The Role Of PurC In The Meningitis Caused By Streptococcus Equi Subsp.zooepidemicus

Streptococcusequi subsp.zooepidemicus(SEZ)belongs to the group C streptococci beta-hemolytic streptococcus,which can cause sepsis,arthritis,endocarditis and meningitis.The strain has a broad host spectrum that infects pigs,horses,dogs,cats,and other animals closely related to humans.In China,SEZ is an important porcine pathogen causing swine streptococcal disease,and it is one of the major pathogens causing swine meningitis.Meanwhile,a large number of cases aboutmeningitis caused by SEZ in humans have been reported worldwide.Most cases are caused by close contact with infected animals or eating contaminated foods.Therefore,the zoonotic infection and meningitis disease means SEZ is a great threat to human health.1.Screening of the interaction protein between SEZ PurC and HEK 293TPhosphoribosylaminoimidazole-succinocarboxamide synthetase(SAICAR synthetase,PurC,EC 6.3.2.6)has been known as a catalyzing enzyme participating in the seventh step in the de novo purine biosynthetic pathway.In our previous research,we sequenced the whole genome of a SEZ virulent strain ATCC35246.After comparative genome analysis,a SEZ ATCC35246-specific purC gene was discovered in its pathogenicity island II.In this study,SILAC and LC-MS/MS were used to screen proteins that may interact with PurC in 293T cells.We used SILAC medium to label HEK 293T cells.SEZ PurC eukaryotic expression vector PurC-pAcGFP was transfected into heavy labeled HEK 293T cells.At the same time,we transfected light-labeled 293T cells with pAcGFP plasmid.The cell lysates of two group were mixed equally and CO-IP was used to enrich the binding protein,followed by mass spectrometry detection.19 differential proteins were detected.We selected some of these proteins for mutual validation.The validation results confirmed the existence of interactions between PurC and Ezrin,CIP4 or Elmod2.2.The PurC of SEZ disrupts BBB through modulating phosphorylation of Moesin in cerebral endothelial cellPenetration of the blood-brain-barrier(BBB)is the precondition for SEZ to cause meningitis in human.Moesin was verified as PurC interacting protein through Co-IP and SPR.The interaction site of Moesin is C-ERMAD domain,which is related to Thr558 phosphorylation activation under PurC treatment.Phosphorylated Moesin bind Dbl,a RhoA activator,and lead to RhoA activation.The abnormal activation of RhoA induces stress fiber formation and cytoskeleton dynamic disorder,causing cell shape to fail and BBB integrity to collapse,thereby allowing SEZ to penetrate the BBB.3.The PurC of SEZ modulate p-Ezrin in neutrophil to promote uropod formation and migrationInflammation is a defensive reaction of the immune system against pathogenic bacteria,but excessive immune response can cause serious inflammation damage,especially the inflammatory reaction in brain tissue will be life-threatening.Our previous study found that purC gene is significantly related to the virulence of SEZ,and PurC may interact with Ezrin.In this study,PurC was confirmed to interact with Ezrin and increase the phosphorylation of EzrinThr567 in neutrophils.Binding of p-Ezrin to Dbl causes activation of Rac1,resulting in uropod formation in neutrophils.In addition,p-Ezrin recruit and localize the selectin receptors CD44 and PSGL-1 in the uropod,promoting a large number of neutrophils penetrate the BBB and cause inflammation damage.4.SILAC and LC-MS/MS identification of SEZ proteins that contribute to mBMEC infectionSEZ causes meningitis in both humans and animals.Some dissociative proteins of SEZ are cytotoxic to mouse brain microvascular endothelial cells(mBMECs)and may contribute to the penetration of SEZ across the BBB.In this study,the ability of SEZ to penetrate across an in vitro BBB model was confirmed.We used SILAC to label SEZ proteins with heavy or light isotopetagged amino acids,along with LC-MS/MS to determine which SEZ proteins were involved in interactions with mBMECs.The efficiency of SEZ protein isotope labeling was 94.7%,which was sufficient for further analysis.Fortynine labeled peptides were identified as binding to mBMECs,which matched to 25 SEZ proteins.Bioinformatic analysis indicated that most of these proteins were cytoplasmic.These proteins may have functions in breaching the host BBB,and some of them are known virulence factors in other bacteria.Indirect immunofluorescence results indicated that SEZ enolase had binding activity toward mBMECs.Protective test results showed that enolase was a protective antigen against SEZ infection.