结核分枝杆菌喹诺酮耐药基因功能研究
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摘要
结核病是由结核分枝杆菌引起的一种世界性传染病,近年来随着人口流动增加、HIV与结核分枝杆菌伴发感染以及结核分枝杆菌多重耐药性菌株的出现等原因,使全球结核病疫情再度回升,其中耐药菌株的出现是治疗失败的主要原因。结核分枝杆菌由于其特殊的细胞壁结构,对多种药物有天然的耐药性,耐药机制复杂。1998年,Cole等发布了结核分枝杆菌H37Rv全基因测序结果,使人们能从基因水平研究结核分枝杆菌的耐药机制。
随着耐多药结核分枝杆菌感染的出现,人们期待更加有效的药物出现,从而把更多的目光投向了二线药物,其中喹诺酮类药物尤其受到青睐。喹诺酮类药物为一类全合成的抗菌制剂,自1962年发现了奈啶酸(Nalidixic acid)以来,已有四代喹诺酮类药物上市,但只有第三代的喹诺酮类抗菌药具有抗分枝杆菌活性。喹诺酮类药物主要作用于结核杆菌DNA促旋酶,该酶由A、B亚单位组成,药物的耐药性主要由于作用靶位的改变,A亚单位突变常常导致高耐药,B亚单位突变常常导致低耐药,而有的低水平的耐药常常缺乏喹诺酮类药物靶位的改变,有时高水平的耐药又不能单一地用靶位的改变来解释,所以还有其他的耐药机制的存在。随着喹诺酮类药物的广泛使用,耐药菌株也日渐显现,严重影响其疗效和临床应用。研究喹诺酮耐药产生机制,就具有重要意义。
本研究利用生物信息学的方法对结核分枝杆菌全基因组进行分析发现,结核分枝杆菌基因组中大约有20种外排蛋白编码基因,其中多数与
Tuberculosis is the world's leading cause of mortality owing to an infectious bacterial agent, Mycobacterium tuberculosis. The HIV/AIDS pandemic has been attributed to the global resurgence of TB, with increased mobility owing to mass movement of population and the emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MDR-TB). The emergence of MDR-TB is one of the major causes of the treatment failure. The complete genome sequence and annotation of Mycobacterium tuberculosis H37Rv was published in 1998.This has provided researchers important information to gain insight into the mechanism of the drug resistance.The emergence of multidrug-resistant strains of Mycobacterium tuberculosis has resulted in an increased interest in the second-line antituberculosis agents, and Quinolones, one class of the synthetic antibacterial agents, are being used in selected tuberculosis patients. Quinolones have possessed the activity of antituberculosis since the third generation. The DNA gyrase composed of two A and two B subunits is the only target of quinolones in mycobacteria. Mutation in the target often confers quinolones resistance. Mutation in a particular region of gyrA,
which encodes the gyrase A subunit, has been associated with a high level of quinolone resistance. Mutations conferring low-level resistance have also been found in gyrB, which encodes the B subunit of the DNA gyrase. However, these mutations do not account for all resistant strains, indicating that other mechanisms have contributed to the resistance in mycobacteria.Notably, most quinolones are only moderately active against mycobacteria, and unfortunately, quinolone-resisitant clinical isolates of Mycobacterium tuberculosis have already appeared. If more can be learned about what determines the effectiveness of a particular quinolone against the mycobacteria and the mechanisms by which the resistance develops, new agents or strategies may be designed to prevent or circumvent this resistance.In this study, using bioinfomatics method to analyze the genome of Mycobacterium tuberculosis, we discovered that the genome of Mycobacterium tuberculosis H37Rv has 20 open reading frames encoding putative efflux proteins. Most of them associate with resistance. Furthermore, Rv2994 gene may be the efflux bump of the quinolones.First, we amplified the gene Rv2994 from the genome of Mycobacterium tuberculosis H37Rv to make an attempt to investigate the function of it. A recombinant fused expression vector pGEX-2994 was constructed. The recombinant plasmid could express the fusion protein GST-Rv2994 stably, thus provided the basis for the further study of the gene Rv2994.Then, a second recombinant plasmid bearing Rv2994 was constructed with the shuttle vector pMV261 and electroporated into avirulent Mycobacterium smegmatis me2155. The transformants were induced to
express a 47KDa protein of Rv2994. The MIC and accumulation of ofloxacin for the strains of mc2155, mc2155-261 and mc2155-2994 are determined with and without the membrane deenergizer carbonyl cyanide m-chlorophenylhydrazone (CCCP) to clarify the Rv2994 gene function.Finally, the study of quinolone-resistant genes in Sichuan Province confirmed a strong correlation between the quinolone-resistance and the mutation of gyrA gene, which might be a major molecular mechanism of quinolone-resistance in Mycobacterium tuberculosis .In summary, the majority of the results in this study support our hypothesis that the protein encoded by Rv2994 gene is a drug efflux bump, conferring resistance to ofloxacin. The fact that the Rv2994 protein could export drugs and confer multidrug-resistance extends a promising field for future works on Mycobacterium tuberculosis.
随着耐多药结核分枝杆菌感染的出现,人们期待更加有效的药物出现,从而把更多的目光投向了二线药物,其中喹诺酮类药物尤其受到青睐。喹诺酮类药物为一类全合成的抗菌制剂,自1962年发现了奈啶酸(Nalidixic acid)以来,已有四代喹诺酮类药物上市,但只有第三代的喹诺酮类抗菌药具有抗分枝杆菌活性。喹诺酮类药物主要作用于结核杆菌DNA促旋酶,该酶由A、B亚单位组成,药物的耐药性主要由于作用靶位的改变,A亚单位突变常常导致高耐药,B亚单位突变常常导致低耐药,而有的低水平的耐药常常缺乏喹诺酮类药物靶位的改变,有时高水平的耐药又不能单一地用靶位的改变来解释,所以还有其他的耐药机制的存在。随着喹诺酮类药物的广泛使用,耐药菌株也日渐显现,严重影响其疗效和临床应用。研究喹诺酮耐药产生机制,就具有重要意义。
本研究利用生物信息学的方法对结核分枝杆菌全基因组进行分析发现,结核分枝杆菌基因组中大约有20种外排蛋白编码基因,其中多数与
Tuberculosis is the world's leading cause of mortality owing to an infectious bacterial agent, Mycobacterium tuberculosis. The HIV/AIDS pandemic has been attributed to the global resurgence of TB, with increased mobility owing to mass movement of population and the emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MDR-TB). The emergence of MDR-TB is one of the major causes of the treatment failure. The complete genome sequence and annotation of Mycobacterium tuberculosis H37Rv was published in 1998.This has provided researchers important information to gain insight into the mechanism of the drug resistance.The emergence of multidrug-resistant strains of Mycobacterium tuberculosis has resulted in an increased interest in the second-line antituberculosis agents, and Quinolones, one class of the synthetic antibacterial agents, are being used in selected tuberculosis patients. Quinolones have possessed the activity of antituberculosis since the third generation. The DNA gyrase composed of two A and two B subunits is the only target of quinolones in mycobacteria. Mutation in the target often confers quinolones resistance. Mutation in a particular region of gyrA,
which encodes the gyrase A subunit, has been associated with a high level of quinolone resistance. Mutations conferring low-level resistance have also been found in gyrB, which encodes the B subunit of the DNA gyrase. However, these mutations do not account for all resistant strains, indicating that other mechanisms have contributed to the resistance in mycobacteria.Notably, most quinolones are only moderately active against mycobacteria, and unfortunately, quinolone-resisitant clinical isolates of Mycobacterium tuberculosis have already appeared. If more can be learned about what determines the effectiveness of a particular quinolone against the mycobacteria and the mechanisms by which the resistance develops, new agents or strategies may be designed to prevent or circumvent this resistance.In this study, using bioinfomatics method to analyze the genome of Mycobacterium tuberculosis, we discovered that the genome of Mycobacterium tuberculosis H37Rv has 20 open reading frames encoding putative efflux proteins. Most of them associate with resistance. Furthermore, Rv2994 gene may be the efflux bump of the quinolones.First, we amplified the gene Rv2994 from the genome of Mycobacterium tuberculosis H37Rv to make an attempt to investigate the function of it. A recombinant fused expression vector pGEX-2994 was constructed. The recombinant plasmid could express the fusion protein GST-Rv2994 stably, thus provided the basis for the further study of the gene Rv2994.Then, a second recombinant plasmid bearing Rv2994 was constructed with the shuttle vector pMV261 and electroporated into avirulent Mycobacterium smegmatis me2155. The transformants were induced to
express a 47KDa protein of Rv2994. The MIC and accumulation of ofloxacin for the strains of mc2155, mc2155-261 and mc2155-2994 are determined with and without the membrane deenergizer carbonyl cyanide m-chlorophenylhydrazone (CCCP) to clarify the Rv2994 gene function.Finally, the study of quinolone-resistant genes in Sichuan Province confirmed a strong correlation between the quinolone-resistance and the mutation of gyrA gene, which might be a major molecular mechanism of quinolone-resistance in Mycobacterium tuberculosis .In summary, the majority of the results in this study support our hypothesis that the protein encoded by Rv2994 gene is a drug efflux bump, conferring resistance to ofloxacin. The fact that the Rv2994 protein could export drugs and confer multidrug-resistance extends a promising field for future works on Mycobacterium tuberculosis.
引文
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6 http://w3.whosea.org/tb/publichealth.htm
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?conformation analysis for rapid detection of gyrA mutations in fluoroquinolone-resistant Mycobacterium tuberculosis clinical isolates. Antimicrob Agents Chemother. 2004 Feb; 48(2):596-601
2 Rodriguez JC, Cebrian L, Ruiz M, et al. Mutant Prevention Concentration of Isoniazid, Rifampicin and Rifabutin against Mycobacterium tuberculosis Chemotherapy. 2005 May 4; 51(2-3):76-79
3 Sharma SK, Mohan A, Kadhiravan T. HIV-TB co-infection: Epidemiology, diagnosis & management. Indian J Med Res. 2005 Apr; 121(4):550-67
4 Pereira M, Tripathy S, Inamdar V, et al. Drug resistance pattern of Mycobacterium tuberculosis in seropositive and seronegative HIV-TB patients in Pune, India. Indian J Med Res. 2005 Apr;121(4):235-9
5 Frieden TR, Sterling TR, Munsiff SS, et al. Tuberculosis. Lancet. 2003 Sep 13; 362(9387):887-99
6 http://w3.whosea.org/tb/publichealth.htm
7 端木宏谨。第四次全国结核病流行病学抽样调查报告。中华结核呼吸杂志,2002:25(1):3-7
8 Cole ST, Brosch R, Parkhill J, et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 1998, 393(6685):537-544
9 Camus JC, Pryor MJ, Medigue C, et al. Re-annotation of the genome sequence of Mycobacterium tuberculosis H37Rv. Microbiology, 2002, 148: 2967-2973
10 Beeching NJ. Tuberculosis into the next century. February 4, 1995, Liverpool School of Tropical Medicine, U.K. [Congresses] Journal of Infection. 1996 Jan, 32(1):67-8
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