定量蛋白质组学分析链霉素耐药和敏感结核分枝杆菌临床分离株

[目的]发现结核分枝杆菌(Mycobacterium tuberculosis)链霉素耐药相关的潜在菌体蛋白.[方法]以结核分枝杆菌临床分离链霉素敏感株01105和结核分枝杆菌H37Rv为对照,采用iTRAQ技术和生物信息学鉴定并相对定量结核分枝杆菌临床分离链霉素耐药株01108菌体蛋白,并通过WEGO功能注释聚类分析01108菌株差异表达蛋白的细胞组分、分子功能和生物进程.[结果]01108菌株分别与01105菌株和H37Rv菌株比较差异表达蛋白为194个和146个,01108菌株与01105菌株和H37Rv比较均差异表达蛋白121个(共同差异表达蛋白).差异表达蛋白理论相对分子量和等电点分布广泛,其生物进程主要参与中间代谢、呼吸作用和脂质代谢,分子功能主要为催化活性功能和结合功能.共同差异表达蛋白:7个核糖体蛋白(Rv2785c,Rv0056,Rv0641,Rv0652,Rv0701,Rv1630和Rv2442c)在01108菌株中表达下调;7个蛋白在01108菌株中显著差异表达(上调大于1.20倍或下调小于0.55倍),分别为巯基过氧化物酶(Rv1932)、酰基载体蛋白脱氢酶(Rv0824c)、30S核糖体蛋白S15 (Rv2785c)、丙酮酸脱氢酶E2部分(Rv2215)、双组份转录调控蛋白(Rv3133c)以及假定未知蛋白(Rv2466c和Rv2626c).[结论]iTRAQ发现了链霉素耐药结核分枝杆菌相对于链霉素敏感结核分枝杆菌和H37Rv共同差异表达蛋白,为进一步探讨结核分枝杆菌链霉素耐药机制奠定了基础.     

结核分枝杆菌对异烟肼耐药的分子机制

抗结核一线药物异烟肼是应用最广泛的抗结核药物之一,自1952年应用于临床以来,异烟肼就成了治疗结核和潜在感染的基础药物.有报道,我国异烟肼耐药已排在首位.结核分枝杆菌对异烟肼耐药的分子机制十分复杂,涉及katG、inhA、kasA、ndh、axyR等多种基因,本研究仅就此方面的研究作一综述.     

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.     

应用4种方法检测结核分枝杆菌临床分离株对链霉素的耐受性

通过DNA测序、SSCP、RFLP和反向斑点杂交技术分析167株结核分枝杆菌临床分离株的耐药基因型,评价结核分枝杆菌rpsL或rrs基因突变与链霉素（SM）耐受性之间的关系,比较4种分子方法检测SM耐受性的临床价值。98株耐SM分离株中,78株（79．6％）rpsL 43位或88位密码子错义突变导致赖氨酸置换为精氨酸,6株（6．1％）rrs 513位碱基A突变为C或T或516位C突变为T,14株（14．3％）未发现突变;69株SM敏感的分离株未发现这两个基因突变。应用SSCP、RFLP和RDBH方法分析上述突变和野生序列的结果与DNA测序完全一致,RDBH方法可从98株耐SM分离株中正确鉴定出84株（85．7％）分离株的5种突变基因型。结果表明,应用分子技术分析rpsL和rrs基因突变可快速检测大多数结核分枝杆菌对SM的耐受性,反向斑点杂交方法是一个快速、简便和可靠地检测药物耐受性的分子方法。     

猫爪草提取物对临床分离结核分枝杆菌蛋白质组表达的影响

猫爪草已经临床治疗耐药结核病,但其作用机理和有效成分尚不清楚。为研究其可能的作用靶标,采用双向电泳技术比较分析猫爪草提取物作用前后结核分枝杆菌临床分离株的全细胞蛋白表达差异。发现22个蛋白质斑点具有明显差异,对其中3个表达明显下调的蛋白质斑点进行基质辅助激光解吸电离飞行时间质谱分析,获得了肽质量指纹图谱。数据库检索分析确定这3个点代表的蛋白质分别为硫代硫酸硫转移酶,延长因子Ts和热休克蛋白X,分别参与厌氧硫代谢、蛋白质翻译和蛋白质折叠分泌、转录调控等过程。这有助于深入研究猫爪草对结核分枝杆菌的作用机理,也为发现新的抗结核病治疗药物靶标提供了线索。     

结核分枝杆菌耐异烟肼的分子机制

结核分枝杆菌对抗结核化疗药物异烟肼的耐药机制较为复杂,其中katG和inhA基因是主要的耐药基因,katG的丢或突变导致其编码的过氧化氧－过氧化物酶活性丧失或下降,而inhA基因突变减弱了异烟肼对分枝菌酸生物合成的抑制作用。此外,kasA和ahpC基因也与异烟肼的耐药有关,本文对近年来异烟肼耐药基因katG、inhA,kasA和oxyR-ahpC的进展作一综述。     

基于蛋白质组芯片的结核分枝杆菌系统生物学研究进展

近些年全球结核病疫情愈发严重,耐药性结核病使其雪上加霜。一个重要原因是结核病新药的匮乏以及结核分枝杆菌相关基础研究的不足。因此迫切需要开发新的技术以促进结核病系统生物学基础研究,并在此基础上研究新机制,发现新靶标,开发新药物。结核分枝杆菌功能蛋白质组芯片的出现旨在促进结核病相关研究工作。考虑到结核分枝杆菌高毒力、复制周期长和需要在生物安全三级实验室中开展研究等特点和难点,该工具为结核病相关研究人员提供了一个强有力的武器。目前这一技术手段的应用已经使我们对结核分枝杆菌-宿主相互作用、小分子-蛋白结合以及抗生素耐药性机制等关键生物过程有了更深入的了解。为了更好地帮助同行了解这一有效的工具,本文综述了结核分枝杆菌功能蛋白组芯片的几种主要应用,期望同行专家能更好地将其应用于结核病相关的基础研究中。     

一种新型抗结核先导化合物S28 对结核分枝杆菌蛋白质组表达的影响

目的 探讨从化合物库中高通量筛选得到的、可有效抑制结核分枝杆菌生长和繁殖的新型活性化合物S28 的作用机制及其可能的作用靶点。方法 采用双向电泳技术, 比较分析活性化合物作用于结核分枝杆菌H37Ra 前、后的全细胞蛋白表达差异。结果 13 个蛋白质斑点表达下调, 对其中6 个改变明显的蛋白质斑点进行基质辅助激光解吸/ 电离飞行时间质谱分析, 成功测定2 个蛋白质斑点。数据库检索分析确定这2 个差异蛋白点分别为延长因子Tu 和短链脱氢酶, 是参与蛋白质翻译和氧化呼吸、能量代谢等生理过程的重要蛋白。结论 为 进一步深入探索新型抗结核活性化合物的作用机制和可能的靶点提供研究基础和方向。     

快速方法检测结核分枝杆菌耐药基因突变

快速准确地鉴定结核分枝杆菌与结核分枝杆菌对利福平和异烟肼耐药基因突变的快速检测,对结核病人的诊断与治疗具有重要指导意义。本次根据结核分枝杆菌标准株H37RV序列,利用覆盖rpoB、katG、inhA基因突变区的系列寡核苷酸探针,并检测临床样品中结核分枝杆菌的基因突变情况,以此来判断耐药结果,并对其进行方法学评价。     

结核分枝杆菌耐链霉素和乙胺丁醇分子机制的研究

目的:研究结核分枝杆菌耐链霉素和乙胺丁醇的rpsL和emb B基因突变情况,探讨耐药基因突变与耐药性的关系。方法:通过传统药敏实验和聚合酶链反应(PCR)--单链构象多态性(SSCP)技术初步鉴定62株临床分离株的药敏和rps L、emb B基因。结果:与结核菌标准株H37Rv对照,分析30例TB菌耐链霉素(SM)的rps L基因,发现其突变率为70.0%(21/30),分析29例耐乙胺丁醇(EMB)的emb B基因,该基因的突变率为65.5%(19/29)。结论:部分结核分枝杆菌耐SM和EMB是由于其rps L、emb B基因突变所致,PCR-SSCP银染技术可能成为测定部分结核分枝杆菌耐药的简便、快速的方法。     

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.     

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...     

Simplified amplified-fragment length polymorphism method for genotyping Mycobacterium tuberculosis isolates

A simplified amplified-fragment length polymorphism (AFLP) method was developed and applied to genotype 52 Mycobacterium tuberculosis isolates. This method can be carried out using only one restriction enzyme (XhoI), one double strand adapter, and one PCR primer. The amounts of DNA and DNA polymerase, and the concentrations of primer and Mg²⁺ in the PCR step were optimized using the Basic Sequential Simplex method. AFLP analysis of the isolates generated a total of 24 differently sized bands ranging from 1537 to 121 bp, and 52 different band patterns, with a minimum of 2 and a maximum of 13 bands. The results were compared with the well-established IS6110 restriction fragment length polymorphism (IS6110-RFLP) typing method, which rendered a total of 32 differently sized bands from 1 to 12 kbp, and 52 different band patterns, with a minimum of 3 and a maximum of 15 bands. Therefore, both genotyping methods showed a discriminatory power of samples of 100%. Nevertheless, pairwise comparisons of the 1326 similarity indexes calculated for both typing methods showed a total absence of correlation between the similarity indexes of the two methods. The simplified AFLP method is expected to be more useful for genotyping M. tuberculosis isolates compared to the IS6110-RFLP method, since the former evaluates genetic variations throughout the M. tuberculosis genome. Furthermore, the relatively rapid and low-cost simplified AFLP method compares favorably to the IS6110-RFLP or conventional AFLP methods, and shows great promise for genotyping M. tuberculosis isolates, especially in developing countries or for preliminary screening.     

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。     

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。     

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.     

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.