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

Bingqing Sun,Xiaohua He,Haoyang Liu,Nazrul Islam,Qinghai Hu,Xiangqiang Tian,Zhonghao Hu,Xiaoli Du,Zuocheng Zou,Yafei Xue

doi:10.1186/s13567-021-00900-6

Abstract

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.

Highlights

Riemerella anatipestifer, the type species of the genus Riemerella in the family Flavobacteriaceae, mainly infects domestic ducks, geese, turkeys, and other birds
Screening of the hemolytic activities of the R. anatipestifer SX mutant strains A random Tn4351 transposon mutagenesis library of R. anatipestifer strain SX containing 4000 mutants was constructed with biparental mating of E. coli strain BW19851 with R. anatipestifer strain SX
The results showed that the bacterial loads of blood of ducklings infected with the complemented strain M2 or M18 were significantly restored as compared to those infected with M2 or M18

Summary

Introduction

Riemerella anatipestifer, the type species of the genus Riemerella in the family Flavobacteriaceae, mainly infects domestic ducks, geese, turkeys, and other birds. R. anatipestifer infection primarily causes an acute septicemic disease in younger birds and more chronic and localized lesions in older birds, resulting in significant economic losses to the duck industry worldwide [1]. The mechanisms underlying the pathogenesis of R. anatipestifer infection remain unclear. Some R. anatipestifer strains caused hemolysis on 5% bovine blood agar over a 14-day examination period [2] and on Columbia agar base with 7% defibrinated sheep blood [3]. A previous study by our group showed that 29 (55.77%) of 52 R. anatipestifer strains caused hemolysis on duck blood agar and observations by phase contrast microscopy showed that all of the strains generated pores in the membranes of duck erythrocytes in suspension [4].

Methods

Results

Conclusion