Adaptation of signature-tagged mutagenesis to Escherichia coli K1 and the infant-rat model of invasive disease

With the exception of the polysialic acid capsule (K1 antigen). little is known about other virulence factors needed For systemic infection by Escherichia coli K1, the leading cause of Gram-negative neonatal meningitis in humans. In this work, the functional genomics method of signature-ragged mutagenesis (STM) was adapted to E. coli K1 and the infant-rat model to identify nun-capsule virulence genes. Validation of the method was demonstrated by the Failure to recover a reconstructed acapsular mutant from bacterial pools used to systemically infect 5-day-old rats. Three new genes required for systemic disease were identified from a total of 192 mutants screened by STM (1.56% hit rate). Gut colonization, Southern blot hybridization. mixed-challenge infection, and DNA sequence analyses showed that the attenuating defects in the mutants were associated with transposon insertions in rfaL (O antigen ligase). dsbA (thiol:disulfide oxidoreductase), and a new gene, puvA ((p) over bar reviously (u) over bar nidentified (v) over bar irulence gene (A) over bar), with no known homologues. The results indicate the ability of STM I to identify novel systemic virulence factors in E. coli K1. (C) 2001 published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.