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Infection and Immunity, September 2000, p. 5056-5061, Vol. 68, No. 9
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Application of Signature-Tagged Mutagenesis for Identification of Escherichia coli K1 Genes That Contribute to Invasion of Human Brain Microvascular Endothelial Cells

Julie L. Badger,1 Carol A. Wass,1 Scott J. Weissman,1,dagger and Kwang Sik Kim1,2,*

Division of Infectious Diseases, Childrens Hospital Los Angeles,1 and University of Southern California School of Medicine,2 Los Angeles, California 90027

Received 7 March 2000/Returned for modification 31 May 2000/Accepted 12 June 2000

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.

* Corresponding author. Mailing address: Division of Infectious Diseases, MS 51, Childrens Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027. Phone: (323) 669-2509. Fax: (323) 660-2661. E-mail: kskim{at}chla.usc.edu.

dagger Present address: Department of Pediatrics, Kaiser Foundation Hospital, Los Angeles, CA 90027.

Infection and Immunity, September 2000, p. 5056-5061, Vol. 68, No. 9
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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