1887

Abstract

Cytotoxic necrotizing factor 1 (CNF1), a Rho GTPase-activating bacterial toxin, has been shown to contribute to invasion by meningitis-causing K1 of human brain microvascular endothelial cells (HBMEC), which constitute the blood–brain barrier. However, CNF1 is a cytosolic protein and it remains unclear how its secretion occurs, contributing to invasion of HBMEC. To investigate the genetic requirement for CNF1 secretion in K1 strain RS218, we performed mini-Tn mutagenesis and constructed a transposon mutant library of strain NBC, in which β-lactamase was fused to the C-terminus of CNF1 in the chromosome of strain RS218. We identified a transposon mutant (NBC-1E6) that exhibited reduced β-lactamase activity in its culture supernatant and had the transposon inserted into the gene. When was deleted from the genome of strain RS218 (Δ), the translocation of CNF1 into HBMEC was impaired. Subcellular localization analysis of CNF1 demonstrated that YgfZ, a periplasmic protein, contributes to secretion of CNF1 into outer-membrane vesicles (OMVs). The Δ mutant was significantly defective in invasion of HBMEC compared to the parent K1 strain. The defects of the Δ mutant in CNF1 secretion into OMVs and translocation into HBMEC as well as invasion of HBMEC were abrogated by complementation with . Taken together, our findings demonstrate that YgfZ contributes to CNF1 secretion into OMVs in meningitis-causing K1.

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2012-03-01
2019-10-20
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