Identification Of The T9SS Secretory Proteins Of Riemerella Anatipestifer And Study On The Pathogenesis Of SspA

Riemerella anatipestifer is a Gram-negative,rod-shaped bacterium,nonmotile and nonspore forming,and it is a member of the family Flavobacteriaceae and belongs to the phylum Bacteroidetes.R.anatipestifer can infect ducklings,turkeys,gooses and other birds,and is a major pathogenic agent of duck septicemic and exudative diseases.Recent studies show that R.anatipestifer type IX secretion system（T9SS）acts as a crucial virulence factor.We previously identified two T9SS component proteins Gld K and Gld M,which play important roles in the bacterial virulence.In this study,19 T9SS secretory proteins,which harbor a T9SS conserved C-terminal domain（CTD）,were predicted in the R.anatipestifer strain Yb2 by searching CTD sequence in the whole genome,and confirmed using liquid chromatography–tandem mass spectrometry analysis of the bacterial culture supernatant.Nine of them have been reported in our previous research.Furthermore,recombinant proteins and mice antisera of the19 predicted proteins were generated to validate their expression in the bacterial culture supernatant and in the bacterial cells.Results showed that quantities of 14 proteins were significantly decreased in the T9SS mutant Yb2Δgld M culture but increased in the bacterial cells using Western blotting analysis.q RT-PCR indicated no expression difference of these genes between the wild-type strain Yb2 and T9SS mutant Yb2Δgld M.Moreover,19 mutant strains were successfully constructed for determination of the bacterial virulence and proteolytic activity,indicating that seven proteins were determined to be associated with the bacterial virulence,and two proteins AS87＿RS04190 and AS87＿RS07295 were protease activity-associated virulence factors.In summary,we revealed at least 19 T9SS secretory proteins in the R.anatipestifer strain Yb2 genome,which play multifunction on the bacterial virulence and proteolytic activity.A bioinformatic analysis indicated the AS87＿RS04190 protein contains a T9SS C-terminal domain sequence and encodes a putative subtilisin-like serine protease（SspA）.To determine the role of the putative SspA protein in R.anatipestifer pathogenesis and proteolysis,we determined sspA mutation and complementation median lethal doses,bacterial loads in infected duck blood,and their adherence to and invasion of cells.Our results demonstrate that the SspA protein functions in bacterial virulence.It is also associated with the bacterial protease activity,and has a conserved catalytic triad structure（Asp126,His158,and Ser410）,which is necessary for protein function.The optimal reactive p H and temperature were determined to be 7.0 and 50°C,respectively,and K_m and V_max were determined to be 10.15 m M and 246.96 U/mg,respectively.The enzymatic activity of SspA is activated by Ca²⁺,Mg²⁺,and Mn²⁺,and inhibited by Cu²⁺and EDTA.SspA degrades gelatin,fibrinogen,and bacitracin LL-37.These results demonstrate that SspA is an effector protein of T9SS and functions in R.anatipestifer virulence and its proteolysis of gelatin,fibrinogen,and bacitracin LL-37.Protease has been reported to destruct the host blood-brain barrier（BBB）and be associated with bacterial meningitis.R.anatipestifer infection can also cause meningitis.R.anatipestifer SspA is a protease,which can degrade a variety of cellular components such as gelatin.In vivo（animal experiments）and in vitro（BBB model）experiments confirmed that SspA deletion R.anatipestifer mutant strain Yb2ΔsspA can not destroy BBB and enter brain tissue to cause meningitis as the wld-type strain Yb2 did,suggesting that SspA is related to meningitis caused by R.anatipestifer.Western blotting showed that the expression levels of claudin-5,Occludin,and CollagenⅣin brain microvascular endothelial cells（BMEC）were significantly reduced and immunofluorescence assay further showed that the fluorescence intensity of these proteins was similarly significantly reduced during R.anatipestifer wild-type strain Yb2 infection,compared to mutant strain Yb2ΔsspA infection.q RT-PCR indicated no expression difference of these genes between the wild-type strain Yb2 and mutant strain Yb2ΔsspA.The subsequent co-transfection test showed that SspA hydrolyzed Occludin in a dose-dependent manner and the co-incubation tests confirmed that the secreted proteins of wild strain Yb2 and complementation strain c Yb2ΔsspA as well as r SspA could effectively degrade Claudin-5,Occludin and CollagenⅣ.However,the secreted proteins of the mutant strain Yb2ΔsspA could not.These results demonstrated that SspA is necessary for the host blood-brain barrier damage and meningitis occurrence by proteolytic hydrolysis during R.anatipestifer infection.Through identification of the R.anatipestifer T9SS effectors and study the function of SspA will conducive to in-depth understanding of the pathogenesis of R.anatipestifer,and provide theoretical basis for R.anatipestifer vaccine development,clinical diagnosis and treatment.