| Background:Staphylococcus aureus（S.aureus）is one of the important pathogens that causes clinical infections.According to the latest release by China Antimicrobial Surveillance Network,S.aureus ranked third in prevalence among the 301917 isolated pathogens,accounting for about 8.93%of clinical infections in China in 2021.S.aureus can cause various infective diseases including skin and soft-tissue infection（SSTIs）,bloodstream infection,pneumonia,infective endocarditis,burn and war wound infection,septicemia and toxic shock syndrome.S.aureus is the most frequent organism in SSTIs which accounting for more than 30%of adult patients or more than 80%of pediatric patients.In addition to infectious diseases,the colonization of S.aureus is also related to the occurrence and development of chronic inflammatory diseases.Studies have shown that S.aureus colonizes about 20%of the human population asymptomatically in the nares,skin,pharynx,and gastrointestinal tract.Compared with healthy people,higher prevalence of S.aureus colonization in patients with chronic inflammatory diseases such as diabetes,atopic rash,psoriasis,chronic obstructive pulmonary disease and chronic nephritis.Especially,the prevalence of S.aureus carriage was more than 50%in patients with chronic inflammatory skin diseases such as psoriasis and atopic dermatitis.β-hemolysin（Hlb）,is an exotoxin of S.aureus,which has neutral sphingomyelinase activity and DNA binding activity.It is believed that Hlb protein hardly plays a role in the process of S.aureus infection due to the lack of Hlb protein in most clinical isolated.The bacteriophage Sa3mw inserts into the hlb gene in human strains,inactivating it in the majority of S.aureus clonal groups.Hence,most strains do not secrete Hlb protein.However,plenty evidence had suggested that during chronic infections,host pressure and antibiotic treatment favors phage excision with consequent production of Hlb protein,possibly benefiting S.aureus survival and disease progression.Epidemiological investigation showed that over 40%of isolates from SSTIs patients and over 50%of isolates from atopic dermatitis patients were positive for Hlb protein.The high prevalence of Hlb protein in skin focus indicates that Hlb protein may be involved in the process of S.aureus skin infection.This study was aimed to investigate whether Hlb involved in the skin infection induced by S.aureus and the relevant molecular mechanism so as to provide candidate drug targets and a new strategy for the prevention and treatment of S.aureus skin infection.Methods:Recombined Hlb protein was expressed by E.coli expression system.The endotoxin of purified Hlb protein was removed by endotoxin removing resin,and bacteria were removed through bacterial filters.The mouse skin inflammation model was induced by subcutaneous injection of Hlb protein.The phosphorylated membrane receptors which induced by Hlb protein were screened by Proteome Profiler Human Phospho-RTK Array Kit.The change of phosphorylated epidermal growth factor receptor（EGFR）was detected with the specific antibody by Western Blot.HE（hematoxylin-eosin）staining was used to observe the skin tissue of mice,and IHC（immunocytochemistry）was used to detect the types of inflammatory cells.ELISA and GST pull down were used to detect the binding of Hlb protein to EGFR.ELISA was used to detect the expression of IL-8.Flow cytometry was used to detect the expression of CD107a.ELISA was used to detect the expression of IFN-γ.RT-PCR was used to detect the change of FOSB and JUN m RNA expressions level in NK-92 cells.Virtual screening method was used to screen compounds that binding to Hlb protein.Hemolysis assays were used to screen compounds that block the activity of Hlb protein.Results（including the following six parts）:First,Hlb protein successfully provoked the mouse skin inflammation.The Hlb protein subcutaneous injection site showed welling and redness,epilation and ulceration.HE stained section showed an accumulation of leukocytes in the thickened epidermis and dermis at the injection site.Second,although Hlb protein is a hemolysin,most host cells cannot be lysed even under’hot-cold’conditions.And“hot-cold”cannot exist in the physiological state.It was confirmed that epidermal cells（Ha Ca T）were not sensitive to Hlb protein,indicating that the skin inflammation was induced independently on the lysis function.Receptor tyrosine kinase（RTK）,located in the cell membrane,is a kind of important regulatory enzyme-linked receptor.RTK activation is triggered by binding ligands,which leads to auto-phosphorylation and activation of downstream signaling pathways.The changes of abundance and activation level of RTK are closely related to inflammation,diabetes,cardiovascular diseases and tumors.By detecting the phosphorylation level of RTKs on Ha Ca T（Human epidermal cells）and HFF-1（Human skin fibroblasts）,it was found that EGFR was activated by Hlb protein.It was further confirmed that Hlb protein promoted the phosphorylation of EGFR at the cellular level and in vivo.And erlotinib,an inhibitor of EGFR,could block this activation.Moreover,blocking EGFR with erlotinib,the skin inflammation of mice was significantly relieved.These data suggest that abnormal activation of EGFR is involved in skin inflammation provoked by Hlb protein.Third,in order to explore the molecular mechanism of Hlb protein activating EGFR,recombined Hlb H-288-N、Hlb H-149-Nand Hlb H-161-A mutants were expressed.Hlb H-288-Nand Hlb H-149-N mutants,which abolished neutral sphingomyelinase activity,could not activate EGFR while Hlb H-161-A mutant,which abolished DNA binding activity,still activated EGFR.And the addition of sphingomyelin in Ha Ca T and A549 cell culture system can block the activation of EGFR induced by Hlb protein.Theoretically,Hlb protein has neutral sphingomyelinase activity,which cleaves sphingomyelin to generate ceramide and soluble choline phosphate.After A549 cells were incubated with Hlb protein,choline phosphate was detected in the supernatant,indicating that Hlb protein degraded sphingomyelin on the cell membrane in the current culture system.However,neither exogenous cell membrane permeable ceramide（C2,C6 ceramide）nor choline phosphate could activate EGFR,indicating that the activation of EGFR induced by Hlb protein independent of the products of sphingomyelin degradation.EGFR is usually activated by its specific ligand.At present,there are seven kinds of EGFR ligands:epidermal growth factor（EGF）and transforming growth factorα（TGFα）,amphiregulin（AR）,epiregulin（EPR）,heparin binding EGF-like growth factor（HB-EGF）,epigen（EPN）and betacellulin（BTC）.The EGFR ligands are synthesized as type I transmembrane proteins,which are mainly hydrolyzed into soluble mature bodies by ADAM（a disintegrin and metalloprotease）proteins.Although we were unable to find the expression of these seven endogenous ligands in Ha Ca T cells with their specific antibodies,broad-spectrum metalloproteinase inhibitors and ADAM17 inhibitors partially blocked the phosphorylation of EGFR mediated by Hlb protein,which suggesting that Hib protein may promote the cleavage of ligand by ADAM.Further studies found that Hlb protein could bind to EGFR and could promote the phosphorylation of EGFR in cell-free system,indicating that Hlb protein may act as an exogenous ligand to directly activate EGFR.Fourth,recombined Hlb H-288-N,Hlb H-149-Nand Hlb H-161-A mutants were injected subcutaneously into mice skin.It was found that Hlb H-288-N mutant could not induce skin inflammation in BALB/c mice.Further study found that Hlb protein can induce skin inflammation in C57BL/6N mice and BALB/c Nude mice.BALB/C Nude mice are T lymphocyte deficient mice,but there are still plenty inflammatory cells at the injection site of Hlb protein,indicating that T cells may not be involved in skin inflammation induced by Hlb protein.Further,anti-CD11b（targeting mononuclear macrophages,granulocytes and DCs）,anti-Ly6G（targeting neutrophils）and anti-NKp46（targeting natural killer cells）were used to identify the cell types in inflammatory sites.It was found that the cells in the inflammatory part of the skin were mainly CD11b+cells and NK cells,and there were a small amount of neutrophils in the early stage of inflammatory reaction.Furthermore,the secretion of chemokine IL-8 was up regulated by Hlb protein in Ha Ca T cells,suggesting that Hlb protein may recruit inflammatory cells by promoting the secretion of chemokines.Fifth,NK cells were detected in the inflammatory skin tissue of mice induced by Hlb protein,and our previous studies have found that Hlb protein induced CD56brightNK cells to secrete IFN-γ.Therefore,the activation on NK cells induced by Hlb protein was further studied.It was further confirmed that Hlb protein up-regulated the expression of CD107a in NK-92 cells and primary NK cells,and also promoted the killing of K562cells by NK-92 cells,indicating that Hlb protein induced the activation of NK cells.Further studies found that the mechanism might include two aspects.On the one hand,Hlb protein upregulate the expression of Fos B and Jun may by activating JNK.Fos B and Jun,two subunits of transcription factor AP-1,dimerize into AP-1,and then upregulate IFN-γexpression.On the other hand,through calcium signal,Hlb protein may promote the transport of CD107a from lysosomal to the surface of cell membrane.Finally,Hlb protein plays an important role in the colonization of S.aureus and the progression of several chronic inflammation.Therefore it may become a drug candidate target for the prevention and treatment of S.aureus skin infection.59 compounds binding to Hlb protein were screened and synthesized,and further 2 compounds that can block the hemolytic activity of Hlb protein were confirmed.Conclusion:S.aureus Hlb protein can cause skin inflammation in mice.The mechanism might be that Hlb protein causes abnormal activation of EGFR and its downstream signal pathways through two ways in skin tissue cells,resulting in the overexpression of chemokines.Chemokines recruit inflammatory cells and induce skin inflammation in mice.This study enriches the mechanism of the interaction between Hlb protein and the host,as well as provides candidate drug targets and a new strategy for the prevention and treatment of S.aureus skin infection caused by S.aureus. |
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