FIG 8.

Adhesion, invasion, intracellular replication, and exit from infected HeLa cells of MenB and MenC strains. HeLa cells were infected at a multiplicity of infection (MOI) of 50 for 1 h. (a) Adherence of viable bacteria was evaluated by the CFU method after removing nonadherent bacteria by sequential PBS washes. Values are relative to B1940 adherence, and the data are expressed as the means ± SD from at least three independent experiments with triplicate samples. (b) Relative invasion rates (determined as the normalized CFU ratio between intracellular and adherent bacteria) of strains B1940 (MenB) and 93/4286 (MenC) are reported. The number of viable intracellular bacteria was determined after 1 h of infection, followed by 30 min of gentamicin treatment to kill extracellular/adherent bacteria and lysis with 0.1% saponin to release intracellular bacteria. Lysates were serially plated on GC agar to determine the CFU. Values are means ± SD from at least three independent experiments with triplicate samples and are relative to B1940 with a ratio between intracellular and adherent B1940 bacteria equal to about 1.59%. (c) To monitor bacterial intracellular replication, HeLa cells were infected for 1 h, treated with gentamicin for 30 min, washed to remove gentamicin, reincubated for different time intervals (0 to 7 h) in DMEM, and lysed with 0.1% saponin. Lysates were serially plated on GC agar to determine the CFU. (d) To count the number of extracellular bacteria, HeLa cells were infected as described for panel c. Medium was collected at different time intervals after infection and serially plated onto GC agar. In panels c and d, values are relative to B1940 at time zero (0 h). The data are expressed as the means ± SD from at least three independent experiments with triplicate samples. (e) Ratios between extracellular and intracellular bacteria. (f) The numbers of infected cells at two time intervals (0 and 7 h) after 1 h of infection and 30 min of gentamicin treatment. Data were obtained by IFM by analyzing at least 100 microscope fields for each strain in a single experiment. Values are expressed as the means ± SD. In panels a to f, asterisks indicate statistically significant differences (P < 0.05). (g) HeLa cells were infected with strain 93/4286. After application of gentamicin treatment to selectively kill extracellular bacteria, infected cells were analyzed by CLSM. Images were taken 7 h after infection. To detect intracellular bacteria, antibody against MenC capsule was used after permeabilization with saponin, in combination with FITC-conjugated secondary antibody. Actin was stained by Alexa 568 phalloidin. Merged images of the different channels are shown. Bars, 10 μm.