Preliminary Study On Pathogenicity And Immunoprotection Of Kbvr-deletion Strain Of Kebsiella Pneumoniae

Background:Klebsiella pneumoniae is a common opportunistic pathogen in clinical practice,which can not only cause pneumonia,urinary system infection,etc.,but also cause liver abscess,meningitis and endophthalmitis.The emergence of virulent strains and antibiotic-resistant strains has made Klebsiella pneumoniae a conditional pathogen after E.coli,especially K1 and K2 serotype Klebsiella pneumoniae are more common in clinic due to their strong invasiveness and anti-phagocytosis ability.The pathogenic process of bacteria is a process that senses various host environments to regulate the expression of related genes.In this process,bacteria are affected by a variety of regulatory factors,including transcriptional regulatory factors and post-transcriptional regulatory factors.Among them,the protein encoded by KP1＿RS12260（kbv R）gene of Klebsiella pneumoniae is a type of transcription regulator.After the kbv R gene of Klebsiella pneumoniae was knocked out in the research team in the early stage,it was found that the formation of bacterial capsules and biofilms was affected.Objective:This subject will continue to study the role of Klebsiella pneumoniae kbv R gene in the pathogenicity of bacteria and the process of bacteria adapting to the environment of the body,and initially assess the immune protection of kbv R gene knockout.Methods:The research team has constructed kbv R gene knockout strain（△kbv R）and kbv R gene replacement strain（C-kbv R）in the early stage.The subject first infected mice with kbv R gene knockout strains（△kbv R）and wild strains（WT）by subcutaneous,intraperitoneal and digestive tract routes.By observing the survival of mice or calculating the bacterial load of related organs,the kbv R gene was analyzed in pneumonia Role of Klebsiella in Pathogenesis.Analyze the role of kbv R gene in bacterial adaptation to various microenvironments of the body（including bile salts,acidic environment,H₂O₂and hypertonic environment）by comparing growth curves and viable bacterial counts,and use macrophages and bacteria interaction experiments in vivo and in vitro to evaluate the role of the kbv R gene in bacterial anti-macrophage phagocytosis.RNA-Seq sequencing was used to understand the difference in gene expression between the kbv R gene knockout strain and the wild strain from the perspective of the whole genome,and select some pathogenic genes that may be regulated by Kbv R.The E.coli expression system was used to clone and express the Kbv R protein.On this basis,mice were immunized with kbv R gene knockout strains and their immunoprotective effects were preliminarily analyzed to evaluate the possibility of kbv R gene knockout strains as live attenuated vaccine strains.Results:1.kbv R gene knockout does not affect the growth rate of bacteria,but in mouse subcutaneous,abdominal and digestive tract infection models,the pathogenicity of△kbv R was significantly lower than that of WT control group.After subcutaneous and intraperitoneal infections of 10⁷ cfu/m L and 10⁴cfu/m L of WT,respectively,the mice died within two days,while△kbv R-infected mice still survived 7 days after infection.In the digestive tract infection model,the bacterial load of the large intestine,small intestine,and liver of mice infected with 10⁸ cfu/m L WT was significantly higher than that of mice infected with△kbv R 5 days after infection.2.In the acid environment and H₂O₂,there is no significant difference between the growth of△kbv R and WT.However,the growth of kbv R knockout strains was significantly higher than that of WT The growth of the kbv R gene knockout strain is significantly better than WT in bile salt environment and hypertonic environment.3.When the kbv R knockout strain was infected intraperitoneally in mice for 24hours,the blood of the mice was cultured and stained by smear of peritoneal lavage fluid.As a result,the presence of Klebsiella pneumoniae was not observed,and the WT strain infected mice Afterwards,bacteria can be detected in both blood and peritoneal lavage fluid.In vitro macrophage invasion experiments found that when the kbv R gene was knocked out,the adhesion of bacteria to macrophages was significantly enhanced,while the ability to resist phagocytosis decreased.4.The results of RNA-Seq sequencing showed that the transcription of 1410genes was regulated by Kbv R,including bacterial capsules,fimbriae,lipopolysaccharides,and ion acquisition related genes.5.Preliminary immunoprotection experiments demonstrated that subcutaneous immunization with 10⁴ cfu/m L of△kbv R increased the survival rate of mice in challenge experiments by 50%,indicating that△kbv R can provide partial protection for mice.Conclusion:Kbv R is a global regulatory factor,which is closely related to bacterial pathogenicity and plays a negative regulatory role in bacteria sensing external bile salt environment and hypertonic environment.kbv R gene affects bacteria’s anti-phagocytosis function and intestinal colonization ability of macrophages,and then affects bacterial pathogenicity.Preliminary immunoprotection experiments have shown that△kbv R can play a certain protective role,providing new ideas for the next step in the development of a highly attenuated Klebsiella pneumoniae live attenuated vaccine.