Identification Of Contributing Factors To Hemolytic Activities And Their Roles In Virulence Of Uropathogenic Escherichia Coli

As one of the Extraintestinal pathogenic E.coli(ExPEC)pathotypes,uropathogenic E.coli(UPEC)can cause cystitis,pyelonephritis and hemolytic uremic syndrome.It has been estimated that about 80% of urinary tract infections are caused by UPEC,which is particularly common in women.As a typical ExPEC pathotype,the molecular pathogenesis has been well studied by domestic and foreign scholars.In addition to its typical adhesion and invasion functions,UPEC also produces some toxins,such as CNF1,HlyA and Chu.Hemolysin(HlyA)is one of the important virulence factors of UPEC,named for its ability to lyse red blood cells.Regarding its pore-forming activities,some scholars have studied its phenotypes in recent years.Many studies have shown that,HlyA can induce cell pyroptosis by activating canonical inflammasome.However,the regulatory mechanism of HlyA expression is only partially understood.In this study,using a blood agar plate assay,we demonstrated that the hemolysis halo is more pronounced in anaerobiosis compared to that in aerobiosis.Based on that,we prepared a mini-Tn5 transposon-based random mutagenesis library to screen for the regulatory factors of HlyA in anaerobiosis.Transposon insertions were mapped to 14 genes,including the hemolysin operon,Molybdenum Cofactor(Moco)biosynthesis gene(mobA moeB mogA),global regulator FNR,membrane transporter tatB and Fe-S cluster assembly chaperone protein hscA,etc.We have noticed that several genes involved in Moco biosynthesis contribute to anaerobic hemolysis.Through RNA-Seq analysis,we demonstrated that Moco affect hemolysin expression primarily via c3566-67-68,which is co-transcribed with the hlyCABD operon.Further,we showed that the moco pathway modulates uroepithelial cell death,and it is an important fitness factor for UPEC colonization in a murine model.In order to understand the mechanism by which Moco modulates c3566-67-68 and hlyCABD expression,we sought to identify suppressor mutations of mobA,which would lead to enhanced production of hemolysin in mobA mutants.We selected eight transposon insertion mutants,which displayed enhanced hemolysis.Four of the eight genes are either functionally or genetically related to nitrogen metabolic factors(Ntr response pathway).Our results demonstrated that deletion of mobA resulted in increased expression of ntrC,which in turn functions as a repressor of c3566-67-68 and hlyCABD expression.Additionally,we found that FNR TatB HscA affect hemolysin expression primarily via c3566-67-68,thus suggesting that the c3566-67-68 gene cluster may be as an important hub of hemolysin regulation in anaerobiosis.In conclusion,our findings provides valuable information for understanding and controling of ExPEC,and may aid in the development of vaccines and antimicrobials.