Analysis Of Capsular Polysaccharide Biosynthesis Locus Of R.anatipestifer And The Study Of The Function Of Gene G148＿RS04320

Riemerella anatipestifer(RA)is a widely distributed pathogen that causes infectious serositis in ducks.It can cause sepsis and exudative diseases in ducks,and bring huge economic losses to the duck industry.Capsules which envelop bacteria protect bacteria against from serum complement-mediated bactericidal effect and phagocytic cell-mediated phagocytosis.The previous studies of constructing mutants of genes wza,wzc or wzy in our laboratory have proved that those mutants affect the synthesis of the capsule and attenuate the bacterial virulence.It predicted that genes G148＿RS04320 in the same gene cluster are involved in the synthesis of the monosaccharide D-Quip NAc4NAc(2,4-diacetamido-2,4,6-trideoxy-D-glucose,acid),but the molecular basis of capsular polysaccharide biosynthesis is unclear,moreover,there are few reports on genes related to the biosynthesis of capsular polysaccharide of RA.In this study,first we used the Pfam database and HMMER3 3.1b1 retrieved key proteins such as Wza 、 Wzb 、 Wzc 、 ABC transpoter 、 Wzx flippase 、 Wzy polymerase 、glycosyltransferase,then located the capsular polysaccharide gene cluster in RA CH-2 strain;The first gene G148＿RS04320 in this gene cluster was deleted by natural transformation,compare the characteristics between wild-type strain CH-2 and CH-2ΔG148＿RS04320 mutant strain,including bacterial morphology observation,Indian ink staining,transcription level determination,growth curve determination,slide agglutination test,dry environment tolerance test,etc.,to identify whether the gene G148＿RS04320 will influent the production of capslue and the virulence of RA CH-2.The results show that the predicted capsular polysaccharide biosynthesis gene cluster in RA CH-2 exists between the genes rec X and rim O,and there are 22 genes in total,which are similar to the composition of gene clusters in A.baumannii and K.pneumoniae.And gene G148＿RS04320 may be involved in the biosynthesis of capsular polysaccharides.Colonies of this mutant strain were dry,flat,and lost their mucous phenotype.Capsule staining showed that the capsule was significantly thinner;the determination of transcription level proved that the deletion of the gene G148＿RS04320 did not affect the transcription of its downstream genes,and the growth rate of the mutant was significantly faster than wild-type strain;The bacterial pellet of the ΔG148＿RS04320 strain was ‘stickier’ and more compact compared to the wild-type strain.Slide agglutination test showed that the mutant can agglutinate with14-serotypes serum in our study;Infect 7 days ducklings with CH-2 and mutant showed that the LD50 of the mutant had been increased,and the colonization ability was significantly reduced.There was no obvious pathological damage in the tissues of the duckling infected with mutant.The experimental results of this study indicate that the deletion of gene G148＿RS04320 affects the synthesis of capsular polysaccharide of R.anatipestifer CH-2strain and the surface antigen of the strain,reduce the virulence of the strain.