The Correlation And Mechanism Of Action Between PhoQ/PhoP And OprH In The Resistance Of Pseudomonas Aeruginosa To Aminoglycoside Antibiotics

Objective:To analyse the sequences of two component signaling system PhoP/PhoQ and OprH encoding gene of Pseudomonas aeruginosa strains sensitive or resistant to aminoglycoside antibiotics, and to determine the correlation between the resistance.Methods:The segments of entire phoQ/phoP and oprH genes of P.aeruginosa were obtained by PCR and then sequenced after T-A cloning. phoQ/phoP and oprH were constructed and the target recombinant expression products rphoQ, rphoP and rOprH were extracted by Ni-NTA chromatography. Rabbits were intracutaneously immunized to obtain antisera and double immunodiffusion test was used to detect the titers of antisera. The phoQ, phoP and oprH genes of aminoglycoside antibiotics-resistant P.aeruginosa strains were knocked out by using Red Recombination System, phoQ phoP- and oprH- mutants 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. The transcription levels of other genes in mutant strains were monitored by real-time fluorescence quantitative RT-PCR.Results:In comparison with the corresponding sequences in GenBank, the similarities of nucleotide and putative amino acid sequences of the cloned phoQ, phoP, oprH genes were 98.4%-99.8% and 99.1%-100.0%,98.7%-99.6% and 98.7-100%,97.3%-99.5% and 96.0%-100.0%,97.3%-99.5% and 96.0-100%, respectively. rphoQ, rPhoP and rOprH was successfully expressed using pET42a and E.coliBL21DE3 system, and their rabbit antisera with 1:4,1:8 and 1:8 double immunodiffusion titers was also obtained. The deletion of phoQ, phoP and oprH genes and absence of the products in the phoQ-, phoP- and oprH- mutants were confirmed by PCR, sequencing and Western Blot assay. MIC values of the four different aminoglycoside antibiotics to the phoQ-,phoP- and oprH- were 1/512-1/2048,1/512-1/1024 and 1/256-1/1024 as those of their wild strains. The transcription levels of other genes in mutant strains were obviously lower than aminoglycoside antibiotics-resistant P.aeruginosa strain 160756.Conclusion:PhoQ/phoP is a sequence conserved of P.aeruginosa, and this system mediates resistance of the microbe to aminoglycoside antibiotics.