SUMMARY Cryptococcus neoformans (C. neoformans) penetration into the central nervous system (CNS) requires traversal of the blood-brain barrier that is composed of a single layer of human brain microvascular endothelial cells (HBMEC), but the underlying mechanisms of C. neoformans traversal remain incompletely understood. C. neoformans transcytosis of HBMEC monolayer involves rearrangements of the host cell actin cytoskeleton and small GTP-binding Rho family proteins such as Rac1 are shown to regulate host cell actin cytoskeleton. We, therefore, examined whether C. neoformans traversal of the blood-brain barrier involves host Rac1. While the levels of activated Rac1 (GTP-Rac1) in HBMEC increased significantly upon incubation with C. neoformans strains, pharmacological inhibition and down-modulation of Rac1 significantly decreased C. neoformans transcytosis of HBMEC monolayer. Also, Rac1 inhibition was efficient in preventing C. neoformans penetration into the brain. In addition, C. neoformans phospholipase B1 (Plb1) was shown to contribute to activating host cell Rac1, and STAT3 was observed to associate with GTP-Rac1 in HBMEC that were incubated with C. neoformans strain but not with its Δplb1 mutant. These findings demonstrate for the first time that C. neoformans Plb1 aids fungal traversal across the blood-brain barrier by activating host cell Rac1 and its association with STAT3, and suggest that pharmacological intervention of host-microbial interaction contributing to traversal of the blood-brain barrier may prevent C. neoformans penetration into the brain.

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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 A 2 ␣ (cPLA 2 ␣) 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 cPLA 2 ␣.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 Gramnegative 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 b...