Flotillin acts as a regulatory cellular barrier against invasion by Escherichia coli K1

Abstract

Pathogenic microorganisms that cause neonatal meningitis must cross the blood-brain barrier to reach the central nervous system. Escherichia coli K1 remains a leading pathogen in cases of neonatal bacterial meningitis and invades the endothelium in the brain microvasculature. Signalling through a cascade of sphingosine kinase activity and the epidermal growth factor receptor (EGFR) has been implicated in this process, but how it is coupled to membrane organisation is unclear. Here we show that invasion of human brain microvascular endothelial cells by E. coli K1 can also be regulated by flotillins through the sphingosine/EGFR axis. Loss of flotillin 1 markedly increased bacterial invasion, whereas flotillin overexpression suppressed it, and the hyperinvasive phenotype of flotillin-deficient cells remained dependent on cholesterol. Depletion of sphingosine kinases by siRNA also increased invasion, in contrast to pharmacological inhibition, suggesting that the kinase proteins, and not only their catalytic activity, may help restrain bacterial entry. In a neonatal mouse infection model, flotillin 1 knockout animals had lower bacteraemia but equivalent brain bacterial burdens, indicating increased penetration of the blood-brain barrier. We propose that flotillin-dependent membrane organisation restrains an EGFR- and sphingolipid-dependent route of E. coli K1 entry, defining a host barrier to meningitic infection.