Mechanism Of Brucella Virulence Attenuated Caused By Lon And ClpP Protease Mutation

Brucellosis is an allergic zoonotic infectious disease caused by Brucella.Since 2000,with the continuous growth of cattle and sheep breeding in China,and the frequent transportation among regions,the epidemiology of brucellosis in human and livestock populations has become more and more serious in China,posing a huge threat to human health and animal husbandry.Molecular pathogenesis of Brucella is one of the important research directions of brucellosis.ATP-dependent proteases Lon and Clp P had been reported to involve in the virulence of various pathogenic bacteria.However the biological functions of this two proteases in Brucella is still poorly understood.In this paper,the biological functions of Lon and Clp P in Brucella were studied,and the results provide basis for revealing the pathogenic mechanism of Brucella,developing noval Brucella vaccine and searching for potential drug targets.To study the biological function of Lon protein in Brucella,a lon gene mutant strain was constructed,the biological properites of lon mutant was studied,and the transcriptome changes caused by the lon gene mutation was analyzed by RNA-seq.The results showed that the growth rate,the sensitivity to stress environment and beta-lactam antibiotics,and the virulence of the lon mutant were significantly impaired,compared with parents strain.Transcriptionomic data showed that a total of 604 genes were expressed differently,which were mainly related to the pathways of iron biosynthesis,microbial metabolism in different environments,nitrogen metabolism,carbon metabolism,two component regulatory system and amino acid metabolism.To understand the function of another important ATP-dependent protease Clp P in Brucella,a clp P gene deletion strain were constructed by homologous recombination,and investigated the biological characteristics of clp P mutant,meanwhile the transcriptome changes in clp P mutant was identified by RNA-seq.The results showed that the growth rate,sensitivity to stress environment and virulence of clp P mutant were significantly impaired,compared with parents strain.We also found that,1769 differentially expressed genes mainly related to the pathways of ABC transporters,chloroalkanes and chloroalkenes degradation,tryptophan metabolism,cationic antimicrobial peptide resistance,β-lactams resistance,lysine degradation,valine,leucine and isoleucine degradation,bacterial secretion system,limonene and pinene degradation,naphthalene degradation,and vancomycin resistance.The residual virulence of clp P mutant was significantly lower than that of vaccine strain A19,whereas it could only provide weak immune protection in mice model,the immunoprotection of clp P mutant is less effective than A19.To understand the differentially expressed protein in lon mutant strain,isobaric tags for relative and absolute quantitation proteomics was used to analyze the protein expression profile of lon mutant strain.The results showed that there were 422 differential proteins mainly involved in metabolic pathways,microbial metabolism in adverse environment,secondary metabolites synthesis,amino acid metabolism,ABC transport system,fatty acid metabolism and pyruvate metabolism.On the basis of quantitative proteomic analysis,5downstream target proteins interacting with Lon protein were identified by bacterial two hybrid assay.Comprehensive analysis the results of transcriptomics and proteomics,q PCR and western blots detection indicated that the virulence of lon mutant strain at the early stage of infection was attenuated by down regulating the expression of transcriptional regulator Vjb R and type IV secretion system.It was also proved that Lon protein could be induced to express in acid nutrition deficient environment.In conclusion,our data suggest that Brucella ATP-dependent proteases Lon and Clp P play an important role in regulating bacterial division cycle,tolerance to stress environment,transcriptional regulators,bacterial secretion system and virulence.Our results provide valuable reference for Brucella pathogenic mechanism,vaccine development and screening of potential targets for drug therapy.