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Escherichia coli K1 invasion of brain microvascular endothelial cells (BMEC) is a prerequisite for penetration into the central nervous system. We previously have shown that outer membrane protein A (OmpA) and cytotoxic necrotizing factor-1 (CNF1) contribute to E. coli K1 invasion of BMEC. In this study we constructed a double-knockout mutant by deleting ompA and cnf1. We demonstrated that the double-knockout mutant was significantly less invasive in human BMEC as compared with its individual Delta ompA and Delta cnf1 mutants, suggesting that the contributions of OmpA and CNF1 to BMEC invasion are independent of each other. In addition, we showed that OmpA treatment of human BMEC resulted in phosphatidylinositol 3-kinase (PI3K) activation with no effect on RhoA, while CNF1 treatment resulted in RhoA activation with no effect on PI3K, supporting the concept that OmpA and CNF1 contribute to E. coli K1 invasion of BMEC using different mechanisms. This concept was further confirmed by using both PI3K inhibitor (LY294002) and Rho kinase inhibitor (Y27632), which exhibited additive effects on inhibiting E. coli K1 invasion of BMEC. We isolated a 96KD OmpA interacting human BMEC protein by affinity chromatography using purified OmpA, which was identified as gp96 protein, a member of the HSP90 family. This receptor differed from the CNF1 receptor (37LRP) identified from human BMEC. Taken together, these data indicate that OmpA and CNF1 contribute to E. coli K1 invasion of BMEC in an additive manner by interacting with different BMEC receptors and using diverse host cell signaling mechanisms.