Fig. 4

In vitro interaction between HrpA-M3 and the DYNLT1, and co-localization of N. meningitidis with DYNLT1 in infected HeLa and HBMEC cells. A Interaction between histidine-tagged HrpA-M3 (His-HrpA-M3) and Myc-tagged DYNLT1 (Myc-DYNLT1) was analyzed by pull-down experiments. Purified His-HrpA-M2 or His-HrpA-M3 were bound to Ni–NTA resin and incubated with a HeLa cell lysate overexpressing Myc-DYNLT1. The negative control was represented by Ni–NTA resin alone. Eluted proteins were detected by western blotting using mouse antibodies against poly-His (α-poly-His) or Myc (α-Myc). B HeLa cells were infected with N. meningitidis strain B1940. After infection, cells were exposed to gentamicin to kill remaining extracellular bacteria, washed to eliminate the majority of dead extracellular bacteria, and analyzed by immunofluorescence microscopy. Green labeled DYNLT1 and red stained N. meningitidis are shown in merged images in the panels on the right. Bars, 10 µm. C, D HeLa cells were infected with N. meningitidis B1940 strain and B1940ΩhrpA derivative mutant. Bacteria were immunostained before permeabilization (blu) and after permeabilization (red) to visualize extracellular (purple) and intracellular (red) bacteria. DYNLT1 was immunostained also after permeabilization and showed in green. White dashed squares show zoomed areas. Bars, 10 µm. E HBMEC cells were infected with wild type bacteria, ΩhrpA mutant derivative strain and the hrpA-complemented strain B1940 ΩhrpA/hrpA + . DYNLT1 (green) and N. meningitidis (red) are shown in merged images. Bars, 10 µm. F Example of colocalization between B1940 wild type strain and DYNLT1 in HBMEC cells using the XYZ orthogonal view. G The degree of colocalization between the three strains of meningococci and DYNLT1 in HBMEC cells was determined using the XYZ orthogonal view. Values are the mean ± SEM of three different experiments where bacteria present in at least 50 cells/sample were scored. *P < 0.05 **P < 0.01 ***P < 0.001