导读与评议（2017年第6期）

精准医疗概念的提出开启了一个医学新时代,其实质包括精准诊断和精准治疗。张文宏课题组围绕结核病治疗中的精准医疗进行了阐述,涉及结核病的精准诊断,包括结核病的临床诊断及结核分枝杆菌的检测(分子检测及耐药检测技术等)、特殊人群的药理学参数与药物代谢相关的分子标记、针对病原体生命周期分子靶点的直接作用药物研发、通过正向调控或负向调控药物的使用实现宿主导向抗结核精准治疗。本期刊登了3篇关于结核病耐药的综述。鉴于耐药结核分枝杆菌的补偿性进化是其传播与流行的基础,高谦课题组从结核分枝杆菌的耐药分子机制、耐药突变的适应性代价与补偿性进化,以及补偿性进化如何影响耐药结核病传播等方面进行了综述。袁莉课题组就近年来结核分枝杆菌“毒素-抗毒素系统”(TAS)与生物膜的研究及抗结核药物对生物膜形成的影响进行综述......     

耐药结核分枝杆菌潜伏-复发感染动物模型的研究进展

潜伏结核感染(latent tuberculosis infection,LTBI)复发是新发结核病的主要来源,其中耐药结核病所占比例较大,使耐药LTBI复发的防控成为结核病研究的重点。耐药结核分枝杆菌潜伏-复发感染动物模型是开展耐药结核病防控相关机制研究、抗耐药结核分枝杆菌药物和疫苗研究的基础。目前耐药结核分枝杆菌感染动物模型缺乏,而已有的结核分枝杆菌标准株H37Rv潜伏-复发感染模型存在缺陷,如小鼠模型的潜伏期荷菌量偏高、复发期变异大,而猴模型的潜伏期和复发期不可预测。模型的可控性差使其应用困难,且缺乏可用的免疫学评价指标,导致远期复发无法预测。因此,基于现有H37Rv潜伏-复发感染动物模型的制备方法,展望耐药结核分枝杆菌潜伏-复发感染动物模型可能存在的缺陷,通过选用新的抑菌剂和诱导剂,制备有稳定潜伏期、潜伏时长适中、复发起点和复发水平变异小的动物模型,是未来耐药结核分枝杆菌潜伏-复发感染动物模型研究的方向。     

生长环境对结核分枝杆菌胁迫作用的研究进展

高传染性结核病是由结核分枝杆菌引起的慢性消耗性疾病,病死率较高,备受全球关注。结核分枝杆菌是一种胞内寄生菌,通过呼吸道感染宿主,感染期间寄居肺部形成肉芽肿。肉芽肿或者免疫系统施加的环境压力,比如低pH、缺乏营养(缺铁)、缺氧等使结核分枝杆菌进入休眠状态,从而影响结核分枝杆菌的正常生长和抗生素的疗效。结核病治疗周期长,易产生耐药性,因此迫切需要研发新的药物以提升治疗效果。本文通过3个主要具有胁迫作用的生长环境因素综述了近年来发现的结核分枝杆菌相关调控因子和药物新靶点,为疫苗研究及新药物设计提供理论基础和研究依据。     

治疗性结核病疫苗研究进展

治疗性结核病疫苗主要用于接种已感染结核分枝杆菌的个体,包括化学药物治疗的患者和潜伏感染者。治疗性疫苗可逆转发生在疾病进展期的非保护性免疫反应,使其向Th1型反应发展;能打破机体的免疫耐受,有效激发宿主针对结核分枝杆菌的以抗原为基础的细胞免疫反应,诱发抗原特异性的细胞毒性T淋巴细胞免疫反应,来清除胞内寄生的结核分枝杆菌。治疗性疫苗将有助于防止潜伏结核病的复发;与药物联合使用以提高药物的治疗效果,尤其是针对耐药结核病的治疗。     

儿茶素类化合物的抗结核活性研究进展

近年来,耐多药结核病(multidrug-resistant tuberculosis,MDR-TB)的出现及人类免疫缺陷病毒(human immunodeficiency virus,HIV)与结核分枝杆菌合并感染日益增多,使结核病的防治面临更加严峻的挑战,人们急需开发新型抗结核药物及天然低毒的辅助治疗药物。绿茶中的儿茶素类化合物具有多种生物活性,对多种疾病有一定的辅助疗效。多项研究显示,儿茶素类化合物也具有抗结核活性,其机制包括抑制二氢叶酸还原酶活性、影响分枝菌酸及细胞壁的合成、下调富含色氨酸天冬氨酸的膜蛋白(tryptophan-aspartate containing coat protein,TACO)基因表达以抑制结核分枝杆菌的胞内寄生,降低氧化应激水平,下调结核分枝杆菌85B蛋白和宿主肿瘤坏死因子α(tumor necrosis factor α,TNF-α)表达,从而改善炎症水平。有研究显示,喝绿茶可降低结核分枝杆菌感染风险,儿茶素类化合物可辅助治疗结核病并与抗结核药物有协同治疗作用,但目前对其作用机制的研究还不够深入,需进一步探讨。     

中性粒细胞在抗结核免疫中的作用

结核病是世界范围内的重要传染性疾病之一,严重威胁人类健康。免疫细胞在抗结核免疫过程中起重要作用,各细胞亚群通过不同作用机制影响结核病的病程及转归。中性粒细胞为机体应对结核分枝杆菌感染的第一道防线,在宿主免疫应答过程中是一把双刃剑。一方面,机体感染结核分枝杆菌后,中性粒细胞于第一时间向感染部位聚集,通过多种方式对抗感染：中性粒细胞吞噬结核分枝杆菌后,通过自身凋亡而杀菌;参与形成肉芽肿,形成胞外陷阱来限制结核分枝杆菌的生长和传播;产生功能性细胞因子,调控宿主的抗结核免疫反应。另一方面,中性粒细胞还参与机体的病理损伤过程,甚至促进体内结核分枝杆菌的生长。本文综述了中性粒细胞在抗结核免疫中作用的最新研究进展。     

结核分枝杆菌持续感染相关基因及其调控网络分析

结核分枝杆菌是结核病的致病菌, 也是迄今最成功的人类致病菌之一. 结核分枝杆菌能逃避宿主免疫攻击, 在人体内持续感染或呈休眠状态. 当人体免疫功能低下时, 持续感染或休眠的致病菌可能被重新激活. 结核分枝杆菌的持续感染是制约结核病控制计划成功的主要障碍之一. 揭示结核分枝杆菌持续感染的分子机制、寻找其中薄弱环节、发现适当的药物靶标并开发全新药物及免疫干预措施, 被认为是遏制结核病蔓延的关键. 结核分枝杆菌持续感染和再激活是众多基因协同的系统适应过程. 本文在全面分析全球结核分枝杆菌持续感染相关基因研究文献的基础上, 通过文本挖掘, 综合本实验室前期研究结果, 提出了结核分枝杆菌持续感染相关基因的调控网络, 为揭示结核分枝杆菌持续感染的机制, 筛选控制结核病的新靶标和免疫干预节点提供研究基础.     

结核潜伏感染动物模型的研究进展

结核潜伏感染(latent tuberculosis infection, LTBI)是机体对结核分枝杆菌抗原持续性免疫反应的状态,既无活动性结核病临床症状,也无结核病影像学表现。LTBI激活是新发结核病的主要来源之一。LTBI动物模型的建立是研究结核的潜伏感染和复发机制,开发诊断试剂,评估抗结核新药、新疫苗的有效性、安全性的基础。建立稳定、成本低、易推广、潜伏期时长适中、复发起点和复发水平变异小的LTBI动物模型,是其未来研究发展的方向。本文就LTBI动物模型的研究进展进行综述,期望为结核病防治工作者提供参考资料。     

两株不含c-di-GMP代谢相关基因的重组卡介苗菌株引起的免疫应答对不同毒力的结核菌株的抵抗作用

菌膜是一种生物膜,作为一种类似于结核分枝杆菌感染过程中出现的某些特征的结构,如抗生素耐药性和感染的长期性,以及作为感染豚鼠的体液反应抗原的来源,在结核病领域重新引起了人们的兴趣。有充分的证据表明,在其他细菌中,第二信使c-di-GMP调节从浮游细胞到生物膜形成的转变。在这项工作中,作者使用牛分枝杆菌活疫苗BCG来确定与c-di-GMP代谢相关的基因缺失是否会影响与巨噬细胞的相互作用,在小鼠细胞系和小鼠中诱导免疫应答的能力,以及在表面膜的生长过程中如何改变蛋白谱。     

结核分枝杆菌的免疫学研究进展

由结核分枝杆菌引起的结核病多年来依然是世界范围内严重的公共卫生问题。结核杆菌的致病特点是可在体内巨噬细胞中长期存活,形成潜伏感染。本文就结核杆菌感染过程中机体的免疫应答过程,尤其是潜伏感染形成机制等方面进行了综述,以期为结核病新型疫苗的研发提供参考。     

Dormant forms of mycobacteria

Dormant states of bacteria with drastically decreased metabolic activity, enhanced resistance to harmful factors, and absence of cell division is a form for surviving unfavorable environmental conditions. This state does not necessarily imply formation of highly differentiated spores and cysts; it has been demonstrated for non-spore-forming bacteria, including pathogenic ones. The latency of a number of infectious diseases is generally believed to be related to the capacity of bacteria (including Mycobacterium tuberculosis, an infective agent of tuberculosis) to produce dormant forms. Indeed, some results of histological investigation and modeling of latent infections in animals, as well as results obtained with in vitro models, support the hypothesis of production of dormant forms by tuberculosis bacteria. In the present review, existing experimental models of dormant form production in mycobacteria are considered, as well as modern data concerning the mechanisms of their formation and their relation to the “nonculturable” state. The mechanisms of reversion to culturability and the role of extracellular factors in reactivation of dormant forms are discussed in detail.     

Persister cells, dormancy and infectious disease

Several well-recognized puzzles in microbiology have remained unsolved for decades. These include latent bacterial infections, unculturable microorganisms, persister cells and biofilm multidrug tolerance. Accumulating evidence suggests that these seemingly disparate phenomena result from the ability of bacteria to enter into a dormant (non-dividing) state. The molecular mechanisms that underlie the formation of dormant persister cells are now being unravelled and are the focus of this Review.     

Human mesenchymal stem cell based intracellular dormancy model of Mycobacterium tuberculosis

Understanding the biology of the tuberculosis pathogen during dormant asymptomatic infection, called latent tuberculosis is crucial to decipher a resilient therapeutic strategy for the disease. Recent discoveries exhibiting presence of pathogen’s DNA and bacilli in mesenchymal stem cells (MSCs) of human and mouse despite completion of antitubercular therapy, indicates that these specific cells could be one of the niches for dormant Mycobacterium tuberculosis in humans. To determine if in vitro infection of human MSCs could recapitulate the in vivo characteristics of dormant M. tuberculosis, we examined survival, phenotype, and drug susceptibility of the pathogen in MSCs. When a very low multiplicity of infection (1:1) was used, M. tuberculosis could survive in human bone marrow derived MSCs for more than 22 days without any growth. At this low level of infection, the pathogen did not cause any noticeable host cell death. During the later phase of infection, MSC-residing M. tuberculosis exhibited increased expression of HspX (a 16-kDa alpha-crystallin homolog) with a concurrent increase in tolerance to the frontline antitubercular drugs Rifampin and isoniazid. These results present a human MSC-based intracelllular model of M. tuberculosis infection to dissect the mechanisms through which the pathogen acquires and maintains dormancy in the host.     

Construction and evaluation of luciferase reporter phages for the detection of active and non-replicating tubercle bacilli

The luciferase reporter phages (LRP) show great promise for diagnostic mycobacteriology. Though conventional constructs developed from lytic phages such as D29 and TM4 are highly specific, they lack sensitivity. We have isolated and characterized Che12, the first true temperate phage infecting M. tuberculosis. Since the tuberculosis (TB) cases among HIV infected population result from the reactivation of latent bacilli, it would be useful to develop LRP that can detect dormant bacteria. During dormancy, pathogenic mycobacteria switch their metabolism involving divergent genes than during normal, active growth phase. Since the promoters of these genes can potentially function during dormancy, they were exploited for the construction of novel mycobacterial luciferase reporter phages. The promoters of hsp60, isocitrate lyase (icl), and alpha crystallin (acr) genes from M. tuberculosis were used for expressing firefly luciferase gene (FFlux) in both Che12 and TM4 phages and their efficiency was evaluated in detecting dormant bacteria from clinical isolates of M. tuberculosis. These LRP constructs exhibited detectable luciferase activity in dormant as well as in actively growing M. tuberculosis. The TM4 ts mutant based constructs showed about one log increase in light output in three of the five tested clinical isolates and in M. tuberculosis H37Rv compared to conventional lytic reporter phage, phAE129. By refining the LRP assay format further, an ideal rapid assay can be designed not only to diagnose active and dormant TB but also to differentiate the species and to find their drug susceptibility pattern.     

Mycobacterium marinum Causes a Latent Infection that Can Be Reactivated by Gamma Irradiation in Adult Zebrafish

The mechanisms leading to latency and reactivation of human tuberculosis are still unclear, mainly due to the lack of standardized animal models for latent mycobacterial infection. In this longitudinal study of the progression of a mycobacterial disease in adult zebrafish, we show that an experimental intraperitoneal infection with a low dose (∼35 bacteria) of Mycobacterium marinum, results in the development of a latent disease in most individuals. The infection is characterized by limited mortality (25%), stable bacterial loads 4 weeks following infection and constant numbers of highly organized granulomas in few target organs. The majority of bacteria are dormant during a latent mycobacterial infection in zebrafish, and can be activated by resuscitation promoting factor ex vivo. In 5–10% of tuberculosis cases in humans, the disease is reactivated usually as a consequence of immune suppression. In our model, we are able to show that reactivation can be efficiently induced in infected zebrafish by γ-irradiation that transiently depletes granulo/monocyte and lymphocyte pools, as determined by flow cytometry. This immunosuppression causes reactivation of the dormant mycobacterial population and a rapid outgrowth of bacteria, leading to 88% mortality in four weeks. In this study, the adult zebrafish presents itself as a unique non-mammalian vertebrate model for studying the development of latency, regulation of mycobacterial dormancy, as well as reactivation of latent or subclinical tuberculosis. The possibilities for screening for host and pathogen factors affecting the disease progression, and identifying novel therapeutic agents and vaccine targets make this established model especially attractive.     

Vaccine strategies against latent tuberculosis infection

The leading tuberculosis (TB) vaccines currently in clinical trials are all designed as prophylactic vaccines. Although these vaccines are highly active, they will most probably not result in sterilizing immunity and, therefore, will not solve the global problem of latent TB. An attractive strategy is to target the remaining dormant bacteria with vaccines based upon antigens induced as the bacteria change from active multiplication to non-replicating dormancy (latency antigens) or during reactivation as dormant bacteria resume active metabolism (resuscitation antigens). These late-stage antigens might have potential as post-exposure vaccines or could form the basis for a multi-stage vaccine strategy, in which they are combined with prophylactic vaccines based on early antigens from replicating bacteria.     

New approaches in the diagnosis and treatment of latent tuberculosis infection

With nearly 9 million new active disease cases and 2 million deaths occurring worldwide every year, tuberculosis continues to remain a major public health problem. Exposure to Mycobacterium tuberculosis leads to active disease in only ~10% people. An effective immune response in remaining individuals stops M. tuberculosis multiplication. However, the pathogen is completely eradicated in ~10% people while others only succeed in containment of infection as some bacilli escape killing and remain in non-replicating (dormant) state (latent tuberculosis infection) in old lesions. The dormant bacilli can resuscitate and cause active disease if a disruption of immune response occurs. Nearly one-third of world population is latently infected with M. tuberculosis and 5%-10% of infected individuals will develop active disease during their life time. However, the risk of developing active disease is greatly increased (5%-15% every year and ~50% over lifetime) by human immunodeficiency virus-coinfection. While active transmission is a significant contributor of active disease cases in high tuberculosis burden countries, most active disease cases in low tuberculosis incidence countries arise from this pool of latently infected individuals. A positive tuberculin skin test or a more recent and specific interferon-gamma release assay in a person without overt signs of active disease indicates latent tuberculosis infection. Two commercial interferon-gamma release assays, QFT-G-IT and T-SPOT.TB have been developed. The standard treatment for latent tuberculosis infection is daily therapy with isoniazid for nine months. Other options include therapy with rifampicin for 4 months or isoniazid + rifampicin for 3 months or rifampicin + pyrazinamide for 2 months or isoniazid + rifapentine for 3 months. Identification of latently infected individuals and their treatment has lowered tuberculosis incidence in rich, advanced countries. Similar approaches also hold great promise for other countries with low-intermediate rates of tuberculosis incidence.     

Crystal structure of the resuscitation-promoting factor (DeltaDUF)RpfB from M. tuberculosis

Mycobacterium tuberculosis is able to establish a non-replicating state and survive in an intracellular habitat for years. Resuscitation of dormant M. tuberculosis bacteria is promoted by resuscitation-promoting factors (Rpfs), which are secreted from slowly replicating bacteria close to dormant bacteria. Here we report the crystal structure of a truncated form of RpfB (residues 194-362), the sole indispensable Rpf of the five Rpfs encoded in this bacterium genome. The structure, denoted as (DeltaDUF)RpfB, exhibits a comma-like shape formed by a lysozyme-like globular catalytic domain and an elongated G5 domain, which is widespread among cell surface binding proteins. The G5 domain, whose structure was previously uncharacterised, presents some peculiar features. The basic structural motif of this domain, which represents the tail of the comma-like structure, is a novel super-secondary-structure element, made of two beta-sheets interconnected by a pseudo-triple helix. This intricate organisation leads to the exposure of several backbone hydrogen-bond donors/acceptors. Mutagenesis analyses and solution studies indicate that this protein construct as well as the full-length form are elongated monomeric proteins. Although (DeltaDUF)RpfB does not self-associate, the exposure of structural elements (backbone H-bond donors/acceptors and hydrophobic side chains) that are usually buried in globular proteins is typically associated with adhesive properties. This suggests that the RpfB G5 domain has a cell-wall adhesive function, which allows the catalytic domain to be properly oriented for the cleavage reaction. Interestingly, sequence comparisons indicate that these structural features are also shared by G5 domains involved in biofilm formation.     

Resuscitation Promoting Factors: a Family of Microbial Proteins in Survival and Resuscitation of Dormant Mycobacteria

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is an extraordinarily successful pathogen of humankind. It has been estimated that up to one-third of the world’s population is infected with M. tuberculosis, and this population is an important reservoir for disease reactivation. Resuscitation promoting factor (Rpf) is a secretory protein, which was first reported in Micrococcus luteus. There are five functionally redundant Rpf-like proteins found in M. tuberculosis. Rpf promotes the resuscitation of dormant bacilli to yield normal, viable colony forming bacteria. All Rpfs share a conserved domain of about 70 amino acids and possess a lysozyme-like activity. The structural studies of the conserved domain suggest that Rpfs could be considered as a c-type lysozyme and lytic transglycosylases. Recently a novel class of nitrophenylthiocyanates (NPT) inhibitors of the muralytic activity of Rpf were reported which opens a new approach in the study of cell-wall hydrolyzing enzymes. This review describes molecular and structural studies conducted on Rpf proteins, their role in the resuscitation of dormant bacteria, in the reactivation of latent infection and identification of low molecular weight inhibitors of resuscitation promoting factors.     

Functional characterization of the phospholipase C activity of Rv3487c and its localization on the cell wall of Mycobacterium tuberculosis

Mycobacterium tuberculosis survives and persists for prolonged periods within its host in an asymptomatic,latent state and can reactivate years later if the host's immune system weakens. The dormant bacilli synthesize and accumulate triacylglycerol, reputed to be an energy source during latency. Among the phospholipases, phospholipase C plays an important role in the pathogenesis. Mutations in a known phospholipase C, plcC, of M.tuberculosis attenuate its growth during the late phase of infection in mice. Hydrolysis of phospholipids by phospholipase C generates diacylglycerol, a well-known signalling molecule that participates in the activation of extracellular signal-regulated kinases (ERK) through protein kinase C leading to macrophage activation. In the present study, we show that M.tuberculosis possesses an additional cell wall-associated protein, Rv3487c, with phospholipase C activity. The recombinant Rv3487c hydrolyses the substrate phosphatidylcholine and generates diacylglycerol by removing the phosphocholine. Furthermore, Rv3487c is expressed during infection as it exhibits significant humoral immunoreactivity with sera from children with tuberculosis, but not with that from adult patients.