Biological Characterization Of Biotin-synthesis Associated BioF Gene In Riemerella Anatipestifer And Its Application

Riemerella anatipestifer infection is a contagious disease of ducklings,turkeys,gooses and other birds,and occurs in acute or chronic form.This desease causes high morbidity(90%)and mortality(75%)for ducks and resulted in major economic losses in the duck industry.Biotin,also known as vitamin H or coenzyme R,is a cofactor for some essential enzymes in vivo,i.e.pyruvate carboxylase(PC)and acyl coenzyme carboxylase(ACC).Biotin acts as a carbon dioxide carrier in carboxylation,decarboxylation and transcarboxylation reactions that are responsible for biosynthesis of fatty acids,gluconeogenesis and amino acid metabolism.Most microorganisms,plants,and a few fungi are able to synthesize biotin,mammals cannot synthesize biotin but depend on dietary intake or gut microbia.In this study,a mutant strain Yb2?bioF which defected in biotin biosynthesis was screened from a random transposon mutagenesis library.The biological characteristics of the mutant strain Yb2?bioF and protection against R.anatipestifer serotype 2 strains were investigated.This study includes the following two parts:We previously found that R.anatipestifer AS87_RS09170,a putative bioF gene,is important for the bacterial virulence.In this study,we characterized the AS87_RS09170 gene in R.anatipestifer strain Yb2.Sequence analysis indicated that the AS87_RS09170 gene is highly conserved among R.anatipestifer strains,and the predicted protein harbored the conserved pyridoxal 5'-phosphate binding pocket of 8-amino-7-oxononanoate synthase.Western blot analysis demonstrated that two isoforms of biotin-dependent enzyme from mutant strain Yb2?bioF were significantly decreased than those of wide-type strain Yb2,suggesting the biotin biosynthesis was defected.The mutant strain Yb2?bioF displayed a significant decreased growth rate at the exponential phase in tryptic soy broth culture,but recoverd the growth ability when biotin was supplemented.In addition,the mutant strain Yb2?bioF showed an enhanced biofilm formation,as well as increased adhesion and invasion capacities to duck embryo fibroblast.Moreover,the mutant strain Yb2?bioF showed irregular shapes with budding vegetations and quite thickened cell walls under scanning and transmission electron microscope observation and reduced capacity to establish systemic infection in a duck infection model.These results provide the first evidence that R.anatipestifer AS87_RS09170 gene is responsible for biotin biosynthesis,bacterial surface morphology and virulence.In addition,the mutant strain Yb2?bioF was investigated for its potential use as a live attenuated vaccine.Results showed that the mutant strain Yb2?bioF remained attenuated virulence after 30 serial passages in TSB and TSA(Yb2?bioF-F30).No visible histological changes were observed in the heart,liver and spleen samples from the Yb2?bioF-F30-infected ducks.Furthermore,the ducks received one immunization or two immunizations of live Yb2?bioF were 90% or 100% protected from challenge with serotype 2 virulent strain Yb2,respectively.No clinical signs or deaths were observed in the protected ducks.The ducks that received two immunizations of the Yb2?bioF generated not only high antibody titers against wild-type strain Yb2,but also enhanced productions of gamma interferon(IFN-?),interleukin 2(IL-2),IL-4 and IL-10.Altogether,our results suggest that the mutant strain Yb2?bioF could be used as a novel live vaccine candidate to protect ducks against R.anatipestifer infection.