SphK2-S1P-S1P<sub>2</sub> mediates meningitic <i>E</i>. <i>coli</i> penetration of the BBB <i>in vitro</i> and <i>in vivo</i>.

<p>(A) S1P generation was significantly higher in HBMEC incubated with wild-type RS218 compared with the triple mutant deleted of <i>ompA</i>, <i>fimH</i> and <i>nlpI</i>. Correspondingly, the sphingosine level was lower in HBMEC incubated with wild-type RS218 than those incubated with the triple deletion mutant. * <i>p</i><0.05. (B) Structures of (<i>S</i>)-FTY720-vinylphosphonae (SphK1 and SphK2 inhibitor), (<i>R</i>)-FTY720-methyl ether (selective SphK2 inhibitor), RB-032 and RB-033 (selective SphK1 inhibitors), and RB-034 (inactive analogue). (C) Both (<i>S</i>)-FTY720-vinylphosphonae (SphK1 and SphK2 inhibitor, shown as (<i>S</i>)-FTY-Pn) and (<i>R</i>)-FTY720-methyl ether (SphK2 inhibitor, shown as ROME) significantly inhibited RS218 invasion of HBMEC, while the SphK1 inhibitors (RB-032 and RB-033) and inactive analogue (RB-034) did not exhibit any inhibition. ** <i>p</i><0.01. The inhibitors were all used at 10 μM. (D) (<i>R</i>)-FTY720-methyl ether inhibited <i>E</i>. <i>coli</i> RS218 invasion of HBMEC in a dose-dependent manner. ** <i>p</i><0.01. (E) <i>E</i>. <i>coli</i> RS218 activated SphK2 in a time-dependent manner in HBMEC, while such activation was abolished by pretreatment with 10 μM (<i>R</i>)-FTY720-methyl ether. ** <i>p</i><0.01. (F) <i>E</i>. <i>coli</i> penetration into the brain was significantly less in SphK2 <sup>−/−</sup> mice compared with wild-type mice. In contrast, the levels of bacteremia did not differ between the two groups of mice. (G) JTE-013 (S1P<sub>2</sub> antagonist) significantly inhibited <i>E</i>. <i>coli</i> invasion of HBMEC, while VPC23019 (S1P<sub>1</sub> and S1P<sub>3</sub> antagonist) did not exhibit any inhibition. ** <i>p</i><0.01. (H) The mutants with deletion of <i>ompA</i>, <i>fimH</i>, or <i>nlpI</i> as well as the triple mutant (<i>ΔompAΔfimHΔnlpI</i>) induced significantly lower levels of SphK2 activation in HBMEC, compared with wild-type RS218. ** <i>p</i><0.01.</p>