Riemerella Anatiptesitfer AS87₀8785 Gene Encodes A Functional Component GldK Of The Type IX Secretion System

Riemerella anatipestifer is an important pathogen of water fowl,causing septisemic and exudative diseases.Determination of virulence factors will help us to understand the pathogenic mechanism of this bacterium and to develop an effective strategy for its control.R.anatipestier has a protein secretion system known as type IX secretion system,we previously described that deletion of AS87₀8785 gene significantly decreased the bacterial virulence of R.anatipestifer strain Yb2,to explore whether the type IX secretion system component R.anatipestifer gldK?AS87₀8785?gene is involved in the virulence through secretion of factors associated with pathogenesis,in this study,R.anatipestifer AS87₀8785 gene mutation and complementation strains were constructed and characterized.Sequence analysis indicated that the AS87₀8785 gene encodes putative GldK protein,which was identified to be localized at the membrane fraction by western blotting analysis.Liquid chromatography-tandem mass spectrometry analysis revealed four proteins,which had typical T9 SS CTDs,were absent from the Yb2?gldK culture.A further assay revealed that mutant strain Yb2?gldK was defective in the digestion of proteins,supporting the notion that enzymes involved in the digestion of these polymers are secreted by T9 SS.The proteolytic activity of complementation strain cYb2?gldK was restored.The mutant strain Yb2?gldK presented a defective gliding motility on the agar plates,decreased protease activity and decreased protein secretion ability.RNA-sequencing and qPCR analysis indicated that transcription of thirteen genes were down-regulated in the mutant Yb2?gldK.Animal experiments showed that bacterial loads in blood of Yb2?gldK infected ducks were significantly decreased compared with those of wild-type strain Yb2-infected ducks.The complementation strain cYb2?gldK recovered most of the defected biological properties.Together our results indicated that R.anatipestifer AS87₀8785 gene encodes a key component GldK of the type IX secretion system,which functions on the bacterial gliding motility,protein secretion and bacterial virulence.