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 haematoge nous spread, however there is a limited understanding of the mechanisms by which circulating E. coli K1 cross the blood-brain barrier. We have previou sly shown that environmental growth conditions both positively and negative ly influence the capabilities of E. coli K1 to invade brain microvascular e ndothelial 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 t his study, differential fluorescence induction (DFI) was used to identify E . coli K1 genes involved in this differentially expressed invasion phenotyp e. E. coli K1 promoter libraries were constructed and screened for gfp expr ession in a manner analogous to the above growth conditions. Twenty-four cl ones were isolated that showed fluorescence induction when grown under the invasion-enhancing condition (i.e. NBS). Four of these clones also demonstr ated repression or no induction of fluorescence when grown under the invasi on-repressing condition (i.e. 0.2 M NaCl). One such clone, containing a ygd P 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 n o homology to other known genes. When ygdP, finPtraJ and several of the uni que 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 transcr iptional level. This is the first demonstration of using DFI to identify E. coli K1 genes contributing to HBMEC invasion.