Application of signature-tagged mutagenesis for identification of escherichia coli K1 genes that contribute to invasion of human brain microvascular endothelial cells

Abstract

Escherichia coli K1 is the leading cause of gram-negative bacterial meningitis in neonates. It is principally due to our limited understanding of the pathogenesis of this disease that the morbidity and mortality rates remain unacceptably high. To identify genes required for E. coli K1 penetration of the blood-brain barrier (BBB), we used the negative selection strategy of signature-tagged transposon mutagenesis (STM) to screen mutants for loss or decreased invasion of human brain microvascular endothelial cells (HBMEC) which comprise the BBB. A total of 3,360 insertion mutants of E. coli K1 were screened, and potential HBMEC invasion mutants were subjected to a secondary invasion screen. Those mutants that failed to pass the serial invasion screens were then tested individually. Seven prototrophic mutants were found to exhibit significantly decreased invasive ability in HBMEC. We identified traJ and five previously uncharacterized loci whose gene products are necessary for HBMEC invasion by E. coli K1. In addition, cnf1, a gene previously shown to play a role in bacterial invasion, was identified. More importantly, a traJ mutant was attenuated in penetration of the BBB in the neonatal rat model of experimental hematogenous meningitis. This is the first in vivo demonstration that traJ is involved in the pathogenesis of E. coli K1 meningitis.

FIG. 1

Outline of experimental design utilizing STM selection. Primary HBMEC tissue culture invasion (TCI) screens were performed on input pools of 96 random mutants (total of 35; 20 in wild-type [w.t.] and 15 in ompA mutant background). Bacteria (10⁸) were added to wells containing HBMEC; invasive bacteria were recovered and combined (output pool). Chromosomal DNA was prepared from input and output pools. PCR-generated probes of the unique transposon tags were prepared and used to hybridize colony blots of the original 96 transposon mutant pools. Mutants with a significant reduction or loss of hybridization signal in the output pool compared to the input pool were reassembled into new 96-well microtiter dishes. Mutants that hybridized to both the input and output pools were included as positive controls. These new pools were subjected to a secondary screen, as described above. Those mutants that repeatedly showed a significant reduction or loss of hybridization signal when probed with the output pool compared to the input pool were further tested in quantitative HBMEC invasion assays.

FIG. 2

Wild-type DNA supplied in trans complements mutant invasion phenotype. HBMEC invasion assays were performed with strains indicated. Results are presented as relative invasion, where the value for wild-type E44 harboring each cloning vector was arbitrarily set at 100%. The data shown are from a single experiment performed in duplicate and are representative of several experiments with similar results.