CTX-M型杂合酶生化特性及遗传特征研究

发布时间：2018-06-07 12:12

本文选题：超广谱β-内酰胺酶 + CTX-M杂合体　；参考：《华南农业大学》2016年博士论文

【摘要】：近年来,CTX-M型超广谱β-内酰胺酶(ESBLs)的出现给临床治疗带来了极大的挑战,三种CTX-M杂合酶(CTX-M-64、-123和-132)已经在我国动物源大肠杆菌中检测到,它们对大多数头孢菌素类表现出增强的水解活性,其基因序列暗示了这些杂合基因可能是通过bla_(CTX-M-14)和blaCTX-M-15的重组形成。本文旨在比较杂合酶及其母体酶的动力学参数,结合蛋白空间结构分析其催化作用机制,并探索杂合基因的可能形成途径,为CTX-M酶的进一步研究提供科学依据及参考数据。本研究通过PCR方法扩增出杂合酶(CTX-M-132、-123和-64)及其母体酶(CTX-M-55、-15和-14)的编码基因完整序列(876bp)及不含信号肽的序列,并将其插入p ET-28b表达载体。重组质粒转入E.coli BL21(DE3),分别用于MIC值的测定及蛋白表达。在蛋白表达水平一致的情况下,测定携带blaCTX-Ms全长的六株重组菌对各种β-内酰胺类抗生素的MIC值。结果显示,产CTX-M-14的菌株对除了氨苄西林外的所有受试抗生素的MIC值最低,产CTX-M-64、CTX-M-132、CTX-M-123和CTX-M-55的菌株对头孢噻肟和头孢他啶的MIC值高于CTX-M-15,其中CTX-M-64对头孢噻肟的MIC值最高。整体来看,六种blaCTX-Ms基因的耐药性水平从低到高的顺序为:bla_(CTX-M-14)、bla_(CTX-M-15)、bla_(CTX-M-55)、bla_(CTX-M-132)、bla_(CTX-M-123)和bla_(CTX-M-64)。β-内酰胺类抑制剂(克拉维酸、他唑巴坦和舒巴坦)对六种酶都有较好的抑制作用,其中克拉维酸和他唑巴坦的抑制作用较强,与头孢噻肟联用后的MIC比单独使用头孢噻肟时减小高达近70000倍。SDS-PAGE结果显示,六个目的蛋白的大小均约为28k Da,最佳表达条件是IPTG1.0mmol/L,30℃诱导培养5h。通过亲和层析和凝胶过滤层析方法得到纯度大于99%的目的蛋白,用于酶动力学参数的测定。结果显示,这些酶的催化活性(kcat/Km)与MIC结果相似,CTX-M-14对除了头孢噻吩外的所有β-内酰胺类抗生素的催化活性最低,而CTX-M-64对头孢硝基噻吩、头孢呋辛、头孢噻呋、头孢曲松和头孢噻肟的催化活性最高。CTX-M-15对除了氨苄西林外的β-内酰胺类抗生素的催化活性均低于CTX-M-55及三个杂合酶CTX-M-64、-132和-123的催化活性。另外,三种抑制剂对六种CTX-Ms酶的抑制水平均在纳摩级,其中舒巴坦的IC50和Ki值高于克拉维酸,而克拉维酸略高于他唑巴坦。结合酶动力学参数,对六种CTX-Ms酶的氨基酸序列及空间结构进行比较分析。结果发现,CTX-M-55与CTX-M-15相差仅一个氨基酸残基A77V,但CTX-M-55对超广谱头孢菌素类药物表现出相对较强的催化活性。结构分析显示,第77位氨基酸位于活性位点远端,CTX-M-55更可能是通过V77与不同α螺旋上的关键氨基酸形成疏水键,从而稳定蛋白空间结构中螺旋群的核心架构并促成更加稳定的活性部位构象。稳定构象的形成促成了CTX-M-55的较高结构稳定性,进而表现出了较高的催化效率和对温度变化的耐受性。从CTX-M-15、CTX-M-132、CTX-M-123到CTX-M-64的演变是通过向CTX-M-15中逐渐引入活性中心远端残基的过程,并且在演变过程中它们对超广谱头孢菌素类药物的催化活性也在逐渐增强。与CTX-M-14相比,CTX-M-64对头孢菌素类增强的催化活性及对β-内酰胺酶抑制剂增强的敏感性大部分也是由于活性中心远端残基的引入。这些结果表明,活性中心远端的氨基酸残基增强了CTX-M酶对超广谱头孢菌素类药物的催化活性。通过PCR测序的方法,对实验室保存的分离自2010~2013年的大肠杆菌检测blaCTX-M杂合体基因,并对阳性大肠杆菌进行多位点序列分型。通过接合转移或化学转化的方法获得携带杂合酶基因和其可能来源基因bla_(CTX-M-15)和bla_(CTX-M-55)单一质粒的接合子或转化子,S1-PFGE鉴定质粒大小,并对质粒进行复制子分型。对分别携带bla_(CTX-M-15)、bla_(CTX-M-64)、bla_(CTX-M-123)和bla_(CTX-M-132)的四个质粒p HNY2-1、p HNAH46-1、p HNAH4-1和p HNLDH19进行高通量测序并分析其结构。结果发现,携带bla_(CTX-M-15)的Inc I2质粒p HNY2-1、携带bla_(CTX-M-55)的质粒p HN1122-1、携带bla_(CTX-M-64)的质粒p HNAH46-1和携带bla_(CTX-M-132)的质粒p HNLDH19都含有相同的插入序列ISEcp1-blaCTX-M-Inc A/C,并且质粒骨架几乎一致,说明杂合基因可能起源于bla_(CTX-M-15)。携带bla_(CTX-M-123)的质粒p HNAH4-1为Inc I1型(ST108),含有ISEcp1-bla_(CTX-M)-Inc A/C-Inc I2片段,提示ISEcp1介导blaCTX-M-Inc A/C-Inc I2转移到Inc I1型质粒上。采用PCR测序及限制性内切酶片段长度多态性分析(RFLP)等方法进一步研究p HNAH46-1和p HNAH4-1的相似质粒在动物源大肠杆菌中的扩散情况。结果从不同省份、不同农场和不同动物中分别检测出5株携带有与p HNAH46-1相似的Inc I2质粒和9株携带有与p HNAH4-1相似的Inc I1(ST108)质粒,这表明携带杂合基因bla_(CTX-M-64)和bla_(CTX-M-123)的质粒已经在动物源大肠杆菌中广泛传播。
[Abstract]:In recent years, the emergence of CTX-M type broad-spectrum beta lactamase (ESBLs) has brought great challenges to clinical treatment. Three kinds of CTX-M heterozygases (CTX-M-64, -123 and -132) have been detected in our animal source Escherichia coli, which exhibit enhanced hydrolysis activity for most cephalosporins. The gene sequences suggest that these heterozygous genes are available. It can be formed by the recombination of bla_ (CTX-M-14) and blaCTX-M-15. This paper aims to compare the kinetic parameters of heterozygase and its maternal enzyme, analyze its catalytic mechanism by combining the spatial structure of protein, and explore the possible formation pathway of heterozygous gene, and provide scientific basis and reference data for further research of CTX-M enzyme. This study through the PCR method The complete sequence of the encoding gene (876bp) and its maternal enzyme (CTX-M-55, -15 and -14) and its maternal enzyme (CTX-M-55, -15 and -14) were amplified and inserted into the P ET-28b expression vector. The recombinant plasmid was transferred into E.coli BL21 (DE3) and used to determine the value and the protein expression respectively. In the case of the same protein expression level, the recombinant plasmid was used to determine the protein expression. The MIC value of various beta lactam antibiotics was carried by six recombinant strains of blaCTX-Ms full length. The results showed that the MIC value of all the tested antibiotics except ampicillin was the lowest, and the MIC value of the strains producing CTX-M-64, CTX-M-132, CTX-M-123 and CTX-M-55 to cefotaxime and ceftazidime was higher than CTX-M-15, including CTX-M-64. The MIC value of cefotaxime is the highest. Overall, the order of the resistance of the six blaCTX-Ms genes from low to high is bla_ (CTX-M-14), bla_ (CTX-M-15), bla_ (CTX-M-55), bla_ (CTX-M-132), bla_ (CTX-M-123) and the inhibitor of beta lactam (clavulanic acid, tazobactam and sulbactam), which have a better inhibition on the six enzymes. The inhibitory effect of clavulanic acid and tazobactam was stronger. The MIC of cefotaxime compared with cefotaxime was nearly 70000 times higher than that of cefotaxime. The results showed that the size of six target proteins was about 28K Da, the best expression was IPTG1.0mmol/L, and 5h. was induced by affinity chromatography and gel filtration at 30 C. The target protein with purity greater than 99% was obtained by chromatography and used for the determination of enzyme kinetic parameters. The results showed that the catalytic activity of these enzymes (kcat/Km) was similar to that of MIC, and CTX-M-14 had the lowest catalytic activity for all beta lactam antibiotics except cephalothiophene, while CTX-M-64 was the cephalosporin, cefuroxime, ceftif, head, and ceftathiophene. The catalytic activity of cefotaxime and cefotaxime was the highest.CTX-M-15 activity of the beta lactam antibiotics except ampicillin was lower than that of CTX-M-55 and three heterozygous enzymes CTX-M-64, -132 and -123. In addition, the inhibition levels of the three inhibitors on the six CTX-Ms enzymes were at the namo level, and the IC50 and Ki values of the sulbactam were higher than those of the sulbactam. Clavulanic acid, while clavulanic acid was slightly higher than tazobactam. The amino acid sequence and spatial structure of the six CTX-Ms enzymes were compared. The results showed that the difference between CTX-M-55 and CTX-M-15 was only one amino acid residue A77V, but CTX-M-55 showed a relatively strong catalytic activity for the hyper broad-spectrum cephalosporin. The analysis shows that the seventy-seventh bit amino acid is located at the far end of the active site, and CTX-M-55 is more likely to form a key by V77 and the key amino acids on the different alpha helix, thus stabilizing the core structure of the helix group in the spatial structure of the protein and facilitating a more stable conformation of the active site. The formation of stable texture contributes to the higher structural stability of CTX-M-55. The evolution of CTX-M-15, CTX-M-132, CTX-M-123 to CTX-M-64 is through the gradual introduction of the distal residues of the active center to CTX-M-15, and their catalytic activity to the hyper broad-spectrum cephalosporin is gradually enhanced during the evolution. And CTX-M Compared with -14, the catalytic activity of the cephalosporins enhanced by CTX-M-64 and the sensitivity to the enhancement of beta lactamase inhibitors are mostly due to the introduction of the distal residues of the active center. These results suggest that the amino acid residues at the distal end of the active center enhance the catalytic activity of the CTX-M enzyme to the Hyper broad-spectrum cephalosporin. By PCR sequencing Methods the blaCTX-M heterozygous gene was detected from the Escherichia coli isolated from 2010~2013 in the laboratory, and the multi point sequence of the positive Escherichia coli was typed. The zygote of the heterozygous gene and the possible source gene bla_ (CTX-M-15) and the bla_ (CTX-M-55) single plasmid were obtained by conjugation transfer or chemical transformation. The plasmid size was identified by S1-PFGE, and the plasmids were typed. The four plasmid P HNY2-1, which carried bla_ (CTX-M-15), bla_ (CTX-M-64), bla_ (CTX-M-123) and bla_ (CTX-M-132), were sequenced and analyzed by high flux sequencing. 1, plasmid P HN1122-1 carrying bla_ (CTX-M-55), bla_ (CTX-M-64) plasmid P HNAH46-1 and plasmid P HNLDH19 carrying bla_ (CTX-M-132) all contain the same insertion sequence, and the plasmid skeleton is almost identical, indicating that the heterozygous gene may be derived from the plasmid. The NC I1 type (ST108) contains ISEcp1-bla_ (CTX-M) -Inc A/C-Inc I2 fragment, suggesting that ISEcp1 mediated blaCTX-M-Inc A/C-Inc I2 is transferred to the plasmids. Results 5 strains of Inc I2 plasmids similar to P HNAH46-1 were detected in different provinces, different farms and different animals, and 9 strains of Inc I1 (ST108) plasmids similar to P HNAH4-1 were carried out. This showed that the plasmid carrying heterozygous gene bla_ (CTX-M-64) and bla_ were widely spread in E. coli from animal sources.
【学位授予单位】：华南农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q55;Q78

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