铜绿假单胞菌phoQ基因敲除对氨基糖苷类抗生素耐药性的影响

Deletion ofphoQ gene decreases the resistance of Pseudomonas aeruginosa to aminoglycoside antibiotics

目的分析氨基糖苷类抗生素敏感或耐药铜绿假单胞菌菌株二元信号系统PhoQ/PhoP编码基因序列并确定该系统与耐药性关系。方法采用PCR获得铜绿假单胞菌菌株全长phoQ和phoP基因片段，T—A克隆后测序。构建phoQ和phoP基因原核表达系统，Ni-NTA亲和层析法提纯目的重组表达产物rPhoQ和rPhoP，皮内注射免疫法制备兔抗血清，免疫双扩散法测定抗血清效价。采用Red重组系统敲除氨基糖苷类抗生素耐药铜绿假单胞菌菌株phoQ基因，采用PCR、测序和Westernblot对phoQ。突变株进行鉴定。采用试管稀释法测定各铜绿假单胞菌野生株和突变株对4种氨基糖苷类抗，丰素的最低抑菌浓度（MIC）。结果与GenBank中相关序列比较，所克隆的phoP和phoQ基因核苷酸和氨基酸相似性分别为98．7％一99．6％和98．7～100％、98．4％～99．8％和99．1％～100％。采用pET-42a和E．coliB121DE3系统成功地表达了rPhoQ和rPhoP。rPhoQ和rPhoP兔抗血清免疫双扩效价分别为1：4和1：8抗血清。经PCR、测序和Westernblot鉴定，两株phoQ^-突变株phoQ基因及产物均缺失。上述phoQ^-突变株对4种氨基糖苷类抗生素的MIC值分别为其野生株的1／512～1／2048。结论PhoQ／PhoP是序列保守的铜绿假单胞菌二元信号转导系统，该系统介导细菌对氨基糖苷类抗生素的耐药性。

Objective To analyze the sequences of two component signaling system PhoP/PhoQ encoding genes of Pseudomonas aeruginosa strains sensitive or resistant to aminoglycoside antibiotics and to determine the correlation between the PhoQ/PhoP and the resistance. Methods The segments of entire phoQ and phoP genes of P. aeruginosa were obtained by PCR and then sequenced after T-A cloning. Two prokaryotic expression systems of phoQ and phoP genes were constructed and the target recombinant expression products rPhoQ and rPhoP were extracted by Ni-NTA chromatography. Rabbits were intracutaneously immunized with rPhoQ and rPhoP to obtain antisera and double immunodiffusion test was used to detect the titers of antisera. The phoQ genes of aminoglycoside antibiotics-resistant P. aeruginosa strains were knocked out by using Red recombination system, and phoQmutants were identified by PCR plus sequencing and Western blot assay. Tube dilution method was applied to determine MIC values of wild and mutant strains of P. aeruginosa to four different aminoglycoside antibiotics. Results In comparison with the corresponding sequences in GenBank, the similarities of nucleotide and putative amino acid sequences of the cloned phoP and phoQ genes were 98.7% -99.6% and 98.7% -100% , and 98.4% -99.8% and 99.1% -100% , respectively. Both rPhoQ and rPhoP were successfully expressed using pET-42a and E. coli BL21 DE3 system, and their rabbit antisera with 1：4 and 1：8 double immunodiffusion titers were also obtained. The deletion ofphoQ genes and absence of the products in the two phoQ- mutants were confirmed by PCR, sequencing and Western blot assay. MIC values of the four different aminoglycoside antibiotics to the two mutants were 1/512- 1/2048 as those of their wild strains. Conclusion PhoQ/PhoP is a sequence conserved two component signaling system of P. aeruginosa, and this system mediates resistance of the microbe to aminoglycoside antibiotics.