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 k nown about other virulence factors needed For systemic infection by Escheri chia coli K1, the leading cause of Gram-negative neonatal meningitis in hum ans. In this work, the functional genomics method of signature-ragged mutag enesis (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 pool s 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 ST M (1.56% hit rate). Gut colonization, Southern blot hybridization. mixed-ch allenge infection, and DNA sequence analyses showed that the attenuating de fects in the mutants were associated with transposon insertions in rfaL (O antigen ligase). dsbA (thiol:disulfide oxidoreductase), and a new gene, puv A ((p) over bar reviously (u) over bar nidentified (v) over bar irulence ge ne (A) over bar), with no known homologues. The results indicate the abilit y 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 Euro pean Microbiological Societies.