Fig 6. The pilE I12A N. meningitidis mutant also showed reduced internalization into HBMEC, the induction of cytoskeleton rearrangements, and interactions with PamA in N. meningitidis.

(A) A I12A mutation in the pilE gene affects N. meningitidis internalization into HBMEC. Adherence (white) and internalization (gray) of N. meningitidis strains in HBMEC. Each value is the mean ± standard error of the mean (CFU per 10⁴ HBMEC) from at least four experiments. The bars indicate the bacterial numbers of N. meningitidis wild-type pamA⁺ (HT1125, left), ΔpamA::spc (HT1822, middle-left), pilE⁺-cat (HT1744 middle-right), and pilE I12A-cat (HT2167, right), respectively (see S1 Table). *P<0.01, **P<0.02, significantly different from the pamA⁺ strain or pilE⁺-cat strain. (B) Immunofluorescence microscopy showing the accumulation of ezrin beneath N. meningitidis strains. The HBMEC monolayer was infected with wild-type pilE⁺-cat (middle) and pilE I12A-cat (right) N. meningitidis strains. A non-infected HBMEC monolayer is also shown in the left panels. Bacteria and HBMEC were observed by phase-contrast microscopy (upper panels). Ezrin was immunostained with anti-ezrin mAb and Alexa Fluor 488-conjugated rabbit anti-mouse IgG (green channel, lower panels). (C) PilE I12A was crosslinked to PamA K278(pBPa) less efficiently than wild-type PilE in N. meningitidis. Black and red arrows show the crosslinked complex between PamA K278(pBPa) and PilE, and the gray arrow indicates an unidentified band that crossreacted with anti-PilE rabbit serum. +/- indicates the presence or absence of the pilE gene, respectively.