系统,其中ESX-1分泌系统负责ESAT-6（early secreted antigenic target of 6 kD）和CFP-10（culture filtrate protein of 10kD）的分泌．这两种蛋白能够形成1：1的二聚体结构,并且是结核分枝杆菌重要的毒力因子．近年来陆续发现其他一些蛋白也由ESX-1分泌系统所分泌．在结核分枝杆菌中还存在其他4种类似ESX-1的分泌系统ESX-2～ESX-5．ESX分泌系统也在其他革兰氏阳性菌和放线菌中被发现,有学者按照公认的术语称其为Ⅶ型分泌系统．本文综述了结核分枝杆菌ESX分泌系统的组成和分子机制,以及分泌蛋白与宿主的相互作用等方面的研究进展．     

牛分枝杆菌32KDa蛋白诱导人细胞毒T细胞的应答

牛分枝杆菌３２ＫＤａ蛋白诱导人细胞毒Ｔ细胞的应答牛分枝杆菌、结核分枝杆菌以及其它分枝杆菌中均存在一高度同源的３２ＫＤ蛋白（Ｐ３２）．人感染结核、麻风病期间及接种ＢＣＧ后均有Ｐ３２表达,提示Ｐ３２是刺激细胞免疫和体液免疫的主要靶抗原,为了验证Ｐ３２在体...     

结核分枝杆菌ABC转运蛋白与物质的跨膜转运

结核分枝杆菌作为一种胞内寄生菌,主要存在于巨噬细胞吞噬体内,并且通过与宿主细胞竞争摄取营养物质、主动排出有毒物质来维持生存。因此,参与上述过程的ABC转运蛋白在结核分枝杆菌的致病中发挥着举足轻重的作用。已有报道结核分枝杆菌基因组编码了38个ABC转运蛋白。这类蛋白质有着广泛的底物结合谱,参与了无机离子、糖类、氨基酸、寡肽、药物等多种物质的跨膜转运。本文将对结核分枝杆菌编码的ABC转运蛋白超家族中的不同成员及其底物特异性、转运机制以及与毒力的关系的研究进展进行综述。     

结核分枝杆菌热休克蛋白GroEL1的研究进展

结核分枝杆菌(Mycobacterium tuberculosis)的GroEL1蛋白是基因复制产物,作为热休克蛋白(HSP)一员,不但行使原有的助折叠分子伴侣功能,而且可作为抗原,在抗原呈递中起作用。另外,由于其独特的结构特点,GroEL1还具有多种生物功能,如作为蛋白分子伴侣,控制细胞因子依赖性肉芽肿的产生;作为DNA分子伴侣,保护DNA免受损害。而在分枝杆菌属的其他物种如耻垢分枝杆菌(Mycobacterium smegmatis)中却行使不同的生物学功能。这些功能产生的原因和机制有待进一步研究,从而为结核病的发生及结核分枝杆菌的进化机制提供更多的依据。     

耻垢分枝杆菌宿主整合因子(IHF)对DNA拓扑结构的影响

结核分枝杆菌(Mycobaterium tuberculosis)是结核病的病原菌,每年导致数百万人死亡.对于分枝杆菌基本生物学特性的研究有助于新的药物及治疗手段的研发.耻垢分枝杆菌(M.smegmatis)是分枝杆菌属中的一种非致病菌,与结核分枝杆菌亲缘关系较近,是实验室常用的研究分枝杆菌的模式菌种.分枝杆菌主要编码三种染色质蛋白,类组蛋白HU、Lsr2和宿主整合因子IHF.为研究IHF在染色体包装中的作用,我们在大肠杆菌中表达、纯化了耻垢分枝杆菌IHF蛋白(MsIHF),并对其影响DNA拓扑结构的性质进行了系统分析.体外研究的结果表明,MsIHF以同二聚体的形式存在,其对负超螺旋DNA具有一定的结合偏好性,同时,该蛋白可以有效地固定DNA负超螺旋.进一步的研究表明,MsIHF可以调控拓扑异构酶的活性.MsIHF的结合明显地抑制拓扑异构酶Ⅰ的松弛活性,而与此相反,该蛋白可以轻微地促进旋转酶引入DNA负超螺旋的能力.以上结果提示,MsIHF可能通过调控拓扑异构酶的活性影响染色体DNA的结构,进而调控其包装.     

Polymorphisms of 20 regulatory proteins between Mycobacterium tuberculosis and Mycobacterium bovis

Mycobacterium tuberculosis and Mycobacterium bovis are responsible for tuberculosis in humans and animals, respectively. Both species are closely related and belong to the Mycobacterium tuberculosis complex (MTC). M. tuberculosis is the most ancient species from which M. bovis and other members of the MTC evolved. The genome of M. bovis is over >99.95% identical to that of M. tuberculosis but with seven deletions ranging in size from 1 to 12.7 kb. In addition, 1200 single nucleotide mutations in coding regions distinguish M. bovis from M. tuberculosis. In the present study, we assessed 75 M. tuberculosis genomes and 23 M. bovis genomes to identify non‐synonymous mutations in 202 coding sequences of regulatory genes between both species. We identified species‐specific variants in 20 regulatory proteins and confirmed differential expression of hypoxia‐related genes between M. bovis and M. tuberculosis.     

Mammalian heterotrimeric G-protein-like proteins in mycobacteria: implications for cell signalling and survival in eukaryotic host cells

Mammalian heterotrimeric GTP-binding proteins (G proteins) are involved in transmembrane signalling that couples a number of receptors to effectors mediating various physiological processes in mammalian cells. We demonstrate that bacterial proteins such as a Ras-like protein from Pseudomonas aeruginosa or a 65 kDa protein from Mycobacterium smegmatis can form complexes with human or yeast nucleoside diphosphate kinase (Ndk) to modulate their nucleoside triphosphate synthesizing specificity to GTP or UTP. In addition, we demonstrate that bacteria such as M. smegmatis or Mycobacterium tuberculosis harbour proteins that cross react with antibodies against the α-, β- or the γ-subunits of heterotrimeric G proteins. Such antibodies also alter the GTP synthesizing ability of specific membrane fractions isolated from glycerol gradients of such cells, suggesting that a membrane-associated Ndk–G-protein homologue complex is responsible for part of GTP synthesis in these bacteria. Indeed, purified Ndk from human erythrocytes and M. tuberculosis showed extensive complex formation with the purified mammalian α and β G-protein subunits and allowed specific GTP synthesis, suggesting that such complexes may participate in transmembrane signalling in the eukaryotic host. We have purified the α-, β- and γ-subunit homologues from M. tuberculosis and we present their internal amino acid sequences as well as their putative homologies with mammalian subunits and the localization of their genes on the M. tuberculosis genome. Using oligonucleotide probes from the conserved regions of the α- and γ-subunit of M. tuberculosis G-protein homologue, we demonstrate hybridization of these probes with the genomic digest of M. tuberculosis H37Rv but not with that of M. smegmatis, suggesting that M. smegmatis might lack the genes present in M. tuberculosis H37Rv. Interestingly, the avirulent strain H37Ra showed weak hybridization with these two probes, suggesting that these genes might have been deleted in the avirulent strain or are present in limited copy numbers as opposed to those in the virulent strain H37Rv.     

Identification of functional Tat signal sequences in Mycobacterium tuberculosis proteins

The twin-arginine translocation (Tat) pathway is a system used by some bacteria to export proteins out from the cytosol to the cell surface or extracellular environment. A functional Tat pathway exists in the important human pathogen Mycobacterium tuberculosis. Identification of the substrates exported by the Tat pathway can help define the role that this pathway plays in the physiology and pathogenesis of M. tuberculosis. Here we used a reporter of Tat export, a truncated β-lactamase, ′BlaC, to experimentally identify M. tuberculosis proteins with functional Tat signal sequences. Of the 13 proteins identified, one lacks the hallmark of a Tat-exported substrate, the twin-arginine dipeptide, and another is not predicted by in silico analysis of the annotated M. tuberculosis genome. Full-length versions of a subset of these proteins were tested to determine if the native proteins are Tat exported. For three proteins, expression in a Δtat mutant of Mycobacterium smegmatis revealed a defect in precursor processing compared to expression in the wild type, indicating Tat export of the full-length proteins. Conversely, two proteins showed no obvious Tat export in M. smegmatis. One of this latter group of proteins was the M. tuberculosis virulence factor phospholipase C (PlcB). Importantly, when tested in M. tuberculosis a different result was obtained and PlcB was exported in a twin-arginine-dependent manner. This suggests the existence of an M. tuberculosis-specific factor(s) for Tat export of a proven virulence protein. It also emphasizes the importance of domains beyond the Tat signal sequence and bacterium-specific factors in determining if a given protein is Tat exported.     

Mycobacterium tuberculosis alters the differentiation of monocytes into macrophages in vitro

This paper shows that in vitro infection of human monocytes by Mycobacterium tuberculosis affected monocyte to macrophage differentiation. Despite the low bacterial load used, M. tuberculosis-infected monocytes had fewer granules, displayed a reduced number of cytoplasmic projections and decreased HLA class II, CD68, CD86 and CD36 expression compared to cells differentiated in the absence of mycobacteria. Infected cells produced less IL-12p70, TNF-α, IL-10, IL-6 and high IL-1β in response to lipopolysaccharide and purified protein M. tuberculosis-derived. Reduced T-cell proliferative response and IFN-γ secretion in response to phytohemagglutinin and culture filtrate proteins from M. tuberculosis was also observed in infected cells when compared to non-infected ones. The ability of monocytes differentiated in the presence of M. tuberculosis to control mycobacterial growth in response to IFN-γ stimulation was attenuated, as determined by bacterial plate count; however, they had a similar ability to uptake fluorescent M. tuberculosis and latex beads compared to non-infected cells. Recombinant IL-1β partially altered monocyte differentiation into macrophages; however, treating M. tuberculosis-infected monocytes with IL-1RA did not reverse the effects of infection during differentiation. The results indicated that M. tuberculosis infection altered monocyte differentiation into macrophages and affected their ability to respond to innate stimuli and activate T-cells.     

Species-specific secretion of ESX-5 type VII substrates is determined by the linker 2 of EccC5

Mycobacteria use type VII secretion systems (T7SSs) to translocate a wide range of proteins across their diderm cell envelope. These systems, also called ESX systems, are crucial for the viability and/or virulence of mycobacterial pathogens, including Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum. We have previously shown that the M. tuberculosis ESX-5 system is unable to fully complement secretion in an M. marinum esx-5 mutant, suggesting species specificity in secretion. In this study, we elaborated on this observation and established that the membrane ATPase EccC₅, possessing four (putative) nucleotide-binding domains (NBDs), is responsible for this. By creating M. marinum-M. tuberculosis EccC₅ chimeras, we observed both in M. marinum and in M. tuberculosis that secretion specificity of PE_PGRS proteins depends on the presence of the cognate linker 2 domain of EccC₅. This region connects NBD1 and NBD2 of EccC₅ and is responsible for keeping NBD1 in an inhibited state. Notably, the ESX-5 substrate EsxN, predicted to bind to NBD3 on EccC₅, showed a distinct secretion profile. These results indicate that linker 2 is involved in species-specific substrate recognition and might therefore be an additional substrate recognition site of EccC₅.     

Crystal structure of DNA polymerase III β sliding clamp from Mycobacterium tuberculosis

The sliding clamp is a key component of DNA polymerase III (Pol III) required for genome replication. It is known to function with diverse DNA repair proteins and cell cycle-control proteins, making it a potential drug target. To extend our understanding of the structure/function relationship of the sliding clamp, we solved the crystal structure of the sliding clamp from Mycobacterium tuberculosis (M. tuberculosis), a human pathogen that causes most cases of tuberculosis (TB). The sliding clamp from M. tuberculosis forms a ring-shaped head-to-tail dimer with three domains per subunit. Each domain contains two α helices in the inner ring that lie against two β sheets in the outer ring. Previous studies have indicated that many Escherichia coli clamp-binding proteins have a conserved LF sequence, which is critical for binding to the hydrophobic region of the sliding clamp. Here, we analyzed the binding affinities of the M. tuberculosis sliding clamp and peptides derived from the α and δ subunits of Pol III, which indicated that the LF motif also plays an important role in the binding of the α and δ subunits to the sliding clamp of M. tuberculosis.     

Three-step purification of lipopolysaccharide-free, polyhistidine-tagged recombinant antigens of Mycobacterium tuberculosis

Previous work has shown that the study of host immune responses against Mycobacterium tuberculosis, the causative agent of tuberculosis, requires the availability of multiple mycobacterial antigens. Since purification of protein from M. tuberculosis cells is extremely cumbersome, we developed a protocol for purifying milligram amounts of ten recombinant antigens of M. tuberculosis from E. coli cells. Purified proteins were immunologically active and free of contaminants that confound interpretation of cell-based immunological assays. The method utilizes a three-step purification protocol consisting of immobilized metal-chelate affinity chromatography, size exclusion chromatography and anion-exchange chromatography. The first two chromatographic steps yielded recombinant protein free of protein contaminants, while the third step (anion-exchange chromatography) efficiently removed E. coli lipopolysaccharide, a potent polyclonal activator of lymphoid cells. The recombinant proteins were immunologically indistinguishable from their native (i.e., purified from M. tuberculosis) counterparts. Thus the method provides a way to utilize recombinant proteins for immunological analyses that require highly purified antigens.     

Physical and functional interactions between 3-methyladenine DNA glycosylase and topoisomerase I in mycobacteria

DNA glycosylases play important roles in DNA repair in a variety of organisms, including humans. However, the function and regulation of these enzymes in the pathogenic bacterium Mycobacterium tuberculosis and related species are poorly understood. In the present study, the physical and functional interactions between 3-methyladenine DNA glycosylase (MAG) and topoisomerase I (TopA) in M. tuberculosis and M. smegmatis were characterized. MAG was found to inhibit the function of TopA in relaxing supercoiled DNA. In contrast, TopA stimulated the cleavage function of MAG on a damaged DNA substrate that contains hypoxanthine. The interaction between the two proteins was conserved between the two mycobacterial species. Several mutations in MAG that led to the loss of its interaction with and activity regulation of TopA were also characterized. The results of this study further elucidate glycosylase regulation in both M. smegmatis and M. tuberculosis.     

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.