Identification of Escherichia coli K1 genes contributing to human brain microvascular endothelial cell invasion by differential fluorescence induction.
Most cases of Escherichia coli K1 meningitis arise as a result of haematogenous spread, however there is a limited understanding of the mechanisms by which circulating E. coli K1 cross the blood-brain barrier. We have previously shown that environmental growth conditions both positively and negatively influence the capabilities of E. coli K1 to invade brain microvascular endothelial cells (BMEC), for example growth in media supplemented with 50% newborn bovine serum (NBS) increased BMEC invasion, whereas growth in media supplemented with 0.2 M NaCl repressed invasion in vitro and in vivo. In this study, differential fluorescence induction (DFI) was used to identify E. coli K1 genes involved in this differentially expressed invasion phenotype. E. coli K1 promoter libraries were constructed and screened for gfp expression in a manner analogous to the above growth conditions. Twenty-four clones were isolated that showed fluorescence induction when grown under the invasion-enhancing condition (i.e. NBS). Four of these clones also demonstrated repression or no induction of fluorescence when grown under the invasion-repressing condition (i.e. 0.2 M NaCl). One such clone, containing a ygdP promoter and an open reading frame (ORF), showed significant homology to Bartonella bacilliformis IalA (invasion associated locus). Among the other NBS-inducing loci, finPtraJ was identified as well as several clones with no homology to other known genes. When ygdP, finPtraJ and several of the unique loci were disrupted in E. coli K1, there was a significant decrease in human BMEC (HBMEC) invasion. RNA transcript analysis determined that these newly identified invasion loci were differentially regulated at the transcriptional level. This is the first demonstration of using DFI to identify E. coli K1 genes contributing to HBMEC invasion.
Badger
J L
JL
Division of Infectious Diseases, Childrens Hospital Los Angeles, CA 90027, USA. University of Southern California School of Medicine, Los Angeles, CA 90027, USA.
Wass
C A
CA
Kim
K S
KS
D001921:Brain / Q000098:blood supply* / Q000166:cytology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003470:Culture Media; D004730:Endothelium, Vascular / Q000166:cytology / Q000382:microbiology*; D004926:Escherichia coli / Q000235:genetics* / Q000472:pathogenicity*; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D020814:Meningitis, Escherichia coli / Q000382:microbiology*; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins; D017422:Sequence Analysis, DNA
D003470:Culture Media; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins