Identification And Functional Characterization Of Riemerella Anatipestifer Nicotinamidase

Riemerella anatipestifer is a Gram-negative,non-spore-forming,capsule-like,rod-shaped bacterium.It is reported worldwide as a cause of epizootic infectious polyserositis in domestic ducks.It is also pathogenic for geese,turkeys,swans,and other birds.R.anatipestifer infection occurs as an acute or chronic septicemia characterized by fibrinous pericarditis,airsacculitis,perihepatitis,caseous salpingitis,and vegetative disorder.We previously reported the identification of 49 virulence-associated genes via random transponson mutagenesis.Transponson Tn4351 disrupted at the AS87₀1735 gene,which encodes a predicted nicotinamidase?PncA?,a key enzyme that catalyzes the conversion of nicotinamide?NAM?to nicotinic acid?NA?.This study describes the expression and identification of the predicted R.anatipestifer PncA-encoding gene,AS87₀1735,as well as the virulence and immunization protection of an AS87₀1735 gene deletion mutant.Conclusion presented in this study can be summarized as follows:-R.anatipestifer AS87₀1735 encodes a protein PncA with nicotinamidase activity.PncA is expressed in the cytoplasm of R.anatipestifer.-In vitro stidies showed RA PncA exihited maximal nicotinamidase activity in pH 6.0 reaction buffer at 40°C.The enzymatic activity was inhibited by Zn2+,Ni2+,Mg2+ and Cu2+,respectively.In addition,D9,F14,D49,H51,H83,K108 and C153 were essential for full nicotinamidase activity of R.anatipestifer.-The intracellular NAD+ concentration is lower in the R.anatipestifer mutant strain Yb2?pncA.-Deletion of the pncA gene significantly attenuates bacterial virulence.-Mutant strain Yb2?pncA immunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2,indicating that mutant strain Yb2?pncA can be used as a novel live vaccine candidate.