Construction And Related Phenotypic Analysis Of Brucella Iron Regulatory Factor Irr And RirA Gene Deletion Strains

As the pathogen of brucellosis,Brucella pathogenesis depends on the ability of the brucellae to survive and replicate within host phagocytic cells.The iron uptake system is one of the key virulence factors of Brucella's pathogenic ability.It provides a precise iron response regulational pathway for Brucella during the pathogenesis of Brucella.Iron response regulator have been reported in Rhizobium(the same strain as Brucella),which plays a key role in the regulation of Rhizobium iron metabolism.irr and rirA genes have been reported that it plays an important role in the pathogenicity of bacteria and the process of intracellular survival.Therefore,studying the function of irr and rirA genes of Brucella will provide a new clues and ideas for revealing the pathogenic mechanism of Brucella.Objective: In order to reveal the function of iron response regulators irr and rirA of Brucella melitensis M5-90 in the pathogenesis of Brucellosis.We constructed the corresponding gene deletion strains,and compared on the growth conditions,the ability of intracellular rsurvival and antibiotic resistance.It can explore the function of these iron response regulator which reveal the molecular pathogenesis of Brucella and lay the foundation for the study of the function of irr and rirA,and provide new clues and ideas for revealing the pathogenic mechanism of Brucellosis.Methods: Iron response regulator irr and rirA of Brucella melitensis M5-90 were acted as the research object,and the genome of heat-inactivated Brucella melitensis M5-90 was used as a template,the upstream and downstream homology arms of the irr and rirA genes were amplified.The fragment of kanamycin resistance was amplified with the pUC19 K plasmid as a template.The upstream and downstream homology arms of the irr and rirA gene were fusioned with the fragment of kanamycin resistance by Overlap PCR respectively.The fusion fragment was directly ligated into the pMD19-T vector which was electrotransformed into Brucella melitensis M5-90.Positive clones were screened by kanamycin resistance and were serially passaged of which the genetic stability were detected by PCR.The growth trend of Brucella melitensis M5-90 and the deletion strains that were cultured under the same initial concentration and nutrient condition was measured and observed.The antibiotic resistance of each strain to different antibiotics was tested by the method of bacteriostasis circle.RAW264.7 cells was infected with Brucella melitensis M5-90 and the deletion strains,and the morphology and intracellular survival capacity of all strains were observed and analysised.Results: The upstream and downstream homology arms and Kana gene fragments of the irr and rirA genes were successfully amplified,and the fusion fragments of the upstream and downstream homologous arms and the Kana gene fragments were successfully obtained.After electroporation of the recombinant plasmid into Brucella cells,Brucella irr and rirA gene deletion strains M5-90-?irr and M5-90-?rirA were successfully obtained,and no deletion occurred within 10 passages after serial passage.Phenomenon;compared with the parental strain,the growth trend of M5-90?irr and ?rirA did not change significantly under the same in vitro culture conditions,but the overall concentration was lower than that of the parental strain;the antibiotic resistance test showed that the irr and rirA genes were deleted.After the resistance tothe corresponding antibiotics decreased,but the sensitivity to neomycin disappeared;adhesion and invasion test showed that at the 15 min,45min and 60 min infection,the missing strains M5-90?irr and ?rirA on RAW264.7 The adhesion invasive ability was significantly lower than that of the parental strain M5-90;the intracellular survival test found that the surviving ability of the deletion strains M5-90?irr and ?rirA in RAW264.7 cells was enhanced compared to the parental strain M5-90Conclusion: The stable inheritance of Brucella deletion strains M5-90-?irr and ?rirA was successfully constructed.The iron response regulators irr and rirA genes affected and participated the reproduction of Brucella melitensis M5-90 in vitro and the intracellular pathogenesis,and regulating the virulence and pathogenic of bacteria.These result lay the foundation for reveal the function of virulence genes of Brucella,and provide a new clues and ideas for reveal;p the pathogenic mechanism of Brucella.