耐药结核分枝杆菌基因组信息挖掘

<正>由于结核分枝杆菌的耐药性问题,使结核病这个古老的传染病死灰复燃,并成为全球性的严重公关问题。从1998年首个结核分枝杆菌(H37Rv)全基因组完成图的获得[1],到近年来采用新一代测序技术对多个菌株进行高通量的基因组测序与分析,对结核分枝杆菌进化及耐药机制的认识不断深入。关于结核分枝杆菌菌株的基因组比较分析有多篇报道,包括对中国12个省来源的161株结核分枝杆菌     

以蛋白激酶B为靶点的新型抗结核药物高通量筛选模型的建立和应用

【目的】建立以结核分枝杆菌蛋白激酶B为靶点的高通量筛选模型,并运用此模型进行化合物的筛选。【方法】克隆和表达结核分枝杆菌蛋白激酶B,并以其为靶酶建立并优化PknB抑制剂高通量筛选模型,利用该模型对化合物样品进行筛选,并对筛选到的阳性化合物进行抗菌和抑酶活性评价。【结果】利用该模型筛选了化合物样品18 000个,得到具有抑酶活性的阳性化合物8个,其中3个化合物具有较好的对结核分枝杆菌、海分枝杆菌、耻垢分枝杆菌的抑菌活性。【结论】建立的以PknB为靶点的抗结核药物高通量筛选模型具有灵敏度高、稳定性强等优点,可成功用于化合物的高效筛选。筛选得到3个在抑酶水平和抗菌方面均具有良好活性的阳性化合物样品,值得进一步研究。     

Factores que afectan la cobertura del programa de tuberculosis en el primer nivel de atención en Honduras

Introduction:

There is a global consensus that early diagnosis and treatment of tuberculosis (TB) can accelerate its control and mitigate its consequences. The gradual increase in the TB mortality rate from 2014 to 2018 in Honduras, the reform of the health system in 2014, and the partial implementation of the “End TB” strategy motivated this study.

Objective:

To analyze barriers to and facilitators of diagnosis and treatment affecting the national TB program coverage using data from 2015 to 2019 and provide tools for the effective implementation of the “End TB” strategy in San Pedro Sula, Honduras.

Materials and methods:

This was an explanatory sequential mixed-methods study on smear-positive pulmonary TB patients older than 18 years of age. TB notification sheets and medical records from two primary health care facilities were reviewed. Semistructured interviews were conducted with health care providers, patients, and their families.

Results:

A total of 74.6% of the cases (297/398) did not receive a timely diagnosis; 62.3% (185/297) were men, 80.8% (240/297) were adults, 53.7% (108/297) had less than high school education, 49.2% (123/297) had some occupation, and 98.2% of participants received timely treatment. Identified barriers included low socioeconomic conditions, lack of coordination between public and private health systems, and boundaries set by gangs. Identified facilitators included good care and attitude of the health care personnel and the availability of medications.

Conclusions:

The lack of opportunity to diagnose the disease affected the coverage of the national TB program due to cultural and health care barriers.     

结核分枝杆菌CFP10和ESAT6对巨噬细胞RAW264.7凋亡及AIM2/ASC/Caspase-8通路的影响

【背景】10 kD培养滤液蛋白(culture filtrate protein 10,CFP10)和6 kD早期分泌性抗原靶蛋白(early secretary antigenic target-6 kD,ESAT6)是结核分枝杆菌(Mycobacterium tuberculosis,Mtb)重要的毒力因子,能引起巨噬细胞的凋亡。【目的】探讨CFP10和ESAT6对巨噬细胞RAW264.7凋亡及AIM2/ASC/Caspase-8信号通路的影响。【方法】利用大肠杆菌表达并纯化获得了CFP10和ESAT6蛋白,重组蛋白处理巨噬细胞RAW264.7后,利用CCK8试剂盒检测细胞存活率,确定重组蛋白处理细胞浓度,利用Western blotting技术检测细胞凋亡相关蛋白及AIM2和ASC炎性小体的变化,利用流式细胞术检测细胞凋亡率。【结果】SDS-PAGE和Westernblotting结果表明重组蛋白CFP10和ESAT6表达正确,不同浓度的CFP10和ESAT6处理RAW264.7后,对细胞的增殖能力具有明显的抑制作用,当CFP10和ESAT6单独处理且浓度为5μg/mL时,细胞存...     

2005～2008年广东省诺如病毒胃肠炎暴发的分子流行病学特征

为研究广东省诺如病毒胃肠炎暴发疫情的分子流行病学特点,我们采集了2005～2008年期间24起急性暴发性胃肠炎患者的粪便和肛拭子标本,使用诺如病毒特异性引物,通过逆转录-聚合酶链反应(RT-PCR)技术进行检测,再经核苷酸序列测定以分析诺如病毒的基因型,同时收集急性暴发性胃肠炎患者的相关流行病学资料。结果显示:24起急性暴发性胃肠炎中19起由诺如病毒引起,时间主要集中在每年的10月至次年2月,2005年病毒胃肠炎暴发疫情是由GⅡ-3基因型病毒引起,主要为幼儿园和小学,发生地主要在内陆山区,2006年秋季疫情以后则均为GⅡ-4型的变异株2006b引起,主要为大学和社区,2007年疫情数比其他年份高1倍,发生地遍及广东全省。广东省诺如病毒变异株2006b在个别特殊的基因位点呈现出高度的地域一致性。随着诺如病毒流行株的基因型由GⅡ-3变为GⅡ-4型,广东省诺如病毒流行的强度大大增加,新出现诺如病毒变异株2006b引起的暴发波及地方多,涉及人群从低幼儿童为主扩展到全年龄组,表明GⅡ-4新变异株比其它毒株具有更高的侵袭力。     

Killing activity and rescue function of genome-wide toxin–antitoxin loci of Mycobacterium tuberculosis

Toxin–antitoxin (TA) loci are typically two-component systems that encode a stable toxin, which binds an essential host target leading to cell growth arrest and/or cell death, and an unstable antitoxin, which prevents the cytotoxic activity of the toxin. The ubiquitous presence of these loci in bacterial genomes, along with their demonstrated toxicity not only in the native but also in heterologous systems, has provided the possibility of their use in wide-spectrum antibacterials. Mycobacterium tuberculosis contains nearly 40 TA loci, most of which are yet to be characterized. Here we report the heterologous toxicity of these TA loci in Escherichia coli and show that only a few of the M. tuberculosis -encoded toxins can inhibit E. coli growth and have a killing effect. This killing effect can be suppressed by coexpression of the cognate antitoxin. This work has identified functional TA pairs for sequences that are presently unannotated in the mycobacterial genome. These toxins need to be further tested for their activity in the native host and other organism backgrounds and growth environments for utilization of their antibacterial potential.     

斑马鱼-海分枝杆菌模型研究对结核病致病机理的启示

全世界约三分之一的人口感染过结核分枝杆菌,其导致的结核病仍然是全球公共卫生的严重威胁。结核菌是典型的胞内致病菌。结核菌的致病性与其成功逃避和利用宿主免疫应答等密切相关。控制结核病需要深入了解致病菌和宿主之间的相互作用。不同的动物模型是揭示致病菌-宿主相互作用的关键。海分枝杆菌-斑马鱼模型是最近才得以发展并获得了不少新见解的研究系统之一。本文总结了该模型揭示的海分枝杆菌毒力因子Erp、Esx-1、pmiA、Mel1和Mel2、KasB等,以及该模型的优缺点。这些结果为大动物模型研究和深入了解结核分枝杆菌感染人体的致病机理提供了线索。     

Structural studies of shikimate 5-dehydrogenase from Mycobacterium tuberculosis

Tuberculosis (TB) remains the leading cause of mortality due to a single bacterial pathogen, Mycobacterium tuberculosis. The reemergence of TB as a potential public health threat, the high susceptibility of human immunodeficiency virus-infected persons to the disease, the proliferation of multi-drug-resistant strains (MDR-TB) and, more recently, of extensively drug resistant isolates (XDR-TB) have created a need for the development of new antimycobacterial agents. Amongst the several proteins and/or enzymes to be studied as potential targets to develop novel drugs against M. tuberculosis, the enzymes of the shikimate pathway are attractive targets because they are essential in algae, higher plants, bacteria, and fungi, but absent from mammals. The mycobacterial shikimate pathway leads to the biosynthesis of chorismate, which is a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Here we report the structural studies by homology modeling and circular dichroism spectroscopy of the shikimate dehydrogenase from M. tuberculosis (MtSDH), which catalyses the fourth step of the shikimate pathway. Our structural models show that the MtSDH has similar structure to other shikimate dehydrogenase structures previously reported either in presence or absence of NADP, despite the low amino acid sequence identity. The circular dichroism spectra corroborate the secondary structure content observed in the MtSDH models developed. The enzyme was stable up to 50 degrees C presenting a cooperative unfolding profile with the midpoint of the unfolding temperature value of approximately 63-64 degrees C, as observed in the unfolding experiment followed by circular dichroism. Our MtSDH structural models and circular dichroism data showed small conformational changes induced by NADP binding. We hope that the data presented here will assist the rational design of antitubercular agents.     

Immune Responses and Protective Efficacy Induced by 85B Antigen and Early Secreted Antigenic Target-6 kDa Antigen Fusion Protein Secreted by Recombinant Bacille Calmette-Guérin

In an attempt to improve immune responses and protective efficacy, we constructed two recombinant bacille Calmette-Guérin （rBCG） strains expressing an 85B antigen （Ag85B） and early secreted antigenic target-6 kDa antigen （ESAT6） of Mycobacterium tuberculosis （MTB） fusion protein. Both rBCG strains have the same protein insertion but in a different order （Ag85B-ESAT6 and ESAT6-Ag85B）. The cultured supernatant of rBCG strains and the sera from the mice immunized with the fusion protein Ag85B- ESAT6 or ESAT6-Ag85B formed a band with a fraction size of 37 kDa, equalivalent to the sum of Ag85B and ESAT6. Six weeks after BALB/c mice were immunized with BCG or rBCG, spleen lymphocytes showed significant proliferation in response to culture filtrate protein of MTB. Compared with the BCG group, mice vaccinated with rBCG elicited a high level increase of immunoglobulin G antibodies to culture filtrate protein in the serum. The T-interferon levels in the lymphocyte culture medium supernatants increased remarkably in the rBCG1 group, significantly higher than that of the BCG immunized group （P〈0.05）. Four weeks after vaccination, mice were infected with M. tuberculosis H37Rv and a dramatic reduction in the numbers of MTB colony forming units in the spleens and lungs was observed in the two rBCG immunization groups. Although these rBCG strains were more immunogenic, their protective effect was comparable to the classical BCG strain, and there were no significant differences between two rBCG groups （p〉0.05）.     

结核分枝杆菌ESX-1分泌蛋白ESAT-6增强巨噬细胞吞噬功能

【目的】研究结核分枝杆菌(Mycobacterium tuberculosis)分泌蛋白ESAT-6(early secreted antigenictarget of 6 kDa)对巨噬细胞吞噬功能的影响。【方法】用重组质粒pFLAG-ESAT-6和pFLAG-EGFP转染RAW264.7细胞,经G418筛选,PCR、RT-PCR和Western blot鉴定,获得稳定表达flag-ESAT-6和flag-EGFP的RAW细胞系,然后用流式细胞术观察各稳转细胞系吞噬荧光微球的能力,并用共聚焦显微镜和菌落计数法检测稳转细胞系吞噬大肠杆菌(Escherichia coli)的能力。【结果】获得了稳定表达flag-ESAT-6的RAW-E6细胞系和表达flag-EGFP的RAW-EGFP细胞系;流式细胞术检测结果表明RAW-E6吞噬荧光微球的能力显著强于野生型细胞系RAW264.7和对照细胞系RAW-EGFP;菌落计数和激光共聚焦分析表明RAW-E6细胞系吞噬E.coli的能力也显著强于RAW264.7和RAW-EGFP。【结论】通过胞内表达发现结核分枝杆菌分泌蛋白ESAT-6能够增强巨噬细胞的吞噬功能,这将为深入理解结核分枝杆菌的致病机制提供新的思路。