The Mechanism Of GldM Protein In The Pathogenic Process Of Riemerella Anatipestifer

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 world duck industry.R.anatipestifer is a member of the family Flavobacteriaceae and belongs to the phylum Bacteroidetes.Cells crawl rapidly over surfaces in a process known as gliding motility,this form of movement is common throughout the phylum Bacteroidetes.Genetic analysis showed that R.anatipestifer has the genes associated with the gliding motility and has a novel protein secretion system,known as the ‘type IX secretion system'(T9SS).We previously reported that deletion of the AS87_RS08465 gene significantly reduced the bacterial virulence of R.anatipestifer strain Yb2,although the mechanism remained unclear.The AS87_RS08465 gene is predicted to encode protein GldM,a key component of the T9 SS complex.In this study,western blotting analysis demonstrated that R.anatipestifer GldM localizes to the cytomembrane.Cells of R.anatipestifer can remove over the TSA plate,but the muant strain can't.Further study revealed that the adhesion and invasion capacities of the mutant strain RA2281(designated Yb2?gldM)in Vero cells and in bacterial loads in the blood of infected ducks were significantly reduced.RNA-Seq and PCR analyses showed that 15 genes were upregulated(six genes were upregulated > 2-fold)or downregulated(five genes were downregulated > 2-fold)in mutant strain Yb2?gldM,which were mainly involved in the secretion of proteins;furthermore,each of these identified genes was predicted to be involved in T9 SS.These results suggest that AS87_RS08465 regulates genes that are mainly responsible for protein secretion and gliding motility in R.anatipestifer.We collected the secretion proteins of the wild-type strain Yb2,mutant strain Yb2?gldM and the complementation strain cYb2?gldM.The SDS-PAGE analysis found that the amont of secretion proteins was significantly decreased in mutant strain Yb2?gldM.Our results also indicate that mutant strain Yb2?gldM was defective in the digestion of proteins.Furthermore,a liquid chromatography–tandem mass spectrometry analysis revealed that nine of the proteins differentially secreted by Yb2?gldM and Yb2,which had a conserved T9 SS C-terminal domain.Taken together,these results indicate that R.anatipestifer GldM is associated with T9 SS and is important in bacterial virulence.