Bacterial meningitis continues to be associated with high morbidity and mortality despite advances in chemotherapy and supportive care. E. coli is the most common gram-negative organism that causes neonatal meningitis. Most cases develop as a result of hematogenous spread, but it is not clear how circulating E. coli traverse the brain microvascular endothelium, which constitutes the blood brain barrier. The PI has demonstrated that the invasion of brain microvascular endothelial cells (BMEC) by E. coli K1 is mediated by multiple factors. Two separate genetic loci, identified by the PI as pathogenicity islands (paiA and paiB) apparently contribute to E. coli meningitis. PaiA contains the 20 kb E. coli locus identified as the major invasion gene cluster in K1 E. coli strain RS218. A TnphoA insertion mutant of E. coli RS218 was unable to invade BMEC in tissue culture and to cause hematogenous meningitis in a newborn rat model. Encoded on paiA is ibe10, which encodes an 8.2 kDa protein displaying the characteristics of an integral membrane protein with four transmembrane domains. A recombinant Ibe10 protein was able to block invasion of BMEC by E. coli K1. Ibe10 was detected in 30% of clinical isolates of K1 E. coli causing meningitis. Six clones for Ibe10 have been isolated from a lambda Gem-12 library. A recombinant plasmid carrying a 15 kb E. coli fragment was able to complement the non-invasive TnphoA mutant 10A-23. And, in an appendix, the PI reports that he has succeeded in conferring invasiveness on a strain of E. coli K12 using an 18 kb fragment of paiA. The long term goal of this work is to develop novel strategies for the prevention and treatment of E. coli meningitis (such as vaccines) based on a full understanding of the molecular mechanisms responsible for E. coli invasion of the blood-brain barrier. The PI hypothesizes that the phenotype of E. coli invasion of the blood-brain barrier is encoded by the pathogenic E. coli K1-specific genetic determinants, the pai's. The proposal comprises two aims:
Aim 1 : To further characterize the role of the invasion gene cluster paiA and/or paiB in the pathogenesis of E. coli meningitis by using in vitro (BMEC invasion) and in vivo (infant rat) models and molecular and genetic approaches.
Aim 2 : To determine the function and functional domains of invasion protein ibe10 by identification of the BMEC receptor for ibe10, in vitro mutagenesis and epitope mapping.
