Genome-wide Identification And Functional Study Of Virulence Genes In Riemerella Anatipestifer

Riemerella anatipestifer is a well-described pathogen of waterfowl and other avian species which can cause septicemic and exudative diseases. In domestic ducks, R. anatipestifer causes epizootic infectious polyserositis, characterized by lethargy, diarrhea, respiratory and nervous symptoms, which led to high mortality and consequently to great economic losses. However, only limited genomic resources, as well as virulence factors, are available for R. anatipestifer in spite of its veterinary importance. To comprehensively and systematically explore the genetic diversity and virulence evolution of R. anatipestifer strains, a genome-wide profiling is needed.In this study, we sequenced the complete genome of R. anatipestifer strain Yb2 and analyzed it against published genomic sequences of R. anatipestifer strains DSM15868, RA-GD, RA-CH-1 and RA-CH-2. Yb2 genome contains one circular chromosome of 2,184,066 bp with 35.73% GC content, and no plasmid. The genome has 2,073 putative open reading frames, 6 ribosomal RNA operons, 38 t RNA genes and one other nc RNA. Of the ORFs, 2,021 were protein-coding genes. The majority of the protein-coding genes（1,214, 60.07%） were classified into the COG families comprising 20 functional categories. Using KEGG, 1,217 ORFs（60.22%） were assigned to 30 pathways. Among all proteins, 11 proteins involved in lipopolysaccharide（LPS） biosynthesis were predicted, and the majority was involved in lipid A biosynthesis. Collinearity analysis revealed 50, 29, 203 and 42 collinearity blocks between Yb2 and each of DSM15868, RA-GD, RA-CH-1 and RA-CH-2, respectively. Genome organization is highly conserved among R. anatipestifer strains, except for four inversions of sequence segment in Yb2. The phylogenetic tree was built with Mega6 based on 16 S r DNA sequence. The resulting phylogenetic tree reveals a strikingly short genetic distance between Yb2 and RA-GD, indicative of very recent divergence.Tn4351 was introduced into R. anatipestifer wild-type strain Yb2 by conjugation from Escherichia coli BW19851 as previously described. The resulting transcojugants were first selected with kanamycin and erythromycin, and then identified by PCR amplification. A library of 3,175 mutants was constructed by random transposon mutagenesis.Tn4351 insertion mutants were deemed defective in virulence based on the loss of pathogenicity in our animal model. We identified 100 mutants with different virulence levels in the initial screening for transconjugants. Southern blot analysis revealed that 62%（62/100） of the attenuation mutants has a single Tn4351 insertion. The DNA sequences flanking transposon insertions were obtained by inverse PCR or genomic walking. A total of 49 virulence genes were identified. Of them, 25 genes encode cytoplasmic proteins, 6 genes encode cytoplasmic membrane proteins, 4 genes encode outer membrane proteins, and the remaining 14 gene products is currently unknown by subcellular localization analysis. The function classification of orthologous group clusters revealed that 16 genes are associated with metabolism, 6 genes are associated with cellular processing and signaling, and 4 genes are associated with information storage and processing. The function of the other 23 genes is poorly characterized or currently unknown.We identified a mutant strain RA2640 by Tn4351 transposon mutagenesis, in which the AS87₀4050 gene was inactivated by insertion of the transposon. Southern blot analysis indicated that only one insertion was found in the genome of the mutant strain RA2640. q RT-PCR analysis revealed that the expression of both upstream and downstream genes in mutant strain RA2640 showed no significant changes compared to wild-type strain Yb2. SDS-PAGE followed by silver staining showed that the lipopolysaccharide（LPS） pattern of mutant strain RA2640 was different from its wild-type strain Yb2, suggesting the LPS was defected. In addition, the phenotype of the mutant strain RA2640 was changed to rough-type, evident by altered colony morphology, autoaggregation ability and crystal violet staining characteristics. Bacterial LPS is a key factor in virulence as well as in both innate and acquired host responses to infection. The rough-type mutant strain RA2640 showed higher sensitivity to antibiotics, disinfectants and normal duck serum, and higher capability of adherence and invasion to Vero cells, compared to its wild-type strain Yb2. Moreover, the mutant strain RA2640 lost the agglutination ability of its wild-type strain Yb2 to R. anatipestifer serotype 2 positive sera, suggesting that the O-antigen is defected. LD50 for RA2640 was 1.91×1010 CFU, indicating that the virulence of mutant strain RA2640 was approximately 100,000-fold attenuated. After bacterial inoculation, wild-type strain Yb2 was recovered from blood at a quantity of 2.51×104 CFU/m L at 3 h.p.i, and 6.04×106 CFU/m L at 24 h.p.i. However, mutant strain RA2640 were not recovered from infected animals as Yb2 did, and showed significant reductions in its ability to colonize ducks, compared to the wild-type strain Yb2. These results suggested that the AS87₀4050 gene in R. anatipestifer is associated with the LPS biosynthesis and bacterial pathogenicity.In summary, the complete genome of R. anatipestifer virulent strain Yb2 was sequenced and assembled. We compared the Yb2 genome against the published genomes of R. anatipestifer DSM15868, RA-GD, RA-CH-1 and RA-CH-2 strains, and identified 49 virulence genes by genome-wide random transposon mutagenesis screening. This is the first genome-wide identification of genes involved in R. anatipestifer virulence. The AS87₀4050 gene was identified to be associated with the LPS biosynthesis and bacterial pathogenicity of R. anatipestifer.