The Effect Of Transcription Regulator GreA On Environmental Adaptation And Pathogenicity Of Francisella Tularensis

Francisella tularensis is a facultative intracellular Gram-negative bacterium that causes the zoonotic disease tularemia.Hosts can be infected by multiple routes,including the inhalation of bactreria-containing droplets or dust,the consumption of contaminated food or water,wound,and arthropod bite.A large amount of studies about pathogenic mechanisms of F.tularensis has been carried out at home and broad over the years due to its high virulence to human.In our previous study F.tularensis LVS greA mutant failed to efficiently survive in mice,indicating that GreA was required for bacterial infection and pathogenicity.Transcription enlongation factor GreA stimulates nucleolytic activity of bacterial RNA polymerase?RNAP?,by which GreA suppresses pauses and arrests,stimulates RNAP promoter escape,and enhances transcription fidelity.To determine the effect of GreA on the infection and pathogenicity of F.novicida,we constructed ?greA mutant and in trans-complemented strain with wild-type F.novicida U112 by homologous recombination technology.The in vitro experiments showed that?i?deletion of greA gene impacted bacterial environment adaptation,including in vitro growth defect,NaCl sensitivity,and enhanced biofilm,while log-phase bacterial morphology,temperature sensitivity,and pH sensitivity were not affected by GreA.?ii?Loss of GreA severely destroyed bacterial invasion and intracellular growth.Furthermore,mice infection suggested that GreA contributed to virulence of F.novicida to mice inoculated subcutaneously or intranasally,and in vivo?greA mutant could be quickly eliminated from livers,spleens,and lungs of mice compared with wild-type U112,confirming that GreA was required for bacterial pathogenicity.To reveal the molecular mechanism of GreA on physiology and pathogenicity of F.novicida,RNA-seq of AgreA mutant and wild-type U112 was performed.Compared with wild-type U112,196 genes were regulated?Fold change>2?,most of which were categorized into amino acid metabolism,carbohydrate metabolism,and inorganic ion metabolism in the AgreA mutant.77 differentially expressed genes,including almost all of FPI genes,were indentified as virulence factors in the previous studies.Promoter activities of pdpD,pepO,and FTN₁104 were lower in ?greA mutant compared with wild-type U112 by?-galactoside assay.To confirm that GreA regulates the expression of virulence factors involved in cell invasion by F.tularensis,FTN₁186?pepO?FTN₁104,and FTN₁551?ampD?gene mutants were generated.The ampD and FTN₁104 deletion mutants showed reduced invasiveness into host cells.In summary,GreA was able to regulate gene transcription by promoter region in F.novicida,contributed to bacterial environment adaptation,invasiveness into host cells and pathogenicity to mice.Based on its conservative property and effect on bacterial pathogenicity,GreA is a new drug target for prevention and treatment of F.tularensis.