结核分枝杆菌PhoP系统研究进展

Pho P与Pho R组成的Pho PR是结核分枝杆菌重要的双组分调节系统。Pho P作为应答调节子调节基因的表达,这些基因参与细胞壁脂质合成,并对结核分枝杆菌毒力有重要调控作用。本文综述了Pho P的结构、性质以及相关的结核分枝杆菌疫苗研发情况,并提出了未来可能的研究趋势。     

结核分枝杆菌抗原蛋白的研究及其应用新进展

结核病(tuberculosis, TB)是由结核分枝杆菌(Mycobacterium tuberculosis, MTB)感染引起的慢性传染病,是仅次于正在暴发的新型冠状病毒肺炎(COVID-19)的第二大单一感染致死病因。COVID-19的大流行对TB的诊断及治疗造成了破坏性的影响,全球实现终结TB目标的进展偏离了轨道。因此,早诊断、早治疗依然是防控TB蔓延的关键。TB精准诊断一直受MTB抗原特异性、检测技术特异性和灵敏度的影响,因此亟需挖掘高特异性新抗原、开发新检测技术。随着蛋白质基因组学(proteogenomics)和质谱技术的快速发展,从临床体液、组织样本中高效、精准靶向检测MTB特异性已知、甚至新抗原的表达,以及监测治疗过程中的抗原表达量的动态变化,是TB诊断及治疗的发展趋势。在MTB标准菌株H37Rv的4 008个注释基因中(NC＿000 962.3, NCBI),国内外报道的已注释抗原虽有140多个,但仅有极少的抗原应用于TB的筛查及辅助诊断,离世界卫生组织(World Health Organization, WHO)的诊断标准尚远。本文通过对MTB已报道抗原以及基...     

相对定量分析异烟肼、链霉素单耐药与多耐药结核分枝杆菌临床分离株的蛋白质组差异

【目的】探讨异烟肼(isoniazid,INH)、链霉素(streptomycin,SM)单耐药结核分枝杆菌(Mycobacterium tuberculosis,MTB)与INH/SM多耐药MTB蛋白质组差异。【方法】应用i TRAQ结合Nano LC-MS/MS定量蛋白质组学技术,分析临床分离INH、SM或INH/SM耐药MTB与H37Rv标准株间均表达差异蛋白;并以INH/SM耐药MTB与H37Rv比值为对照,相对定量分析单耐药与多耐药MTB蛋白表达差异倍数;运用DAVID 6.7分析差异蛋白生物功能;STITCH 5.0分析差异蛋白与INH和SM相互作用。【结果】与H37Rv标准株比较,58个蛋白在INH、SM耐药与INH/SM耐药MTB间均有表达差异,共同差异蛋白生物功能主要为氧化还原酶活性和转移酶活性;主要参与丙酸代谢信号通路。共同差异蛋白中,与INH/SM耐药MTB比较,Rv2986c和Rv1908c在INH、SM耐药MTB均表达上调1.25倍;Rv3133c和Rv0577则均表达下调0.7倍;生物信息学预测发现以上4种蛋白可直接或间接与INH、SM进行相互作用。【结论】INH、SM单耐药和INH/SM多耐药MTB蛋白表达谱有较大差异,蛋白Rv2986c、Rv1908c、Rv3133c和Rv0577表达水平及相互作用可能与INH和SM耐药有关。     

结核分枝杆菌Rv0081基因编码蛋白的生物信息学分析

运用生物信息学分析软件预测结核分枝杆菌（Mycobacterium tuberculosis, Mtb）Rv0081蛋白的生物学特征及筛选潜在的优势抗原表位。 从NCBI数据库获取Mtb Rv0081蛋白的氨基酸序列，利用生物信息学分析软件ProtParam、ProtScale及TMPRED分析Rv0081蛋白的理化性质及亲疏水性；TMHMM、SignalP-5.0 Server预测蛋白的跨膜区及信号肽；NetNGlyc-1.0 Server、NetPhos 3.1 Server分别预测蛋白的糖基化位点及磷酸化位点；STRING预测能与Rv0081相互作用的蛋白；分别运用SOPMA、SWISS-MODEL预测蛋白的二、三级结构；综合运用softberry、WoLF PSORT预测蛋白的亚细胞定位；运用DNAStar预测蛋白的B细胞抗原表位；综合运用SYFPEITHI、NetCTL 1.2 Server、Net MHC pan 4.1 server预测蛋白的CTL细胞抗原表位；综合运用SYFPEITHI、Net MHCII pan 4.0 server预测蛋白的Th细胞抗原表位。 结果表明，Rv0081蛋白由114个氨基酸组成，相对分子质量为12 356.32，亚细胞定位于细胞质中，为稳定的疏水性蛋白，无跨膜区和信号肽，含有1个糖基化位点及9个磷酸化位点；二级结构主要由α-螺旋和无规则卷曲构成，结构较松散；与hycE、hycP、Rv0088、Rv0083、hycD、hycQ、Rv0082、devR、Rv0080及Rv0079蛋白存在相互作用关系；综合分析各软件预测结果筛选出6个优势B细胞抗原表位、6个优势CTL细胞抗原表位及7个优势Th细胞抗原表位。Mtb Rv0081蛋白具有较多潜在的候选B、T细胞抗原表位，可作为研发新型结核疫苗的候选抗原。     

用小鼠模型分析可溶性表达结核分枝杆菌Ag85A的免疫原性

【目的】可溶性表达结核分枝杆菌Ag85A蛋白,并评价其免疫原性。【方法】利用冷休克表达质粒和含有伴侣质粒的大肠杆菌对Ag85A蛋白进行可溶性原核表达,并进行纯化与鉴定,通过C57BL/6小鼠模型对Ag85A蛋白的免疫原性,包括诱导机体特异性体液免疫应答和细胞免疫应答水平进行分析。【结果】重组菌诱导后裂解上清中检测到可溶性Ag85A蛋白的表达,经过亲和层析纯化收获了纯度在90%以上的Ag85A蛋白,Western blot鉴定显示其具有较好的免疫反应性。Ag85A蛋白免疫小鼠后,血清中可以检测到高水平的Ig G抗体效价,其中Ig G2b水平要高于Ig G1。通过特异性多肽、蛋白刺激脾脏和腹股沟淋巴结细胞可分泌高水平的IFN-γ、TNF-α等Th1型细胞因子。【结论】实现了Ag85A蛋白的可溶性表达,免疫特性评价显示Ag85A蛋白可诱导机体产生强烈的特异性体液免疫应答及Th1型的细胞免疫应答,从而为其进一步免疫学功能的研究奠定了重要基础。     

Detection of streptomycin resistance in Mycobacterium tuberculosis clinical isolates from China as determined by denaturing HPLC analysis and DNA sequencing

China is regarded by the World Health Organization as a major hot-spot region for Mycobacterium tuberculosis infection. Streptomycin has been deployed in China for over 50 years and is still widely used for tuberculosis treatment. We have developed a denaturing HPLC (DHPLC) method for detecting various gene mutations conferring drug resistance in M. tuberculosis. The present study focused on rpsL and rrs mutation analysis. Two hundred and fifteen M. tuberculosis clinical isolates (115 proved to be streptomycin-resistant and 100 susceptible by a routine proportional method) from China were tested to determine the streptomycin minimal inhibitory concentration (MIC), and subjected to DHPLC and concurrent DNA sequencing to determine rpsL and rrs mutations. The results showed that 85.2% (98/115) of streptomycin-resistant isolates harbored rpsL or rrs mutation, while rpsL mutation (76.5%, 88/115) dominated. MIC of 98 mutated isolates revealed no close correlation between mutation types and levels of streptomycin resistance. No mutation was found in any of the susceptible isolates. The DHPLC results were completely consistent with those of sequencing. The DHPLC method devised in this study can be regarded as a useful and powerful tool for detection of streptomycin resistance. This is the first report to describe DHPLC analysis of mutations in the rpsL and rrs genes of M. tuberculosis in a large number of clinical isolates.     

Use of visual loop-mediated isotheral amplification of rimM sequence for rapid detection of Mycobacterium tuberculosis and Mycobacterium bovis

Mycobacterium tuberculosis and Mycobacterium bovis are pathogenic bacterial species in the genus Mycobacterium and the causative agents of most cases of tuberculosis (TB). Detection of M. tuberculosis and M. bovis using conventional culture- and biochemical-based assays is time-consuming and laborious. Therefore, a simple and sensitive method for rapid detection has been anxiously awaited. In the present study, a visual loop-mediated isothermal amplification (LAMP) assay was designed from the rimM (encoding 16S rRNA-processing protein) gene sequence and used to rapidly detect M. tuberculosis and M. bovis from clinical samples in South China. The visual LAMP reaction was performed by adding calcein and manganous ion, allowing the results to be read by simple visual observation of color change in a closed-tube system, and which takes less than 1 h at 65 °C. The assay correctly identified 84 M. tuberculosis isolates, 3 M. bovis strains and 1 M. bovis BCG samples, but did not detect 51 non-tuberculous mycobacteria (NTM) isolates and 8 other bacterial species. Sensitivity of this assay for detection of genomic DNA was 1 pg. Specific amplification was confirmed by the ladder-like pattern of gel electrophoresis and restriction enzyme HhaI digestion. The assay successfully detected M. tuberculosis and M. bovis not only in pure bacterial culture but also in clinical samples of sputum, pleural fluid and blood. The speed, specificity, sensitivity of the rimM LAMP, the lack of a need for expensive equipment, and the visual readout show great potential for clinical detection of M. tuberculosis and M. bovis.     

Physical and functional interaction between d-ribokinase and topoisomerase I has opposite effects on their respective activity in Mycobacterium smegmatis and Mycobacterium tuberculosis

d-ribose is an essential component of multiple important biological molecules and must first be phosphorylated by ribokinase before entering metabolic pathways. However, the function and regulation of ribokinases in Mycobacterium tuberculosis, the causative agent of tuberculosis, and its related species are largely unknown. In this study, we have characterized the activities of two putative ribokinases, Rv2436 and Ms4585, from M. tuberculosis and Mycobacterium smegmatis, respectively. The mycobacterial topoisomerase I (TopA) was found to physically interact with its ribokinase both in vitro and in vivo. By creating two ribokinase mutants that showed defective interactions with TopA, we further showed that the interaction between ribokinase and TopA had opposite effects on their respective function. While the interaction between the two proteins inhibited the ability of TopA to relax supercoiled DNA, it stimulated ribokinase activity. A cross-regulation assay revealed that the interaction between the two proteins was conserved in the two mycobacterial species. Thus, we uncovered an interplay between ribokinase and topoisomerase I in mycobacteria, which implies the existence of a novel regulatory strategy for efficient utilization of d-ribose in M. tuberculosis that may be useful in stressful environments with restricted access to nutrients.     

结核分枝杆菌Rv3194c蛋白的亚细胞定位

【目的】Rv3194c基因编码的是结核分枝杆菌的PDZ信号蛋白,本研究探讨该蛋白的亚细胞定位,为其细胞结合蛋白的筛选奠定基础。【方法】从H37Rv基因组中扩增出编码只含有PDZ结构域的tRv3194c (Rv3194c 1–234 aa)的基因片段,在3′端加T2A和EGFP序列,一并插入真核表达载体构建出pcDNA3.1-tRv3194c-T2A-EGFP。将构建好的质粒瞬时转染L929细胞,并共感染重组痘苗病毒vTF7-3,用间接免疫荧光、流式细胞分选以及Western blotting检测融合蛋白的表达以及亚细胞定位。【结果】成功构建出真核表达载体pcDNA3.1-tRv3194c-T2A-EGFP,瞬时转染L929细胞后融合蛋白tRv3194c定位于线粒体膜上,且重组痘苗病毒vTF7-3的感染有助于靶蛋白表达水平的提高。【结论】Rv3194蛋白的PDZ结构域与线粒体外膜相关蛋白结合,为了解该蛋白在细胞内的致病机制提供重要线索。     

结核分枝杆菌EsxV脂质体纳米颗粒亚单位疫苗的制备及免疫学特性

本研究旨在制备脂质体纳米颗粒（lipid nanoparticles,LNP）为载体的结核分枝杆菌（Mycobacterium tuberculosis,Mtb）抗原EsxV亚单位疫苗,明确该疫苗经黏膜免疫诱导的免疫应答水平。采用薄膜分散法制备包裹蛋白EsxV和c-di-AMP的LNP （EsxV：C：L）,并对其包封率、LNP形态、粒径、表位电荷及多相分散指数进行了检测。EsxV：C：L滴鼻免疫BALB/c小鼠,检测免疫后血清和黏膜抗体、肺或脾细胞因子转录及分泌水平、肺T细胞亚群细胞比例。结果成功获得大小均一、呈球状、带负电的EsxV：C：L LNP亚单位疫苗。与EsxV：C相比,EsxV：C：L鼻黏膜接种可诱导小鼠呼吸道黏膜sIgA水平增加,脾细胞因子IL-2分泌水平升高,提高中央记忆T细和组织驻留T细胞的比例。综上,EsxV：C：L经黏膜免疫,可诱导更强的黏膜免疫和记忆性T细胞免疫应答,可能提供更好的抗Mtb感染的保护作用。     

结核分枝杆菌Rv2626c诱导的细胞免疫应答特性

全球有近1/4的人感染结核分枝杆菌(Mycobacterium tuberculosis,M.tb)并长期处于潜伏感染状态。Rv2626c是结核分枝杆菌受DosR调控的重要潜伏感染相关蛋白。本研究对Rv2626c蛋白进行了原核表达和纯化,并以RAW264.7细胞和小鼠为感染模型,对其免疫生物学特性进行了分析。SDS-PAGE及Western blotting鉴定结果表明,Rv2626c-His融合蛋白主要以可溶形式表达,能与兔抗H37Rv多抗血清发生特异性免疫反应。此外,本研究发现Rv2626c蛋白能结合到巨噬细胞RAW 264.7表面并上调细胞NO的生成;显著诱导促炎细胞因子IFN-γ、TNF-α、IL-6和MCP-1的产生;并能诱导小鼠产生更强的Th1免疫应答。上述研究有利于揭示结核分枝杆菌的致病机制,为新型结核病疫苗的研制奠定了理论基础。     

异烟肼耐药结核分枝杆菌感染人源巨噬细胞的蛋白质组学研究

利用双向电泳技术,对人源巨噬细胞U937感染异烟肼耐药结核分枝杆菌前后的全细胞蛋白表达图谱进行差异比较和分析,发现其中产生差异的有32个蛋白质斑点,利用基质辅助激光解吸/电离飞行时间质谱技术,对其中5个表达明显上调的蛋白质斑点进行分析鉴定,获得5个明确的肽质量指纹图谱,通过在数据库中进行检索分析,确定这5个蛋白质分别为热休克蛋白105_β、凋亡抑制蛋白-1、磷酸甘油酸变位酶1、组织蛋白酶B、桥粒胶蛋白3.上述发现有助于了解耐药结核分枝杆菌入侵早期导致的巨噬细胞蛋白质组表达变化,为深入研究耐药结核分枝杆菌-宿主相互作用提供了探索方向.     

A variation of the amplified-fragment length polymorphism (AFLP) technique using three restriction endonucleases, and assessment of the enzyme combination BglII-MfeI for AFLP analysis of Salmonella enterica subsp. enterica isolates

We have performed amplified-fragment length polymorphism (AFLP) fingerprinting on a collection of Salmonella enterica subsp. enterica serovar typhimurium strains with a restriction endonuclease combination (BglII and MfeI) that has previously been used successfully for typing Campylobacter jejuni isolates with high resolution. Additionally, a variation of the AFLP assay in which two rare cutting restriction enzymes (XbaI and BsrGI) in combination with the frequent cutter (HinP1I) was examined. The BglII and MfeI enzyme combination offered low resolution for genotyping Salmonella typhimurium isolates and is not recommended for this common serovar. The three-enzyme combination gave a higher discrimination, and is thus a new alternate way of performing AFLP fingerprinting of S. typhimurium.     

Population structure and genetic diversity of Huperzia serrata (Huperziaceae) based on amplified fragment length polymorphism (AFLP) markers

The levels and pattern of the genetic variation within and among natural populations of Huperzia serrata were investigated using amplified fragment length polymorphism markers. Seven primer combinations used in the study amplified 615 discernible bands with 532 (86.5%) being polymorphic, indicating a considerable high level of genetic diversity at the species level. AMOVA analysis revealed a low level of genetic differentiation among the ten populations. The UPGMA cluster of all samples showed that individuals from the same population occasionally failed to cluster in one distinct group. A Mantel test showed no significant correlation between genetic distance and geographical distance (r = 0.278, P = 0.891), suggesting that the gene flow was not restricted geographically. A number of factors that might affect the genetic profiles of H. serrata included clonal growth, selective effect of niche and outcrossing, as well as the effective wind-dispersal of spores.     

表达结核分枝杆菌CFP10基因的重组腺病毒对A549细胞炎性因子的影响

通过构建表达结核分枝杆菌（Mycobacterium tuberculosis,Mtb）CFP10基因的重组腺病毒,探讨CFP10蛋白对A549细胞炎性因子表达的影响。采用酶切、连接的方法将CFP10编码基因插入到腺病毒穿梭质粒pShuttle-AdV4中,构建重组穿梭质粒pShuttle-AdV4-CFP10,重组穿梭质粒经测序验证后和骨架质粒pGP-Ad-Pac Vector经限制性内切酶PacⅠ酶切线性化后共转染至HEK 293A细胞中进行病毒包装,获得重组腺病毒AdV4-CFP10,经定量PCR和Western blot验证后进行病毒扩增,CsCl密度梯度离心纯化获得高纯度的重组腺病毒。AdV4-CFP10感染Ⅱ型肺泡上皮细胞A549,利用荧光定量PCR和ELISA技术检测A549细胞中细胞因子IL-1α、IL-6、IL-8和TNF-α等的表达水平,初步探究CFP10对A549细胞炎性因子分泌的影响。成功构建了表达结核分枝杆菌CFP10基因的重组腺病毒AdV4-CFP10,AdV4-CFP10转染至A549细胞后,与空病毒相比,过表达CFP10显著上调A549细胞炎性因子的分泌水平。为进一步深入研究结核分枝杆菌CFP10蛋白对A549细胞炎性反应的调控机制提供参考。     

结核分枝杆菌耐药相关单碱基突变的计算方法比较

[目的] 耐药结核分枝杆菌（drug-resistant Mycobacterium tuberculosis）的产生给结核病（tuberculosis）的治疗带来巨大困难。[方法] 使用基于全基因组测序的关联分析探究耐药强相关的单核苷酸多态性（single nucleotide polymorphism,SNP）突变,主要有GEMMA、phyc、plink。为了阐明其中最优的耐药相关SNP计算方法,本研究下载NCBI上已有的1504株结核分枝杆菌数据,并获取它们对于3种常见的一线抗结核治疗药物（isoniazid、rifampicin、ethambutol）的耐药性检验结果。并使用这3种耐药相关SNP计算方法计算与结核分枝杆菌耐药相关的SNP;并评估计算得到的耐药相关SNP在预测耐药表型的敏感性和特异性。[结果] 发现通过phyc可以预测到最多的已知耐药相关SNP和最少的耐药无关SNP,而且phyc预测的耐药相关SNP的敏感性和特异性恒定大于52.49%。[结论] phyc在预测结核分枝杆菌耐药相关SNP中结果最准确,但考虑到运行时间和表型数据的更新,GEMMA和plink的结果也应作为参考。     

One-step purification and characterization of a fully active histidine-tagged Class II fructose-1,6-bisphosphate aldolase from Mycobacterium tuberculosis

Rv0363c (fba), encoding Class II fructose-bisphosphate aldolase (FBA), is one of the potential drug targets identified in our laboratory based on minimal gene set concept. The wild-type enzyme overproduction in E. coli had been reported. However, the purification procedure was relatively tedious and the yield was low. In this study, five histidine codons were introduced into the 3′ end of the amplified fba fragments. The expressed C-terminal histidine-tagged Class II FBA was produced in E. coli BL21 (DE3) and easily purified using immobilized metal affinity chromatography. The purified his-tagged FBA was characterized. Its biochemical properties were compared to the non-his-tagged enzyme purified according to the previous report. Both FBAs have similar characteristics such as native/subunit molecular mass, kinetic parameters, and temperature/pH optima and stability. The C-terminal his-tagged FBA can be a surrogate for the native enzyme and used for screening of inhibitors of FBA. This developed expression system will pave the way for high-throughput screening and crystallization studies. Moreover, in this study, a colorimetric FBA assay has been simplified to facilitate the mass screening of inhibitor of FBA.     

An improved amplified fragment length polymorphism (AFLP) protocol for discrimination of Listeria isolates

Nine restriction enzyme combinations and 108 different primer combinations were initially tested for suitability for amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes; the combination of HindIII and HpyCH4IV showed consistently strong signals on gels, amplified an adequate number of DNA fragments and detected polymorphism among closely related strains based on AscI macrorestriction profiles. AFLP also distinguished between L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri and L. grayi species. All Listeria species showed species-specific clusters, with less than 33% similarity between different species. A total of 34 L. monocytogenes strains were characterised by using both AFLP and pulsed-field gel electrophoresis (PFGE). The results of AFLP analysis of L. monocytogenes strains were in concordance with those obtained by PFGE. Both methods identified 29 different genotypes of L. monocytogenes and had a high discrimination index (> 0.999). By combining the results of AFLP and PFGE, subtype discrimination was further improved. Numerical analysis of both AFLP and PFGE profiles yielded three genomic groups of L. monocytogenes strains. AFLP was found to be faster and less labour-intensive than PFGE. We conclude that the AFLP protocol is a highly discriminatory, reproducible and valuable tool in characterisation of Listeria strains and may also be suitable for Listeria species identification.     

The Mycobacterium tuberculosis shikimate pathway genes: Evolutionary relationship between biosynthetic and catabolic 3-dehydroquinases

Summary The Mycobacterium tuberculosis shikimate pathway genes designated aroB and aroQ encoding 3-dehydroquinate synthase and 3-dehydroquinase, respectively were isolated by molecular cloning and their nucleotide sequences determined. The deduced dehydroquinate synthase amino acid sequence from M. tuberculosis showed high similarity to those of equivalent enzymes from prokaryotes and filamentous fungi. Surprisingly, the deduced M. tuberculosis 3-dehydroquinase amino acid sequence showed no similarity to other characterised prokaryotic biosynthetic 3-dehydroquinases (bDHQases). A high degree of similarity was observed, however, to the fungal catabolic 3-dehydroquinases (cDHQases) which are active in the quinic acid utilisation pathway and are isozymes of the fungal bDHQases. This finding indicates a common ancestral origin for genes encoding the catabolic dehydroquinases of fungi and the biosynthetic dehydroquinases present in some prokaryotes. Deletion of genes encoding shikimate pathway enzymes represents a possible approach to generation of rationally attenuated strains of M. tuberculosis for use as live vaccines.