Functional Studies Of Glycoside Hydrolases SSU05?1921 And SSU05?1922 From Streptococcus Suis Serotype 2 Strain 05ZYH33

Streptococcus suis is an important zoonotic pathogen,causing serious diseases and death both in swine and human.It is responsible for important economic losses in swine industry and seriously threatens public health.Exploration of virulence factors and their function in S.suis is of great significance to reveal the pathogenesis of S.suis and to prevent its infection.Glycoside hydrolases(GHs)are enzymes that cleave the glycosidic bonds of glycoconjugates,oligosaccharides and polysaccharides.Several studies revealed that GHs from pathogens contributed to nutrient acquisition by degrading the glycans on host glycoproteins.GHs also contributed to immune evasion by targeting host immune components,enabling successful colonization and/or invasion.Therefore,GHs are potential virulence factors.In this study,domain analysis revealed SSU05₁921 and SSU05₁922 from S.suis serotype 2 strain 05ZYH33 contain catalytic domain of families GH92 and GH85,respectively.Protein expression and purification were performed to obtain the purified proteins of SSU05₁921,SSU05₁922 and SSU05₁922Cat.By combination of RNase B deglycosylation and MALDI-TOF/TOF-MS,the activities of SSU05₁921 and SSU05₁922 were investigated in detail.The results revealed that SSU05₁921 and SSU05₁922 sequentially act on high-mannose N-glycan.SSU05₁921 is an a-1,2-mannosidase that is active on the a-1,2-mannose decorations of high-mannose N-glycans,while SSU05₁922 cleaves the chitobiose core to release the glycan from the protein.However,SSU05₁922 is only able to carry out this function on Nglycans that carry five or less mannose residues.When SSU05₁921 and SSU05₁922 together on RNase B,the optimum working pH was 7.0-8.0,and the reaction showed no obvious dependence on metal ion.Gene deletion strains ssu05₁922? and ssu05₁922? were constructed by pheromone-induced compentence and recombination.Growth curve showed that the gene-deletion and the wild type(WT)strains exhibited similar growth rates.RNase B deglycosylation by whole cells of ssu05₁922? strain and the WT strain indicated that SSU05₁922 is the only protein having the endo-N-acetylglucosaminidase enzyme activity on the cell surface of the 05ZYH33.Mouse infection experiment showed that ssu05₁921 gene was a potential virulence factor.However,its product SSU05₁921 could not be detected in any cell fraction either by Western blotting or by RNase B deglycosylation.To detect SSU05₁921 in 05ZYH33,we screened and identified the strong promoter of 05ZYH33,and constructed SSU05₁921 overexpression strain,enabling its detection in 05ZYH33,and further confirming its ?-1,2-mannosidase activity.In conclusion,SSU05₁921 and SSU05₁922 were GHs.These two enzymes sequentially degrade high-mannose N-glycan.Furthermore,SSU05₁921 is a potential virulence factor of 05ZYH33.Therefore,degradation of high mannose N-glycan by S.suis is very possibly correlated with its pathogenesis,and SSU05₁921 is a potential drug target for prevention and treatment of S.suis infection.