耻垢分枝杆菌EF-G敲低菌株的构建和耐药分析北大核心CSCD

GTPase延伸因子G(elongation factor G,EF-G)是蛋白质翻译过程中重要的翻译因子。作为唯一在翻译延伸和核糖体再生2个翻译环节发挥重要功能的翻译因子,EF-G成为潜在的抗菌药物作用靶点。耻垢分枝杆菌(Mycobacterium smegmetics,Msm)和结核分枝杆菌(Mycobacterium tuberculosis,Mtb)基因组中均存在2个EF-G同源编码基因,分别为Msm EFG1(MSMEG_1400)和Msm EFG2(MSMEG_6535),fusA1(Rv0684)和fusA2(Rv0120c)。基因突变库和生物信息学推测Msm EFG1(MSMEG_1400)和fusA1(Rv0684)是生长必需基因。为探究分枝杆菌中EF-G的生物学功能及特点,利用成簇的规律间隔的短回文重复序列干扰(clustered regularly interspaced short palindromic repeats interference,CRISPRi)技术构建了耻垢分枝杆菌中2个EF-G诱导型敲低菌株(Msm-ΔEFG1(KD)和Msm-ΔEFG2(KD)),研究发现EF-G2的敲低对细菌生长无影响,而EF-G1的敲低显著影响分枝杆菌的生长,成膜能力显著减弱、菌落形态显著变化、菌体长度显著增长,推测EF-G可能与细菌的分裂相关。最低抑菌浓度(minimal inhibitory concentration,MIC)实验结果表明,抑制EF-G1的表达可增强分枝杆菌对利福平、异烟肼、红霉素、夫西地酸、卷曲霉素等抗菌药物的敏感性,提示EF-G1可能成为未来抗结核药物筛选的潜在靶标,为探究EF-G在分枝杆菌中的生理功能及作为潜在药物靶标提供基础。     

耻垢分枝杆菌在感染与免疫研究中的应用

耻垢分枝杆菌属革兰阳性腐生菌,具有快速生长,无致病性,与结核分枝杆菌基因高度同源、细胞结构相似等特点,较多应用于分枝杆菌感染及相关免疫学研究,是一种相对理想的实验模型。同时,其在非分枝杆菌感染及其他相关免疫研究中也有拓展性的应用。本文就耻垢分枝杆菌在感染与免疫研究中的应用现状进行综述。     

耻垢分枝杆菌ClpC和ClpX敲低表达菌株的构建及表型分析*

蛋白质平衡稳定对细菌的生长繁殖以及应对宿主免疫压力十分重要。Clp蛋白酶复合体在结核分枝杆菌的蛋白质降解和平衡稳定中发挥重要作用。Clp蛋白酶中负责识别底物蛋白并将其解折叠的蛋白质有两种:ClpC和ClpX。为初步探究分枝杆菌中ClpC和ClpX各自的功能特点,运用CRISPRi的方法成功构建了耻垢分枝杆菌的ClpC和ClpX诱导型敲低表达菌株,并对其生长相关表型进行分析。结果显示:与野生菌株相比,ClpC和ClpX的低表达均能严重影响耻垢分枝杆菌的生长。ClpC低表达可导致菌株丧失生物膜的形成能力,而ClpX低表达则导致菌株无法维持正常细胞形态,电镜显示细胞壁不完整且细胞呈丝状化,提示ClpC和ClpX可能在分枝杆菌中具有不同的生理功能。可为后期深入开展ClpC和ClpX对分枝杆菌生理调控功能研究及新型抗结核药物筛选提供基础。     

CRISPR/Cas9基因编辑技术在致瘤病毒感染研究中的应用

成簇的规律间隔的短回文重复序列及其相关蛋白9〔clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9),CRISPR/Cas9〕基因编辑技术的发现源于真细菌和古细菌中CRISPR/Cas系统介导的适应性免疫机制研究。该技术利用特异性向导RNA识别靶点基因,引导核酸内切酶Cas9对其切割,并通过同源重组或非同源末端连接完成对目的DNA的编辑。某些病毒感染机体后,可将其基因组整合到宿主细胞基因组中或潜伏于组织中而无法被彻底清除,从而引起持续性感染。本文参考2013年以来CRISPR/Cas9基因组编辑技术的最新相关研究报道,重点综述其在人类免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)、人乳头瘤病毒(human papillomavirus,HPV )、乙型肝炎病毒(hepatitis B virus, HBV)、 Epstein-Barr病毒(Epstein-Barr virus,EBV)等致瘤病毒感染相关疾病研究中的应用,并概括其作用于这些病毒的有效靶点。     

成簇的规律间隔的短回文重复序列CRISPR 的研究进展

最近发现,在细菌和古菌中广泛存在的成簇的规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)及其相关蛋白是针对噬菌体、质粒等外源DNA的获得性和可遗传的免疫系统。本文综述了CRISPR系统的基本结构、多样性、作用机理及其区分自我与非我的机制,并对CRISPR研究和应用前景进行了展望。     

结核杆菌eis基因的克隆及其在耻垢分枝杆菌中的表达

目的:构建结核分枝杆菌eis基因的穿梭表达载体,鉴定其在重组耻垢分枝杆菌中的生物活性。方法:采用PCR技术克隆结核分枝杆菌eis基因,构建大肠杆菌-分枝杆菌穿梭表达载体pMV-eis,经酶切和测序鉴定其正确性,用电穿孔法将重组质粒转化至耻垢分枝杆菌mc²155中,采用SDS-PAGE和Western blot检测eis基因在耻垢分枝杆菌中的表达。结果:成功构建结核杆菌eis基因穿梭表达载体pMV-eis;生长曲线说明重组质粒不会影响耻垢分枝杆菌的体外生长;SDS-PAGE 和Western blot检测证实eis在耻垢分枝杆菌中可表达出相对分子量约42kDa的Eis蛋白。结论:成功构建了eis基因穿梭表达质粒pMV-eis,且该重组质粒在耻垢分枝杆菌中具有生物活性,为下一步研究表达产物Eis的功能奠定了一定基础。     

CRISPR在细菌分型和进化中的研究进展

规律成簇的间隔的短回文重复序列（CRISPR）是近年发现的一类存在于古细菌和细菌基因组内的结构,该结构可以使细菌获得对外源DNA如质粒和噬菌体的免疫,同时由于其结构的多态性,也可作为细菌分型和进化研究的位点。简要综述了CRSIPR系统的基本结构,及其在分型和进化应用方面的研究进展。     

肿瘤坏死因子α突变型斑马鱼在海分枝杆菌感染中的应用研究

肿瘤坏死因子α(tumor necrosis factorα,TNF-α)作为重要的炎性细胞因子,在类风湿关节炎、感染性休克等疾病的发病中起重要作用。为探究TNF-α在宿主抵抗海分枝杆菌感染中的作用,本研究采用成簇的规律间隔的短回文重复序列及其相关蛋白9[clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein 9(Cas9),CRISPR/Cas9]技术,使野生型斑马鱼TNF-α基因发生突变,成功构建并筛选出含有移码突变的TNF-α突变型(TNF-α-/-)斑马鱼。利用原位杂交技术,发现TNF-α-/-斑马鱼中TNF-αmRNA表达水平显著降低。与野生型斑马鱼相比,海分枝杆菌在TNF-α-/-斑马鱼体内扩散和增殖更显著。结果提示,本研究成功构建TNF-α-/-斑马鱼,有助于深入探究TNF-α在斑马鱼抵抗海分枝杆菌感染中的作用及其免疫调节机制。     

规律成簇的间隔短回文重复：结构，功能与应用

规律成簇的间隔短回文重复(Clustered regularly interspaced short palindromic repeats,CRISPRs)是一类广泛分布于细菌和古菌基因组中的重复结构.最近研究表明,CRISPR与一系列相关蛋白、前导序列一起,为原核生物提供对噬菌体等外源基因的获得性免疫能力,其作用机制可能与真核生物的RNA干扰过程类似.作为基因组中高度可变的区域,CRISPR非常适合成为研究细菌种内分型和微进化的分子靶标.本文综述了CRISPR系统的结构、功能及其应用概况,并对CRISPR研究的前景进行了展望.     

Molecular and biochemical characterisation of Mycobacterium smegmatis alcohol dehydrogenase C

The gene encoding of an alcohol dehydrogenase C (ADHC) from Mycobacterium smegmatis was cloned and sequenced. The protein encoded by this gene has 78% identity with Mycobacterium tuberculosis and Mycobacterium bovis BCG ADHC. The M. smegmatis ADHC was purified from M. smegmatis and the kinetic parameters of this enzyme showed that using NADPH as electron donor it has a strong preference for aliphatic and aromatic aldehyde substrates. Like the M. bovis BCG ADHC, this enzyme is more likely to act as an aldehyde reductase than as an alcohol dehydrogenase. The discovery of such an ADHC in a fast-growing, and easily engineered mycobacterial species opens the way to the utilisation of this M. smegmatis enzyme as a convenient model for the study of the physiological role of this alcohol dehydrogenase in mycobacteria.     

Increased alanine dehydrogenase activity during dormancy in Mycobacterium smegmatis

The aerobic fast-growing Mycobacterium smegmatis has, like its slow-growing pathogenic counterpart M. tuberculosis, the capability to adapt to anaerobiosis by shifting down to a drug resistant dormant state. Here, we report the identification of the first enzyme, l-alanine dehydrogenase, whose specific activity is increased during dormancy development in M. smegmatis. This mycobacterial enzyme activity was previously identified as the 40-kDa antigen in M. tuberculosis and shows a preference for the reductive amination of pyruvate to alanine at physiological pH. The determination of the temporal profile of alanine dehydrogenase activity during dormancy development showed that the activity stayed at a low baseline level during the initial aerobic exponential growth phase (0.7 mU mg⁻¹ min⁻¹). After termination of aerobic growth, alanine dehydrogenase activity increased rapidly 5-fold. As oxygen becomes more and more limiting, the enzyme activity declined until it reached a level about two-third that of the peak value. The strong induction immediately after deflection from aerobic growth suggests that alanine might be required for the adaptation from aerobic growth to anaerobic dormancy. As alanine synthesis is coupled to NADH oxidation, we propose that the induction of alanine dehydrogenase activity might also support the maintenance of the NAD pool when oxygen as a terminal electron acceptor becomes limiting.     

葡萄球菌CRISPR/Cas系统

成簇的规律间隔的短回文重复序列及其相关蛋白〔clustered regularly interspaced short palindromic repeat(CRISPR)/ CRISPR-associated protein, CRISPR/Cas〕是原核生物在进化过程中形成的获得性免疫系统,能抵抗噬菌体、质粒及可移动遗传因子等外源性DNA或RNA的入侵。目前,在多种葡萄球菌基因组中均发现CRISPR序列存在,其间隔序列通常与葡萄球菌的噬菌体或接合性质粒具有同源性,可能对葡萄球菌的毒力、耐药性传递和生物膜形成等生理学特性有影响。本文在简单介绍细菌CRISPR/Cas系统的基础上,对葡萄球菌CRISPR/Cas系统的构成、防御机制等进行综述。     

CRISPR/Cas9高通量筛选技术与肿瘤治疗

成簇规律间隔短回文重复(clustered regularly interspaced short palindromic repeats, CRISPR),是细菌或古菌在与噬菌体长期生存进化获得的一种免疫系统. 根据Cas蛋白(CRISPR-associated protein)的不同,CRISPR系统可分为3种. 其中II型CRISPR/Cas9已被改造成为一种有效的基因编辑工具,并运用于多种物种基因的改造. 作为1种基因编辑的手段,CRISPR/Cas9技术通过诱导DNA双链断裂损伤,进一步干扰基因的表达. 与传统的基因编辑技术相比,CRISPR/Cas9技术显示出效率高、成本低和易操作等特点. 与此同时,二代测序技术的发展促进全基因组的解析. CRISPR技术结合高通量二代测序手段的使用,在肿瘤的治疗领域中已发挥出了独特的优势. 本文就近年来CRISPR/Cas9高通量筛选技术的发展,及其在肿瘤治疗过程中的应用进行综述.     

基于CRISPR/Cas生物传感原理的病原菌检测技术研究进展*

病原菌的快速准确检测是实现疫情高效防控、疾病精准治疗、污染环境及时处置的关键。而现有的病原菌现场快速检测技术,主要以定性分析为主,假阳性/假阴性受到诟病,检测准确性仍有待提升,亟待发展基于新原理、新方法的病原菌快速检测技术。基于CRISPR(clustered regularly interspaced short palindromic repeats)的生物传感技术因具有高灵活性(对不同的基因靶点只需改变crRNA序列)、高特异性(单碱基分辨)、高灵敏(优于10^-18 mol/L浓度)、可编程、可模块化、低成本、可在各种体外介质中高效稳定运行等独特优势,打破了传统分子诊断与检测技术的局限性,正在成为下一代病原菌检测技术的引领者。在该技术中,Cas效应蛋白被用作高特异性的序列识别元件,结合不同的生物传感机制,即可用于病原菌的高特异性快速灵敏检测。在总结CRISPR/Cas生物传感技术原理的基础上,综述了用于病原菌检测的CRISPR/Cas12和CRISPR/Cas13生物传感技术研究进展。通过阐述CRISPR/Cas生物传感技术在实际应用中面临的挑战,展望其未来的发展前景。     

Identification of novel metronidazole-inducible genes in Mycobacterium smegmatis using a customized amplification library

The incidence of antibiotic resistance in pathogenic bacteria is rising. Bacterial resistance may be a natural defense of organisms, or it may result from spontaneous mutations or the acquisition of exogenous resistance genes. We grew spontaneous metronidazole-resistant Mycobacterium smegmatis mutants on solid medium cultures and employed differential expression using a customized amplification library to analyze the global gene profiles of metronidazole-resistant mutants under hypoxic conditions. In total, 66 genes involved in metronidazole resistance were identified and functionally characterized using the gene role category of M. smegmatis. Overall, genes associated with cell wall synthesis, such as methyltransferase and glycosyltransferase, and genes encoding drug transporters were highly expressed. The genes may be involved in the natural drug resistance of mycobacteria by increasing mycobacterial cell wall permeability and the efflux pumps of active drugs. In addition, the genes may play a role in dormancy. The genes identified in this study may lead to a better understanding of the mechanisms of metronidazole resistance during dormancy.     

Control of the acetamidase gene of Mycobacterium smegmatis by multiple regulators

The acetamidase of Mycobacterium smegmatis is an inducible enzyme which enables the organism to utilise several amides as sole carbon sources. The acetamidase structural gene (amiE) is located downstream of four other genes, of which three form a probable operon with amiE; the fourth (amiC) is divergently transcribed. We constructed deletion mutants in two of these genes in order to determine their role in acetamidase expression. Both AmiC and AmiD were shown to be positive regulators of acetamidase expression required for induction. Combinations of regulatory gene deletions were made which revealed that AmiC interacts with the previously characterised negative regulator AmiA, whereas AmiD does not.