Functional cloning of Bacillus anthracis dihydrofolate reductase and confirmation of natural resistance to trimethoprim
Functional cloning of Bacillus anthracis dihydrofolate reductase and confirmation of natural resistance to trimethoprim
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DOI：
10.1128/aac.48.12.4643-4649.2004
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发表时间：
2004-12-01
影响因子：
4.9
通讯作者：
Barrow, WW
中科院分区：
文献类型：
--
作者：
Barrow, EW;Bourne, PC;Barrow, WW
据报道，炭疽杆菌对甲氧苄啶（TMP）具有天然耐药性，甲氧苄啶是一种抑制二氢叶酸还原酶（DHFR）（叶酸途径中的关键酶）的药物。微量肉汤稀释法测定TMP对B.炭疽菌Sterne> 2，048但小于或等于4，096 μ g/ml。从B中扩增出一个推定的DHFR序列。炭疽菌Sterne基因组DNA。将PCR产物克隆到Invitrogen pCRT 7/CT-TOPO载体中，然后转化到大肠杆菌TOP 10 F ′化学感受态细胞中。将来自克隆的质粒DNA转化到E. coli BL 21星星（DE 3）pLysS感受态细胞中表达6个组氨酸标签的融合蛋白并在His-Bind树脂柱上纯化。在已建立的酶试验中证实了纯化Sterne重组DHFR（Sterne rDHFR）的功能。发现TMP和甲氨蝶呤对Sterne rDHFR的50%抑制浓度分别为77，233和12.2 nM。结果表明，E. coli BL 21星星（DE 3）pLysS感受态细胞转化Sterne DHFR基因。氨基酸序列分析表明，Sterne DHFR基因与B强毒株的同源性为100%。炭疽病这些结果证实了B的天然抗性。炭疽菌对TMP的抗性，并阐明该抗性与染色体编码的基因产物缺乏选择性相关。这些发现将有助于开发用于治疗炭疽的窄谱抗菌剂。
Bacillus anthracis is reported to be naturally resistant to trimethoprim (TMP), a drug that inhibits dihydrofolate reductase (DHFR), a key enzyme in the folate pathway. A microdilution broth assay established that the MIC of TMP for B. anthracis Sterne is >2,048 but less than or equal to4,096 mug/ml. A putative DHFR sequence was amplified from B. anthracis Sterne genomic DNA. The PCR product was cloned into the Invitrogen pCRT7/CT-TOPO vector, followed by transformation into Escherichia coli TOP10F' chemically competent cells. Plasmid DNA from a clone showing the correct construct with a thrombin cleavage site attached downstream from the terminus of the cloned PCR product was transformed into E. coli BL21 Star (DE3)pLysS competent cells for expression of the six-histidine-tagged fusion protein and purification on a His-Bind resin column. Functionality of the purified Sterne recombinant DHFR (Sterne rDHFR) was confirmed in an established enzyme assay. The 50% inhibitory concentrations of TMP and methotrexate for the Sterne rDHFR were found to be 77,233 and 12.2 nM, respectively. TMP resistance was observed with E. coli BL21 Star (DE3)pLysS competent cells transformed with the Sterne DHFR gene. Alignment of the amino acid sequence of the Sterne DHFR gene revealed 100% homology with various virulent strains of B. anthracis. These results confirm the natural resistance of B. anthracis to TMP and clarify that the resistance is correlated to a lack of selectivity for the chromosomally encoded gene product. These findings will assist in the development of narrow-spectrum antimicrobial agents for treatment of anthrax.