SILAC and LC-MS/MS identification of Streptococcus equi ssp. zooepidemicus proteins that contribute to mouse brain microvascular endothelial cell infection

Streptococcus equi ssp. zooepidemicus (SEZ) 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 blood-brain barrier (BBB). In this study, the ability of SEZ to penetrate across an in vitro BBB model was confirmed. We used stable isotope labeling with amino acids in cell culture (SILAC) to label SEZ proteins with heavy or light isotope-tagged 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. Forty-nine 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. This research is the first application of SILAC combined with LC-MS/MS to identify SEZ proteins that may contribute to the infection of mBMECs and potentially show functions related to breaching the BBB. The outcomes provide many future avenues for research into the mechanism of SEZ-induced meningitis.