NlpI Contributes to Escherichia coli K1 Strain RS218 Interaction with Human Brain Microvascular Endothelial Cells

Escherichia coli K1 is the most common Gram-negative bacillary organism causing neonatal meningitis. E. coli K1 binding to and invasion of human brain microvascular endothelial cells (HBMECs) is a prerequisite for its traversal of the blood-brain barrier (BBB) and penetration into the brain. In the present study, we identified NlpI as a novel bacterial determinant contributing to E. coli K1 interaction with HBMECs. The deletion of nlpI did not affect the expression of the known bacterial determinants involved in E. coli K1-HBMEC interaction, such as type 1 fimbriae, flagella, and OmpA, and the contribution of NlpI to HBMECs binding and invasion was independent of those bacterial determinants. Previous reports have shown that the nlpI mutant of E. coli K-12 exhibits growth defect at 42°C at low osmolarity, and its thermosensitive phenotype can be suppressed by a mutation on the spr gene. The nlpI mutant of strain RS218 exhibited similar thermosensitive phenotype, but additional spr mutation did not restore the ability of the nlpI mutant to interact with HBMECs. These findings suggest the decreased ability of the nlpI mutant to interact with HBMECs is not associated with the thermosensitive phenotype. NlpI was determined as an outer membrane-anchored protein in E. coli, and the nlpI mutant was defective in cytosolic phospholipase A₂α (cPLA₂α) phosphorylation compared to the parent strain. These findings illustrate the first demonstration of NlpI''s contribution to E. coli K1 binding to and invasion of HBMECs, and its contribution is likely to involve cPLA₂α.Neonatal Gram-negative bacillary meningitis continues to be an important cause of mortality and morbidity (12, 26, 35). The key aspect of pathogens associated with neonatal bacterial meningitis is related to their ability to traverse the blood-brain barrier (BBB), but the microbe-host interactions involved in microbial traversal of the BBB remain incompletely understood (17). Escherichia coli K1 is the most common Gram-negative bacillary organism causing neonatal meningitis (12, 26, 35), and most cases of neonatal E. coli meningitis develop via hematogenous spread, but it is incompletely understood how circulating E. coli K1 traverses the BBB (16).The BBB is a structural and functional barrier formed by brain microvascular endothelial cells to protect the brain from microbes and toxins circulating in the blood. The in vitro BBB model has been developed by isolation and cultivation of human brain microvascular endothelial cells (HBMECs) (31). This in vitro model makes it feasible to study the mechanisms on how meningitis-causing pathogens cross the BBB. It has been shown that E. coli K1 binding to and invasion of HBMECs is a prerequisite for its penetration into the brain (3, 13, 14, 17, 18), but the microbe-host interactions involved in HBMEC binding and invasion remain incompletely understood.NlpI is a lipoprotein that was first identified in E. coli K-12 (22). The nlpI mutant of E. coli K-12 exhibited growth defect at 42°C on low-salt medium. This phenotype can be suppressed by a mutation on the spr gene. The spr single mutation of E. coli K-12 also exhibited the similar thermosensitive phenotype (32). In addition, NlpI is shown to be an important component of Crohn''s disease-associated E. coli strain LF82 (O83:H1) to interact with intestine epithelial cells (5, 6). Deletion of nlpI in E. coli strain LF82 decreases expression of type 1 fimbriae and flagella (5). However, it is unknown whether NlpI is involved in extraintestinal E. coli infection such as meningitis.While investigating the microbe-host interactions involved in E. coli K1 binding to and invasion of HBMECs, we noted that deletion of nlpI decreased the ability of E. coli K1 to interact with HBMECs. The present study examined and characterized the role of NlpI in E. coli K1 binding to and invasion of HBMECs.