Enzymatic Activity And Function Analysis Of HD-GYP Domain Protein From Leptospira Interrogans

Objective Pathogenic Leptospira species are the causative agent of the most widespread zoonosis,Leptospirosis.Human will be infected through contacting with the water or soil,which contaminated by the urine from infected reservoir host.Chemotaxis and motility play a crucial role in the infection of Leptospira.Genome sequence analysis show that there are several genes that encode methylation receptor chemotaxis proteins cheB and cheR,but the regulation mechanism is still unclear.Methods 1.Real-time PCR was used to determine the mRNA levels of these five genes during the infection of Leptospira.2.Fragments of these genes were amplified and cloned into the expression vector pET28a,respectively,to construct the prokaryotic expression system for them.3.The colony morphologies of E.coli that overexpressed target genes were observed to determine the effect of c-di-GMP on the CheB and CheR.Results 1.60 min after infection,the mRNA level of cheBl increased and reached the peak at 90 min.The level of cheB3 still increased at 60 min,while there were no significant increase of cheB2 and cheR,compared with the control.2.We constructed the expression system and obtained the purified proteins.3.CheB1,CheB3 and CheR2 improved the motility of E.coli and can be inhibited by the inhibitor of DGC or PDE,respectively.Conclusion CheB and CheR effect the swarming motility of E.coli and are controlled by intracellular c-di-GMP.Objective Leptospirosis,caused by pathogenic Leptospira species,is an important reemerging zoonosis and there are more than half a million cases be reported annually.C-di-GMP signal system is an almost ubiquitous intracellular two-component system in bacteria,it is known to sense the environmental signal and regulate complex physiological processes,including mobility,adhesion,virulence and biofilm formation.Diguanylate cyclases(DGCs)containing GGDEF domain and phosphodiesterases(PDEs)containing EAL or HD-GYP domain have been identified as the enzymes modulating intracellular c-di-GMP levels.Bioinformatics analysis showed that there are two genes that encode HD-GYP domain proteins,LA2383 and LA2847,but their functions are still unclear.Method 1.Determining the mRNA levels of LA2383 and LA2847 during the infection of Leptospira interrogans on host cells by Real-time PCR.2.Constructing the prokaryotic expression system of LA2383 and LA2847 by molecular biological methods such as T-A coloning.3.Detecting the phosphodiesterase activities of puurified recombinant proteins to specific substrates bis-pNPP,c-di-GMP,c-di-AMP,cGMP,cAMP and pGpG by Microplate Reader and HPLC.4.Constructing a report plasmid to detect the intercellular PDE activity of LA2383 protein in E.coli.Result 1.12h after infection,the mRNA level of LA2383 increased significantly while the mRNA level of LA2847 increased appreciably.2.We successfully constructed the prokaryotic expression system of LA2383 and LA2847 and got the purified recombinant proteins.3.The bis-pNPP could be completely degraded by LA2383 protein,and pGpG could be hydrolysed by LA2383 protein much easilier than c-di-GMP.But LA2847 protein couldn't degrade any substrate used in this experiment,4.The intracellular level of c-di-GMP decreased after LA2383 and LA2847gene was overexpressed in E.coli BL21(DE3).Conclusion During the infection,the mRNA level of LA2383 and LA2847 decreased first and then jumped to a peak at 12h.The HD-GYP domain protein LA2383 and LA2847 are phosphodiesterases,which can decrease the level of c-di-GMP in vivo.So the HD-GYP domain proteins must play an important role during the infection of Leptospira interrogans.