PPE protein (Rv3425) from DNA segment RD11 of Mycobacterium tuberculosis: a novel immunodominant antigen of Mycobacterium tuberculosis induces humoral and cellular immune responses in mice

Subtractive DNA hybridization of pathogenic M. bovis and BCG, and comparative genome-wide DNA microarray analysis of M. tuberculosis H37Rv and BCG identified several RD, designated as RD1 to RD16, between M. tuberculosis and M. bovis on the one hand and BCG on the other. These regions cover 108 ORF of M. tuberculosis H37Rv, and are deleted from all 13 BCG sub-strains currently used as anti-tuberculosis vaccines in different parts of the world. In this study, we evaluated cellular and humoral immune response in C57BL/6 mice immunized with the PPE protein Rv3425, encoded by an ORF found in RD11 of M. tuberculosis. Rv3425 protein induced an increased Th1/Th2 type immune response in mice, characterized by an elevated concentration of IFN-gamma in antigen stimulated splenocyte culture and a strong IgG(1) antibody response. These results provide evidence on the immunogenicity of the PPE protein Rv3425 which, together with its reported immunodominant characteristics, imply that it may be a candidate for development of a vaccine for the control of TB.     

Advances in antibody-mediated immunity against Mycobacterium tuberculosis: implications for a novel vaccine strategy

Cell-mediated immunity is considered to be the major component of the host response against Mycobacterium tuberculosis, whereas antibody-mediated immunity historically has been considered inconsequential. In recent years, studies from several groups have challenged the traditional dogma and demonstrated that monoclonal antibodies can modify various aspects of mycobacterial infections. This review describes the experimental evidence supporting a role for antibodies in defense against mycobacterial infections and outlines future challenges to the field of antibody-mediated immunity against M. tuberculosis, with particular emphasis on the implications of these findings for a novel vaccine strategy.     

Mycobacterium thermoresistibile as a source of thermostable orthologs of Mycobacterium tuberculosis proteins

The genus Mycobacterium comprises major human pathogens such as the causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), and many environmental species. Tuberculosis claims ~1.5 million lives every year, and drug resistant strains of Mtb are rapidly emerging. To aid the development of new tuberculosis drugs, major efforts are currently under way to determine crystal structures of Mtb drug targets and proteins involved in pathogenicity. However, a major obstacle to obtaining crystal structures is the generation of well-diffracting crystals. Proteins from thermophiles can have better crystallization and diffraction properties than proteins from mesophiles, but their sequences and structures are often divergent. Here, we establish a thermophilic mycobacterial model organism, Mycobacterium thermoresistibile (Mth), for the study of Mtb proteins. Mth tolerates higher temperatures than Mtb or other environmental mycobacteria such as M. smegmatis. Mth proteins are on average more soluble than Mtb proteins, and comparison of the crystal structures of two pairs of orthologous proteins reveals nearly identical folds, indicating that Mth structures provide good surrogates for Mtb structures. This study introduces a thermophile as a source of protein for the study of a closely related human pathogen and marks a new approach to solving challenging mycobacterial protein structures.     

建立液相芯片方法检测鉴别结核分枝杆菌复合群、鸟分枝杆菌与副结核分枝杆菌

运用液相芯片技术原理,以分枝杆菌菌种(群)特异基因序列IS6110、IS1081、IS1245和F57为目标基因,设计筛选4套扩增引物和杂交探针,建立同时检测鉴别结核分枝杆菌复合群、鸟分枝杆菌和副结核分枝杆菌的四重液相基因芯片检测方法。对13种共54株分枝杆菌菌株以及23种常见微生物样品的检测结果显示,四重液相芯片方法可特异检测鉴别目标菌种(群),与其它分枝杆菌菌种或微生物无非特异交叉反应;检测敏感性达2.1×101-2.5×102基因拷贝或0.06-0.74 fg DNA;组内检测变异系数和组间检测变异系数均<10%。采用四重液相芯片方法从临床结核疑似人痰样和牛组织样品中检出结核致病菌,检出率分别达75.6%(99/131)和94.9%(37/39),显著高于培养法(38.9%和53.8%)。对副结核疑似临床样品的检测试验结果显示,四重液相芯片方法与荧光PCR方法的阳性符合率为83%(24/29)。对四重混合模板的检测试验结果显示该液相芯片方法可鉴别不同菌种混合感染。四重液相芯片方法的检测周期<1 d,其中对纯化DNA模板的检测时间可在2-3 h内完成。     

CRISPR/Cas系统与结核病防控新措施相关性研究

结核分枝杆菌(Mycobacterium tuberculosis,MTB,以下简称结核杆菌)感染引起的结核病仍然是严重影响人类健康全球性重大传染病。全球约1/4人口是结核杆菌的潜伏感染者。2019年,世界卫生组织(Worldhealthorganization,WHO)报道全球约150万人死于结核病。深入研究结核杆菌生物学有望为结核病防控提供新工具。成簇规律性间隔短回文重复(Clusteredregularlyinterspacedshort palindromic repeats,CRISPR)/Cas是细菌免疫系统的重要成分,在结核杆菌等分枝杆菌中也广泛存在,同时,也是分枝杆菌基因编辑的重要工具。本文结合课题组研究工作,综述了结核杆菌III-A型CRISPR/Cas系统各组分的生物学功能以及与致病的相关性,CRISPR/Cas编辑工具在诊断治疗耐药结核杆菌和结核病防控新措施中的进展。     

Drug targeted virtual screening and molecular dynamics of LipU protein of Mycobacterium tuberculosis and Mycobacterium leprae

The lipolytic protein LipU was conserved in mycobacterium sp. including M. tuberculosis (MTB LipU) and M. leprae (MLP LipU). The MTB LipU was identified in extracellular fraction and was reported to be essential for the survival of mycobacterium. Therefore to address the problem of drug resistance in pathogen, LipU was selected as a drug target and the viability of finding out some FDA approved drugs as LipU inhibitors in both the cases was explored. Three-dimensional (3D) model structures of MTB LipU and MLP LipU were generated and stabilized through molecular dynamics (MD). FDA approved drugs were screened against these proteins. The result showed that the top-scoring compounds for MTB LipU were Diosmin, Acarbose and Ouabain with the Glide XP score of ?12.8, ?11.9 and ?11.7 kcal/mol, respectively, whereas for MLP LipU protein, Digoxin (?9.2 kcal/mol), Indinavir (?8.2 kcal/mol) and Travoprost (?8.2 kcal/mol) showed highest affinity. These drugs remained bound in the active site pocket of MTB LipU and MLP LipU structure and interaction grew stronger after dynamics. RMSD, RMSF and Rg were found to be persistent throughout the simulation period. Hydrogen bonds along with large number of hydrophobic interactions stabilized the complex structures. Binding free energies obtained through Prime/MM-GBSA were found in the significant range from ?63.85 kcal/mol to ?34.57 kcal/mol for MTB LipU and ?71.33 kcal/mol to ?23.91 kcal/mol for MLP LipU. The report suggested high probability of these drugs to demolish the LipU activity and could be probable drug candidates to combat TB and leprosy disease.     

Occurrence of RD9 region and 500 bp fragment among clinical isolates of Mycobacterium tuberculosis and Mycobacterium bovis

The present investigation dealt with the identification of Mycobacterium tuberculosis and M. bovis by RD9 region and 500 bp fragment PCR assays. Eight M. tuberculosis and 5 M. bovis characterized and identified from 40 human sputum and 41 bovine lung specimens and 20 M. tuberculosis and 9 M. bovis strains maintained at Mycobacteria Laboratory, Indian Veterinary Research Institute were included in this study. In this way, 28 M. tuberculosis and 14 M. bovis strains and, for comparison and control purpose, M. tuberculosis H37Rv, M. bovis BCG, M. canetti, M. smegmatis, M. phlei, M. chelonae, M. kansasii, M. xenopi and M. avium were subjected to RD9 and 500 bp amplification by PCR. All M. tuberculosis strains, M. tuberculosis H37 Rv and M. canetti yielded a product of 333 bp which showed presence of RD9 region in these strains, whereas all M. bovis yielded a product of 206 bp with RD9 PCR assay. There was no ampli-fication product found in M. bovis BCG, M. xenopi, M. smegmatis, M. phlei, M. chelonae, M. kansasii, and M. avium. PCR based on 500 bp fragment showed a product of 500 bp in all M. bovis strains and M. bovis BCG. There was no amplification product of 500 bp found in M. canetti, M. smegmatis, M. phlei, M. chelonae, M. avium, M. kansasii, M. xenopi and was absent in all M. tuberculosis strains. The PCR assay results correlated 100% with the culture and biochemical results of the isolates. Our study suggested that PCR based on RD9 and 500 bp may effectively identify two closely related species of M. tuberculosis and M. bovis.     

Ligand-induced dynamical regulation of NO conversion in Mycobacterium tuberculosis truncated hemoglobin-N

Mycobacterium tuberculosis, the causative agent of human tuberculosis, is forced into latency by nitric oxide produced by macrophages during infection. In response to nitrosative stress M. tuberculosis has evolved a defense mechanism that relies on the oxygenated form of "truncated hemoglobin" N (trHbN), formally acting as NO-dioxygenase, yielding the harmless nitrate ion. X-ray crystal structures have shown that trHbN hosts a two-branched protein matrix tunnel system, proposed to control diatomic ligand migration to the heme, as the rate-limiting step in NO conversion to nitrate. Extended molecular dynamics simulations (0.1 micros), employed here to characterize the factors controlling diatomic ligand diffusion through the apolar tunnel system, suggest that O2 migration in deoxy-trHbN is restricted to a short branch of the tunnel, and that O2 binding to the heme drives conformational and dynamical fluctuations promoting NO migration through the long tunnel branch. The simulation results suggest that trHbN has evolved a dual-path mechanism for migration of O2 and NO to the heme, to achieve the most efficient NO detoxification.     

结核杆菌感染T细胞介导免疫应答研究的新进展

结核病对免疫学家构成了巨大的挑战,因为它是一种慢性传染性疾病,病原体具有持久性特点.在对人和动物进行实验时,检测到结核分枝杆菌适应性免疫应答的特点之一为感染早期T细胞免疫应答延迟.新近研究揭示了此种延迟应答的机制:通过结核杆菌抑制免疫细胞(CD4+和CD8+T细胞及DC)凋亡延迟应答,通过特异性Treg细胞抑制作用延迟应答.结核杆菌慢性感染期间存在IFNγ信号调节网络和ESAT-6抗原的慢性刺激作用,抗原特异性PD-1+ CD4+T细胞具有高度增殖分化为更多终末效应性T细胞的潜能,以此可调节和维持免疫应答.深入了解抗原特异性T细胞调节与维持适应性免疫应答的机制,有益于抗结核疫苗的设计和研制.     

抗结核分枝杆菌疫苗的研究进展

结核病是由结核分枝杆菌感染引起的传染病,细胞免疫中的CD4+T细胞、CD8+T细胞、Th17细胞在对抗结核分枝杆菌感染中发挥重要作用,新近研究显示抗体特定的糖链修饰有助于清除病原体,提示体液免疫也可能参与免疫保护。目前使用的疫苗——卡介苗对婴幼儿重症结核病具有良好的保护力,但是对成人肺结核保护力欠佳,所以需要研发新的疫苗。目前已有数个新型疫苗进入临床试验。本文就结核分枝杆菌的免疫保护机制作一简要介绍,主要阐述现用疫苗——卡介苗及新型疫苗的研究现状,让读者对上述知识的进展有所了解。     

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。     

Recent insights into Mycobacterium tuberculosis through proteomics and implications for the clinic

Introduction: This review aimed at providing an update on the application of proteomics-based approaches to gain recent insights of Mycobacterium tuberculosis (M.tb) and its relevance to clinic. Proteomics and bioinformatics approaches helped in the identification and characterization of novel proteins. Studying M.tb, causative agent of tuberculosis (TB), at the proteomic level can contribute to the identification of proteins which can be considered as potential targets for developed drugs and can help us in better understanding the pathogen physiology.

Areas covered: In this review we have presented a comprehensive literature pertaining to role of proteomics in understanding M.tb. We have also focused on how the development and advancement in technology in the field of proteomics has augmented the research and played a pivotal role in answering many unexplored questions. Lastly, the application of proteomics to clinic has also been discussed.

Expert commentary: We envisage that proteomics has gained remarkable momentum over the years. Proteomics can play an important role in the discovery of biomarkers for TB and other diseases. Also, it can aid in development of effective vaccines and simple, rapid and cost-effective test for the diagnosis of TB which is crucial for the management and control of the disease.     

Screening and Assessing 11 Mycobacterium tuberculosis Proteins as Potential Serodiagnostical Markers for Discriminating TB Patients from BCG Vaccinees

Purified protein derivative (PPD) skin tests often yield poor specificity, so that to develop new serological antigens for distinguishing between Mycobacterium tuberculosis infection and Bacille Calmette-Guerin (BCG) vaccination is a priority, especially for developing countries like China. We predicted the antigenicity for selected open reading frames (ORFs) based on the genome sequences of M. tuberculosis H37Rv and M. bovis BCG, as well as their functions and differences of expression under different stim...     

人结核分枝杆菌特异性核酸探针制备及结核病PCR检测技术的初步临床应用

采用人结核分枝杆菌(Mycobacterium tuberculosis TB)染色体DNA为模板,选择位于插入片段IS6110中884～865和568～588碱基对处的两个片段为引物,扩增出317bp的特异性片段．将其克隆进pUCl9载体。酶切图谱分析和DNA序列测定证实为目的片段。该片段经DIG标记,分别与11种分枝杆菌DNA进行Southern杂交,结果证明只与人型复合分枝杆菌发生杂交反应。利用该对引物建立的PcR检测拄术对74份结核病痰液标本进行检测,并与临床细菌快速培养结果相比较,发现48份临床阳性均为PcR阳性,在26份临床阴性标本中亦发现11份PCR检测阳性。将标本PCR产物与克隆探针进行杂交,显示两者结果完全一致。说明PCR检测体系结果可靠,其灵敏度明显高于目前临床所采用的方法,可作为一种常规技术用于结核病的临床检测。