Role Of Eno And IdeZ In Anti-phagocytosis Of Streptococcus Equisubsp. Zooepidemicus

Enolase(Eno)and immunoglobulin-degrading enzyme(IdeZ)are two virulence factors reported by Streptococcus equi.subsp.zooepidemicus(SEZ),but their biological functions and pathogenesis are not well studied.This study aims to elucidate the pathogenic mechanism of SEZ by elucidating the mechanism of the above two virulence factors in the process of anti-cell phagocytosis.1.Screening of SEZ Eno and Raw264.7 Cell Interacting ProteinsIn this study,cell-stable isotope labeling(SILAC)was used to label mouse alveolar macrophages(Raw264.7 cells).Under in vitro conditions,the total protein of Raw264.7 cells interacted with the recombinant Enolase(rEno)protein expressed in prokaryotic cells,and then the total protein of Raw264.7 cells was fractionated by mass spectrometry.LC-MS/MS analysis showed that 17 proteins possibly interacting with SEZ Eno protein in Raw264.7 cells were screened.After bioinformatics analysis of the screening results,the interaction between proteins was verified by immunoprecipitation(Co-IP).The results confirmed the interaction between Dctn,Itgam,Lgals,Gsdmdc,Snx6 protein and SEZ Eno.It lays a foundation for explaining the role of SEZ Eno protein in the process of bacterial anti-phagocytosis.2.Interaction between SEZ Eno and Dctn 2-encoded protein in Raw 264.7 cellsThe recombinant plasmid pGEX-6P-1-Dctn was constructed using Raw 264.7 cell genome as template,and its expression purity was induced.Dynactin subunit protein 2(Dctn)of about 44 kDa was obtained.Taking this as antigen,anti-Dctn polyclonal antibody was prepared by immunizing Balc/C mice.Serum was obtained by eyeball blood collection.By ELISA Methods,the titers of antibodies were determined and the results showed that the titers of antibodies in group A were higher than those in other groups.The titers of antibodies in group A were determined by Dot Blot and Western Blot,the results showed that the prepared mouse serum had good reactivity.And then Raw 264.7 pretreated with Eno,detection of intracellular Dctn protein level showed that compared with the control group,intracellular Dctn protein expression level was extremely high significant increased.It is presumed that the interaction between Eno and Dctn may lead to the higher expression level of intracellular Dctn in Raw264.7 cells.The changes of Dctn protein expression level lay a foundation for further exploring the mechanism of recombinant Eno protein on Raw264.7 cells.3.Identification of Biological Activity of Antibody Degrading Protein SEZ IdeZPrevious studies found that SEZ ATCC35246 strain IdeZ had high homology with Streptococcus pyogenes antibody degrading protein(IdeS)by genomic sequence alignment.To further understand the biological function of IdeZ encoding protein in SEZ and its possible role in bacterial resistance to phagocytosis.In this study,pCold-SUMO-IdeZ recombinant plasmid was constructed and induced to express and purify recombinant IdeZ protein with a size of about 39 kDa.In vitro,the conditions of antibody degradation were simulated.The results showed that the degradation products of IgG could be detected by 10 minutes in vitro.And the Raw264.7 cell model of macrophages was used to simulate macrophage phagocytosis SEZ test in vitro to detect IdeZ protein antagonism.The results showed that IdeZ protein could effectively inhibit the regulation and phagocytosis of anti-SEZ immune serum or purified IgG,and help SEZ resist phagocytosis.This study provides a new entry point for further revealing the mechanism of SEZ resistance to macrophage phagocytosis.