The Mechanisms By Which Staphopain B From Staphylococci Cleaves Interleukin-33 To Aggravate Diabetic Skin Infection

Wound infection is one of the main reasons to cause impaired wound healing in diabetes.Staphylococcus aureus is a primary pathogen of diabetic wound infection.S.aureus expresses multiple virulence factors,including enterotoxins,cytolytic toxins,nucleases and proteases,to evade host defences.Interleukin-33(IL-33),as a new member of the IL-1 cytokine family,has been shown to be increased in S.aureus-infected skin.Although it is known that IL-33 enhances host antibacterial defenses through inducing NO release and REG3A expression,whether S.aureus could escape from host defenses by inactivating IL-33 remains unknown.In this study,we found that IL-33 was increased in the skin of normal patients infected by S.aureus,but significantly decreased in the skin of diabetic patients infected by S.aureus.We next developed an S.aureus-infected diabetic mice model to detect IL-33 expression in lesional skin.Similary,we found the production of IL-33 and antimicrobial protein Reg?? was decreased in diabetic mice infected with S.aureus compare to those in the skin of S.aureus-infected normal mice.In contrast to decreased production of IL-33 and Reg??,the survial of S.aureus in diabetic mouse skin and proteolytic activity of S.aureus were increased.To determine whether hyperglycemia could regulate the protease activity of S.aureus to cleave IL-33,we next cultured S.aureus with different doses of glucose to mimic hyperglycemia in vitro.We found that the mRNA expression of cysteine proteases Staphopain A and Staphopain B(ScpA,SspB)was dramatically increased by high glucose through the regulation of accessory gene regulator protein A(agrA),thus leading to enhanced proteolytic activity.To our surprise,IL-33 and Reg?? were not decreased in the skin from diabetic mice infected with S.aureus sspp(ScpA)mutant.We thereby focused on investigating the function of S.aureus SspB.To investigate whether Staphopain B would cleave/degrade IL-33 or Reg??,IL-33 or Reg?? was incubated with Staphopain B.IL-33 but not Reg?? was cleaved by Staphopain B.All thses data suggest that Staphopain B cleaves IL-33,thus leading to decreased Reg?? expression.Moreover,N-terminal amino acid sequencing showed that Staphopain B cleaved IL-33 at Gly97,and protein structure prediction further suggested that the binding capacity of cleaved peptide IL-3 3 98-270 with its receptor ST2 was decreased compared to that of mature IL-33,thus resulting in a function-impaired peptide IL-3398-270.Finally,the in vivo infection experiments showed that IL-33 production and Reg?? expression were restored in the skin of diabetic mice infected by S.aureus sspb mutant compared to those in diabetic mice infected by S.aureus wild type.Taken together,our data demonstrate that S.aureus infection increases IL-33 and Reg?? expression in skin of normal mice,while hyperglycemia in diabetic mice increases S.aureus SspB expression,thus cleaving full length IL-33 at Gly97.The induction of REG3A by cleaved IL-3398-270 is decreased,which is associated with decreased host defence but increased S.aureuus infection.Our findings provide new insights into the mechanism by which S.aureus infection impairs wound healing in diabetes,and provide a potential therapeutic target for treatment of S.aureus infection in diabetes.