| Riemerella anatipestifer is an important pathogen of waterfowl,it mainly infected ducklings,turkeys,gooses and other poultry.It can cause septicemia,and fibrinous pericarditis,perihepatic inflammation,meningitis and so on can be seen in necropsy.After infection,the morbidity and mortality were high,which brought huge economic losses to the poultry industry.Studies were found that the biological function of effector molecules of bacterial type Ⅸsecretion system(T9SS)was diverse.They are involved in the life activities of bacteria or are related to the pathogenicity of bacteria.Recently,T9SS has been found in RA.In previous studies,we found that the virulence of the T9SS component gene mutants Yb2ΔgldK and Yb2ΔgldM of RA was significantly lower than that of the wild-type strain Yb2,and 9 effector molecules of T9SS secreted protein were differentially expressed in RA compared with the wild-type strain Yb2.Most of these effector molecules,including Endonuclease I(EndoI),have enzyme activity and may be involved in the life activities of RA or related to its pathogenicity.In this study,we investigated the biological characteristics of the effector molecule EndoI mutant strain Yb2ΔendoI and complementation strain cYb2ΔendoI.The results of growth curve showed that the effector molecule EndoI had no effect on wild-type strain,mutant strain Yb2ΔendoI and complementation strain cYb2ΔendoI.In the virulence test,the bacterial loads of mutant strain Yb2ΔendoI was significantly lower than that of wildtype strain Yb2 at 12,24,36 and 48 h after infection,indicating that the effector molecule EndoI was related to the virulence of RA.The ability of the mutant strain Yb2ΔendoI to adhere to and invade host cells was significantly lower than that of the wild-type strain Yb2 and complementation strain cYb2ΔendoI,indicating that the effector molecule EndoI is crucial to the cell adhesion and invasion of RA.The expression of cellular IL-1β,IL-6,IL-8 and TNF-αwas reduced at infection with Yb2ΔendoI,compared with Y2,suggesting that the deletion of the effector molecule EndoI reduced the ability of Yb2 to stimulate cells to produce an inflammatory response.In previous studies,the effector molecule EndoI was predicted to have nuclease activity.In this study,the recombinant EndoI(rEndoI)was determined to have DNase and RNase activities.Optimal temperature and pH of rEndoI to cleave λDNA were determined as 55 to 60℃ and 7.5 respectively.The DNase activity of the rEndoI was dependent on the presence of divalent metal ions.Presence of Mg2+ at a concentration of 7.5 mM in the rEndoI reaction buffer displayed the highest DNase activity,Mg2+ and Mn2+ have a certain promoting effect on the activity.In addition,the rEndoI displayed RNase activity to cleave ssRN A,either in the absence of divalent cations.Moreover,the DNase activity of rEndoI is competitively inhibited by addition of RNA substrate in the reaction buffer.The E111,H112 and N140 residues are involved in binding the cluster in Endonuclease I domain,which may be the key active site of EndoI,needs further verification.These results indicate that the R.anatipestifer endoI gene encodes an EndoI that is a T9SS effector molecule,displays endonuclease activity and plays an important role in bacterial virulence. |
