Analysis Of Biological Characteristics And Monoclonal Antibody Preparation Of MPPE,an Effector Molecule Of Type Ⅸ Secretion System Of Riemerella Anatipestifer

Riemerella anatipestifer is the pathogen of Riemerella anatipestifer infection,it mainly infected ducklings,turkeys,gooses and other poultry.Riemerella anatipestifer infection is an acute,contact,and septic infectious disease.The main clinical features of this disease are fibrinous pericarditis,perihepatitis,air sacculitis,and meningitis.It is one of the most serious bacterial infectious diseases that endanger the duck industry and resulted in major economic losses.Studies had found that the effector molecules of T9SS have diverse biological functions,and they can degrade or break down nutrients in the host or the environment to provide nutrients for their own growth and reproduction,but also exert pathogenic effects on the host.In recent years,genetic analysis showed that R.anatipestifer has the T9SS genes.In our previous study,we identified three putative T9SS mutant strains by random mutagenesis of transposons and found that their virulence was significantly attenuated.R.anatipestifer genes AS87＿08785 and AS87＿RS08465 encode T9SS Gld K and Gld M,which functioned in bacterial gliding motility,protein secretion,and bacterial virulence.Liquid chromatography–tandem mass spectrometry analysis revealed a total of nine effector molecules were differentially expressed in the culture supernatant of T9SS mutant strains Yb2Δgld K and Yb2Δgld M.Most of these effector molecules have enzyme activities and participate in various life processes of bacteria,as well as associated with bacterial virulence.Metallophosphoesterase（MPPE）is one of such effectors.In this study,we constructed the MPPE mutant strain Yb2Δ00980 and studied its biological characteristics.Determination of growth curves showed no significant difference among the wild-type strain Yb2,the mutant strain Yb2Δ00980 and the complementation strain c Yb2Δ00980.The virulence assessment showed a more than 1,000-folds attenuated virulence for the mutant strain Yb2Δ00980,compared to the wild-type strain Yb2.The LD₅₀of the mutant strain Yb2Δ00980 was 2.21×10⁸CFU,and that of the wild-type strain Yb2 was2.20×10⁵CFU.In addition,bacterial loads in blood of infected ducks for the mutant strain Yb2Δ00980 was significantly reduced.These data suggest the MPPE is associated with the bacterial virulence.We also found that the adhesion and invasion capacities in HD11 cells were all significantly reduced in the mutant strain Yb2Δ00980.These results indicate that MPPE plays an important role in the process of bacterial adhesion and invasion.We found that the recombinant MPPE（r MPPE）could hydrolyze the substrate p-NPP.The secretory proteins of the wild-type strain Yb2 and the complementation strain c Yb2Δ00980 also showed high phosphatase activity,while the mutant strain Yb2Δ00980 had a little phosphatase activity.The above data validated that the effector molecule MPPE is an metallophosphoesterase.The enzyme activity properties of r MPPE were further investigated,p H and temperature of the r MPPE were optimized as 7.0 and 60°C,respectively.Km and Vmax of the r MPPE were determined as 3.53 m M and 198.1U/mg,respectively.The r MPPE activity was activated by Zn²⁺,Cu²⁺,but inhibited by Fe³⁺,Fe²⁺,and EDTA.Bioinformatics analysis found that MPPE has 5 active amino acids,they are asparagine at position 267,histidine at position 268,351,389,and 391.We prokaryotic expressed the five mutant proteins,using r MPPE as a control,the enzymatic activity of the mutant protein Y267-r MPPE was 29.30%of that of r MPPE,the mutant protein Y268-r MPPE was only 19.81%,and the mutant proteins Y351-r MPPE,Y389-r MPPE,and Y391-r MPPE almost completely lost their enzymatic activity indicated that histidine at position 351,389,and 391 are critical for r MPPE to exert enzymatic activity.Monoclonal antibodies to MPPE were prepared,and 6 cell lines stably secreting monoclonal antibodies were obtained and named as 1,2,3,4,5,and 6,respectively.The subtypes of monoclonal antibodies were identified.The heavy chain was determined as Ig G1 for monoclonal antibodies 1,2,3 and 5,and was determined as Ig G2b and Ig G2a for monoclonal antibodies 4 and 6 respectively.The light chain were determined asκfor all 6 monoclonal antibodies.The titers were determined as1:204800 for all 6 monoclonal antibodies indirect ELISA;The specificity of 6 monoclonal antibodies was identified by Western blotting,showed no cross reaction with other avian pathogenic bacteria.Six monoclonal antibodies were successfully developed in this study,which have high specificity and are helpful for the study on R.anatipestifer pathogenic mechanism and diagnostic technology.