Subtilisin-like Serine Protease A Mediated Complement Evasion Of Streptococcus Suis Serotype 2

The innate immune system acts as an initial protective barrier against bacterial pathogens and defends the host until the acquired immune system is activated.The complement system is an essential part of the innate immune system.It can rapidly recognize and opsonize bacteria for phagocytosis by professional phagocytes.Therefore,,pathogenic bacteria have evolved a series of mechanisms of complement evasion to escape complement attack,such as modulation of the initial steps;modulation of convertases;modulation of C3 and its split products;modulation of the terminal pathway;interactions with host regulators;modulation of complement receptors and so on.Streptococcus suis serotype 2?SS2?is an important zoonotic pathogen,can cause swine sepsis,meningitis,pneumonia,arthritis,etc.,can also cause meningitis and toxic shock syndrome in humans.SS2 highly virulent strains can escape from the immune surveillance to survive in host blood-stream and conoloze in tissues for a long term.The mechamisms of immune evasion remain unclear.The complements C3a and C5a are two important chemokines and play important roles in inflammation response and opsonophagocytosis.In order to study the mechanisms of SS2 complement evasion,we used the complements C3a and C5a as baits to screen and identify C3a-and C5a-binding proteins from the gene expression library of SS2 secreted proteins and membrane proteins by Bacterial two-hybrid experiments.Among these C3a-and C5a-binding proteins,two subtilisin-like serine protease A?SspA?were further studied.They can interact with and hydrolyse C3a and C5a,and consequently inhibit the chemotaxis of C3a and C3a.1.Identification of C3a-and C5a-binding proteins from SS2 secreted and cell wall-anchored proteins.The cDNAs of complement C3a and C5a were cloned into the pBT plasmid respectively,and used to screen the gene expression library of SS2 secreted proteins and cell wall-anchored proteins by Bacterial two-hybrid experiments.13 SS2 proteins were identified to be interacted with C3a,they are SSU05₀179 SSU05₀214,SSU05₀246,SSU05₀274,SSU05₀802,SSU05₀812,SSU05₁213,SSU05₁214,SSU05₁539,SSU05₁664,SSU05₂065,SSU05₂100 and SSU05 2104.5 SS2 proteins displayed potential interaction with C5a,they are SSU05₀246 SSU05₀431,SSU05₁213,SSU05 1293 and SSU05₂100.2.Validation of the interactions of SspA to C3a and C5a.The protein domain of SspA-1?NJAUSSU 0853?and SspA-2?SSU05 1982?are analyzed.According to the result,SspA-1 and SspA-2 are piecewised express and purificated as SspA-1-S?SspA-2-S?SspA-1-L and SspA-2-L.Through ELISA test,confirme that both SspA-1 and SspA-2 have interaction with C3a,however,only SspA-2 has interaction with C5a.3.SspA cleavage activity to C3a and C5a.In order to study wether the SspA-1 and SspA-2 can hydrolyze C3a and/or C5a by interacting,we compound fluorescent protein substrates of people C3a and C5a with DABCYL perssad and EDANS perssad.The results of fluorescence resonance energy transfer?FRET?found that C5a peptide enzyme activity structure domain of SspA-1 and SspA-2 can carve C5a,but showed no concentration dependence;SspA-1 and SspA-2 C5a peptide enzyme activity structure domain can also carve C3a,and showed a concentration dependent effect.4.SspA inhibits the chemotaxis activities of C3a and C5a.Through in vitro monocyte chemotactic experiment found that incubated with SspA-1 and SspA-2 respectively can significantly reduce chemotaxis ability of C3a and?p<0.01?,proving that SspA-1 and SspA-2 do have the ability to inhibit C3a,C5a chemokines.In conclusion,SS2 can secrete two kinds of subtilisin-like serine protease A?SspA-1 and SspA-2?,they can carve complement C3a and C5a,as C3a peptidase and C5a peptidase all can inhibite C3a and C5a chemotaxis,escape the monitoring of thecompleent system.