Identification of genetic determinants of hemolytic activity of Riemerella anatipestifer using random transposon mutagenesis.

Riemerella anatipestifer causes epizootic infectious disease in poultry resulting in serious economic losses especially to the duck industry. In our previous study, R. anatipestifer was found to lyse duck erythrocytes in vitro. In the present study, a random Tn4351 mutagenesis library of hemolytic R. anatipestifer strain SX containing 4000 mutants was constructed to investigate the genetic basis of hemolytic activity. Thirty mutants with reduced hemolytic activity and one with increased hemolytic activity were screened and insertions in 24 genes were identified. Of these genes, four were predicted to encode outer membrane proteins, one encoded a cytoplasmic membrane protein, 11 encoded cytoplasmic proteins, and eight encoded proteins with unknown locations. Based on current annotations of the R. anatipestifer genomes, of the 24 genes, 7 (29.17%) were involved in iron utilization. The hemolytic activities of the complemented strains M2 (pRES-Riean_0790) and M18 (pRES-Riean_0653) were restored, indicating that both Riean_0653 and Riean_0790 are involved in the hemolytic activity of strain SX. However, the recombinant proteins rRiean_0317, rRiean_0790, rRiean_0653, rRiean_1027, rRiean_1143, and rRiean_1561 had no hemolytic activity, suggesting that none were hemolysins.

Development of signature-tagged mutagenesis in Riemerella anatipestifer to identify genes essential for survival and pathogenesis.

Riemerella anatipestifer causes epizootic infectious disease in ducks, geese, turkeys and other birds, and serious economic losses especially to the duck industry. However, little is known about the molecular basis of its pathogenesis. In this study, signature-tagged transposon mutagenesis based on Tn4351 was developed in R. anatipestifer to identify genes essential for survival and pathogenesis. Seventeen tagged Tn4351 random mutation libraries of the R. anatipestifer strain WJ4 containing 5100 mutants were screened for survive using a duckling infection model. Twenty mutants that could not be recovered from the infected ducklings, were identified, and 17 mutated genes were identified by inverse PCR or genome-walking PCR. Of these genes, FIP52_03215, FIP52_04350 and FIP52_09345, were inserted into two mutant strains, and FIP52_03215 and FIP52_03175 were found exclusively on the chromosome of serotype 1 R. anatipestifer strains. Twelve out of 17 genes encoding for proteins were predicted to be involved in amino acid, nucleotide, coenzyme, or lipid transport and metabolism, one gene was predicted to be involved in signal transduction, one gene was predicted to be involved in DNA replication, recombination and repair, the other three genes had an unknown function. Animal experiments showed that the virulence of mutants 16-284, 7-295, 24-231, 9-232 and 19-214 were significantly attenuated compared to that of the wild-type WJ4. Moreover, the median lethal dose of mutant 16-284 was greater than 1010 CFU, and its virulence to ducklings was partially restored when it was complemented with the shuttle expression plasmid pRES-FIP52_09345. The results in this study will be helpful to further study the molecular mechanisms of the pathogenesis of R. anatipestifer infection.