【作者】 龙泉鑫；

【导师】 谢建平； 赵西林；

【作者基本信息】 西南大学 ， 微生物学， 2012， 博士

【摘要】 从科赫(Robert Koch)1882年确定结核分枝杆菌(Mycobacterium tuberculosis)是结核病的致病菌以来,全球结核病疫情似乎只是得到短暂的遏制。世界卫生组织预测2011年全球结核病新发病例为980万,创历年新高。严峻的疫情再次凸显了抗结核治疗手段的匮乏。尽管进行了长达半个世纪的化学治疗,世界上仍有三分之一的人口为结核分枝杆菌潜伏感染人群。此外,潜伏感染结核菌的重新激活对于其他疾病也是一个高风险因素,尤其是HIV病人以及糖尿病病人(进行抗肿瘤坏死因子治疗的病人)等免疫缺陷的个体[2]。多耐药以及广泛耐药结核病(Multi and extensively drug resistant tuberculosis, MDR-TB and XDR TB)的出现使结核病的疫情火上浇油,也使几十年以来一直延续的抗结核疗法更加捉襟见肘。一线抗结核药物包括异烟肼(Isoniazid, INH)、利福平(Rifampicin, RIF)、比嗪酰胺(Pyrazinamide, PZA)以及乙胺丁醇(Ethambutol, EMB)。多耐药结核菌指至少同时对异烟肼和利福平耐药的结核分枝杆菌。对MDR-TB的治疗主要依靠二线抗结核药物,主要有氨基糖苷类、多肽类、氟喹诺酮类等。氟喹诺酮药物因其低毒副作用以及充足的衍生物库而尤为受到重视。新一代氟喹诺酮药物莫西沙星和加替沙星与一线抗结核药物联用缩短结核病治疗时长的Ⅲ期临床试验也在进行中,这进一步提示氟喹诺酮药物在抗击结核病中的重要作用。氟喹诺酮药物主要靶向细菌的DNA旋转酶和拓扑异构酶Ⅳ。结核分枝杆菌中只有DNA旋转酶,该酶为氟喹诺酮药物的唯一靶点。结核分枝杆菌耐受氟喹诺酮药物主要也是因为DNA旋转酶相关区域的突变造成的。近年来,不同国家或地区的结核分枝杆菌耐受氟喹诺酮药物的分子特征被陆续报道,但这些研究所采用的样本量小,具有明显的地域局限性。为了解中国大陆地区的氟喹诺酮耐受的结核分枝杆菌的分子机理,我们收集了来自中国大陆各地的氟喹诺酮耐受的结核分枝杆菌临床菌株,对氟喹诺酮耐受决定区进行扩展测序,发现了若干新氟喹诺酮耐受相关位点和突变类型(GyrB:Glu498及Gly551, GyrA:Asp94Asn-Gly112His, GyrB:Thr539Asn-Gly551Arg),可以为结核分枝杆菌药敏芯片提供设计依据；采用数学方法对不同突变类型进行评价,确定了结核分枝杆菌氟喹诺酮耐受决定区中不同突变位点或者突变类型对耐药性的贡献,得到的结果提示该区域的双重突变与高水平药物耐受的相关性,提醒对氟喹诺酮药物的合理使用尤为重要。结合得到的氟喹诺酮药物的耐药位点,我们优化了氟喹诺酮药物作用于结核分枝杆菌DNA旋转酶结构学特征,为新型氟喹诺酮药物的开发提供支持。本研究中第一次对氧氟沙星和左氧氟沙星对多耐药结核分枝杆菌的杀伤效果进行了评价。到目前为止,该研究是样本量最大(177例),地域来源(中国大陆)最为广泛的结核分枝杆菌耐受喹诺酮药物分子特征的调查。在细菌自身进化史中,会发展一系列的抵御外界环境压力的武器,这类环境压力也包括抗生素压力。细菌抵御抗生素的机制包括药物代谢相关酶、药物外排系统以及靶点修饰相关基因等。目前开发新的抗痨药物进展缓慢,故我们尝试寻找分枝杆菌属细菌抵御氟喹诺酮药物的本底机制,将相关基因作为潜在的氟喹诺酮药物增效剂靶点。通过构建分枝杆菌突变库,筛选获得了对氟喹诺酮药物——莫西沙星超敏感的突变菌株,完成插入位点的鉴定以及功能验证,确定了若干分枝杆菌抵御氟喹诺酮药物基因。所获得的基因可以作为氟喹诺酮药物增效剂开发靶点,筛选获得的化合物可以与氟喹诺酮药物联用,提高氟喹诺酮药物使用效率,延长药物使用寿命。虽然喹诺酮药物靶点明确,不同喹诺酮药物杀伤细菌的特征迥异。主要以喹诺酮药物的杀菌作用是否受蛋白合成抑制剂氯霉素所抑制以及杀菌作用是否在低氧条件下消失来区分。不同喹诺酮药物在氯霉素和厌氧条件下的杀伤效果显著的不同。第一代化合物,比如萘啶酸(nalidixic)和奥索利酸(oxolinic acids)在氯霉素处理或者低氧条件下无致死活性,而第二代药物比如诺氟沙星(norfloxacin)虽在氯霉素存在情况下无杀伤活性,但其在高浓度下,却可以在低氧条件下杀伤细菌。第三代喹诺酮药物环丙沙星(ciprofloxacin),可以在氯霉素存在或者低氧条件下杀伤细菌(低氧条件下的杀菌作用需要高浓度药物)；第四代C-8-甲氧基衍生物莫西沙星(moxifloxacin)的杀伤作用几乎不受氯霉素或者低氧条件的影响。这说明不同喹诺酮药物通过不同的途径完成对细菌的杀伤作用。前期研究证实喹诺酮的致死效应分两步进行,第一阶段是DNA-解旋酶-喹诺酮药物形成断裂复合体,该复合体的形成会阻止细菌DNA的复制,导致次级杀伤作用的出现；第二阶段是断裂DNA从断裂复合体中释放出来,形成大量的染色体碎片,导致细菌快速死亡快速杀伤性的氟喹诺酮药物比如莫西沙星的杀菌作用,可能是通过迅速破坏断裂复合体来实现的。研究者猜测细菌本身一些分子可能作为“自杀分子”参与破坏染色体复合体的功能,释放出断裂DNA,导致细菌快速死亡。我们通过实验首次证实：使用氯霉素阻止蛋白合成依赖性杀菌通路后,耻垢分枝杆菌RuvAB缺失菌株,可以明显的抵御莫西沙星的杀菌作用,这直接证明了RuvAB在非依赖蛋白质合成杀菌途径中的重要作用。在实验室直接使用结核分枝杆菌进行实验存在诸多的困难。首先,结核分枝杆菌菌属于3类人体致病菌,需要在3级生物安全实验室及相应动物设施中进行实验；其次,结核分枝杆菌生长缓慢,在液体培养基中倍增时间长达22小时,菌落的形成需要2-3个星期,导致实验时间花费巨大。故很多实验室采用其他一些分枝杆菌模型来研究结核分枝杆菌的致病机制。耻垢分枝杆菌(Mycobacterium smegmatis和结核病的致病菌——结核分枝杆菌(Mycobactreium tuberculosis)同为分枝杆菌属细菌,其生长快,无生物危害性,被广泛的用于结核分枝杆菌致病机制、结核菌疫苗、结核菌药物等方面的替代替代研究。为了更好的理解耻垢分枝杆菌模型在结核分枝杆菌替代研究中的替代性,我们使用系列的比较基因组学手段对耻垢分枝杆菌模型基因组进行了分析,发现耻垢分枝杆菌和结核分枝杆菌具有保守的转录机制、类似的sigma factors和双组分系统,可以使用耻垢分枝杆菌进行调控机制的早期研究；我们还发现耻垢分枝杆菌基因组中所富集的基因类目与其特别的生理特征相适应,比如其生存的腐生的环境、耐受高盐浓度及其相对短暂的代时。更多还原

【Abstract】 Since Robert Koch identified Mycobacterium tuberculosis as the causative agent of TB in1882, a breakthrough in the fighting against this deadly pathogen, the global Tb epidemic seems unmitigated substantially. About a record9.8million new cases were noted in2011. This serious situation highlights the emergency of the newly and effective treatment strategies for TB. Although the anti-TB chemotherapy had lasted for half a century, one-third of the world’s population still asymptomatically harbors a dormant or latent form of M. tuberculosis, with a life long risk of disease reactivation. The reactivation of latent TB represents high risk for other abnormalities, especially for the patient infected with human immunodeficiency viruses, or with diabetes and other disease need anti-tumour necrosis factor therapy. In recently years, the TB epidemic has been further fuelled by the multi and extensively drug resistant tuberculosis (MDR-TB and XDR TB), the classical anti-TB therapy is also undergoing acid test.The first-line anti-tuberculous drug including isoniazid(INH, H), rifampicin(RIF, R), pyramiznamide(PZA, Z) and ethambutol (EMB, E). The MDR-TB is defined as tuberculosis that is resistant at least to isoniazid (INH) and rifampicin (RIF), the two most powerful first-line anti-TB drugs. The treatment of MDR-TB largely relies on the second line anti-TB drugs, including aminoglycosides, polypeptides, thioamides and fluoroquinolones. The fluoroquinolones have bactericidal activity against M.tuberculosis, excellent oral bioavailability, favorable safety profiles, it’s an important second line anti-TB drug. Data from phase Ⅱ trials of fluoroquinolones-containing regimens for shortening the duration of treatment for pulmonary TB are encouraging and phase Ⅲ trials are currently underway, also highlighted the importance of fluoroquinolones in anti-TB therapy.The target of fluoroquinolones in bacteria is DNA gyrase and topoisomerase Ⅳ. M.tuberculosis is unusual in possessing only one type Ⅱ topoisomerase, DNA gyrase. The amino acid substitutions within the quinolone resistance-determining region(QRDR) of GyrAB can dramatically reduce susceptibility to fluoroquinolones. Although a variety of mutation types have been reported in several countries and regions, epidemiologic information on fluoroquinolone-resistant M. tuberculosis in China’s mainland remains obscure. To investigate the mutation types of the genes encoding the A and B subunits of DNA gyrase (gyrA and gyrB, respectively) in ofloxacin-and levofloxacin-resistant M.tuberculosis strains prevalent in China’s mainland,177clinical drug-resistant isolates collected by the National Tuberculosis Reference Laboratory of China were analysed. The GyrB single mutations (Glu498and Gly551) and double mutation (Thr539Asn-Gly551Arg) as well as a GyrA double mutation (Asp94Asn-Gly112His) were reported to be involved in fluoroquinolone resistance for the first time. To simplify quantification of the contribution of each mutation type and mutation site to overall fluoroquinolone resistance, a mathematical method was established by assigning each resistance allele a numerical score between5and50(the larger the number, the higher the resistance level). The score of double mutants is the sum of the scores for the two single alleles. The double mutation types, including Asn538Ile(GyrB)-Asp94Ala(GyrA), Ala543Val(GyrB)-Asp94Asn(GyrA) and Ala543Val(GyrB)-Asp94Gly(GyrA) scored relatively high by this methodology, serve as a cautionary tale that it is imperative to use fluoroquinolones rationally.During millions of years of evolution, bacteria must have encountered a variety of stressful environment, including exposure to antibiotics. Thus, it is not surprising if bacteria have acquired genetic and biochemical systems that protect cells from antimicrobial lethality. Bacterial genes defining intrinsic resistance to antibiotics encode proteins that can be targeted by antibiotic potentiators. Fluoroquinolones is a type of second line anti-tuberculosis drugs, have been widely used in tuberculosis therapy. To find the intrinsic resistance related genes, a transposon inerstion library of Mycoabcterium.smegmatis was screened with subinhibitory concentration of moxifloxacin to find supersusceptible mutants. Transposon insertions3genes were found to cause at least twofold to8folds to moxifloxacin. These included mutants with disruption of genes encoding proteins involved in DNA damage repair (RuvA, Rv2593), FHA proteint(GarA, Rv1827) as well as a proteasome accessory factor (pafC,Rv2095). Electroproration of recombinant plasmids Palace+Rv2592-Rv2593, Palace+Rv1827to M136and M625respectively, partly reverse the fluoroquinolones resistance. The complement results help validate RuvAB as a potential antibiotic combination target for Mycobacterium, other hypersensitive phenotype related genes need further validation.Although the target of quinolones is clear, the killing pathways of different quinolones are strikingly different, distinguished by whether bactercidal effect are effected by the protein synthesis inhibitor(chloramphenicol) or the hypoxic condition. First-generation compounds, such as nalidixic and oxolinic acids, are not lethal in the presence of chloramphenicol or during anaerobic growth; the second-generation agent norfloxacin fails to kill bacterium in the presence of chloramphenicol, but at high concentrations it kills cells growing anaerobically. Ciprofloxacin, a third-generation compound, kills under both conditions but requires higher concentrations during anaerobiosis; the lethal activity of fourth generation C-8-methoxy derivatives, such as moxifloxacin, is affected little by chloramphenicol or anaerobic growth. The early experiment validated the bactericidal process of quinolone is a two steps characteristic. Step1:formed Quinolone-Topoisomerase-DNA complexes, rapid inhibition of DNA replication, bring secondary damage that might contribute to slowly quinolone-mediated cell death; Step2:broken DNA released from Quinolone-Topoisomerase-DNA complex, chromosome fragmentation that contribute to rapid cell death. Researchers are doubt there presence a sucide factor in step2that lead the cleaved complex collapsed. In our experiment, RuvAB mutant strain showed an obvious higher survival rate after moxifloxacin treatment when the protein synthesis depend killing pathway was blocked, suggested the helicase RuvAB is an important factors for protein independent killing pathway of quinolones.The high biosafety level III required to tackle its causative agent Mycobacterium tuberculosis seriously hinders the exploration of its biology and new countermeasures. M. smegmatis is a widely recognized good surrogate of M.tuberculosis, due to their conserved transcriptional machinery, sigma factors and two-component systems. However, their distinct lifestyles often confound the explanation of the results. M.tuberculosis leads both parasitic and free life, while M.smegmatis is largely saprophyte. To make full advantage of this model, it is helpful to discover the genome features associated with M. smegmatis unique niches, such as its saprophytic life, high salt tolerance and relative short generation time. We employed the gene ontology enrichment analysis to characterize the unique lifestyle of M.smegmatis. Gene ontology enrichment analysis provided24terms, most are relevant to the special lifestyle of M. smegmatis, especially the saprophytic niche, high salt tolerance adaptation and short generation time. In-depth functional characterization of these genes will shed new lights on the genetic basis of M.smegmatis saprophytic life and hasten the understanding of the unique biology of M.tuberculosis.更多还原