Escherichia coli is the most common cause of neonatal Gram-negative sepsis and meningitis, occurring in 1 in 1000 live births. Despite the fact that over 100 capsular types have been identified in E. coli, K1 strains predominate, causing 40% of the bacteremia infections and 80% of the meningitis in neonates. Colonization with K1 occurs primarily at parturition with maternal GI tract colonization resulting in newborn colonization. For infants to become bacteremia, the organism must penetrate the GI mucosal barrier. The focus of this proposed research is the pathogenesis of neonatal infections, specifically the mechanisms by which E. coli K1 penetrates the intestinal mucosa. This project will examine the hypothesis that virulent E. coli K1 strains are able to enter the bloodstream of young infants because they have the ability to invade GI epithelial cells. Using a tissue culture model to examine invasion and an infant rat model of bacteremia to confirm virulence, this research will undertake the molecular analysis of the bacterial gene(so involved in the invasion of GI epithelium by E. coli K1. Transposon mutagenesis with Tnpho A has been used to derive mutants of a well-characterized, invasive 018:K1:H7 strain, RS218, which are unable to invade both in the tissue culture and infant rat models. A non- invasive mutant with a single transposon insertion, RS218-B3, has been selected for further characterization: it does not have a mutation within an operon encoding known virulence factors including K1 capsule, LPS or Type 1 pili or S-fimbriae. This and other non-invasive mutants will be used to clone invasion-related determinants: the DNA sequences flanking the TnphoA insertions will be cloned and sequenced and used to identify corresponding sequences from a cosmid library of DNA from the invasive parent strain, RS21. In parallel experiments, direct cloning of the invasion determinant(s) into a non-invasion E. coli strain is underway: a library has been constructed and is being screened for invasion. Complementation of the mutants will be attempted and nucleotide sequence and gene product analysis of putative invasion gene(s) will be performed. These studies will form the basis for the identification of a likely invasion-related structural gene. A deletion mutant of this gene will be constructed and its phenotype will be examined in both the tissue culture and infant rat models of invasion. These studies should help us understand the pathogenesis of E. coli K1 infections in newborn infants. In the future, this understanding may lead to improved strategies for the prevention and treatment of these life-threatening neonatal infections.
NIH ExPorter
NIH ExPorter