In order to better understand the events that allow Escherichia coli K1 to cross the blood-brain barrier we used differential fluorescence induction to identify bacterial genes that are preferentially expressed when associated with human brain microvascular endothelial cells (HBMEC), which comprise the blood-brain barrier. Random gene fusions of E. coli K1 DNA were created in a promoterless gfp vector and gene fusion libraries were incubated with and without HBMEC. The cells were subjected to a series of fluorescence-activated cell sorting screens to identify promoter fusions which lead to fluorescence when bacteria were associated with HBMEC, yet not fluorescent when grown in media alone. Two genes were identified, purA (encodes adenylosuccinate synthetase) and a sorC homologue (encodes a member of the sorC family of transcriptional regulators), whose expression were preferentially induced when bacteria were associated with eukaryotic cells. Individual gene disruption mutants of E. coli K1 purA and sorC demonstrated significantly decreased HBMEC invasion phenotype in vitro, when compared to the wild-type strain, and could be complemented when the respective wild-type sequences were supplied in trans. The purA and sorC mutants were deficient in their ability to grow in defined minimal media, without adenine, and with sorbose as sole carbon source, respectively, yet capable of normal growth in complex media. We have identified novel phenotypes associated with E. coli K1 purA and sorC, which provide evidence that these genes contribute to the invasion of HBMEC.
- Differential fluorescence induction
- Escherichia coli K1
- Human brain microvascular endothelial cells
ASJC Scopus subject areas
- Infectious Diseases