Disease and Carrier Isolates of Neisseria meningitidis Cause G₁ Cell Cycle Arrest in Human Epithelial Cells

Both pathogenic N. meningitidis isolates and carrier strains cause an accumulation of epithelial cells in the G₁ phase. Detroit cells either were left uninfected (control cells) or were infected with N. meningitidis strain MC58, 8013/clone12, α711, or α4 at an MOI of 100. Cells were collected at the time points indicated for propidium iodide (PI) staining and subsequent flow cytometry analysis of the cell cycle. In the left panel, representative histograms (24 h p.i.) for uninfected control cells and cells infected with the different meningococcal isolates are shown. The G₁ and G₂/M phases are indicated. In the right panel, the histograms were analyzed with the software package WEASEL v3.1, and the percentages of the cells in the G₁, S, and G₂/M phases are shown. The results are presented as the means and the SD from three independent experiments. The statistical significance was determined for comparison between uninfected control cells and N. meningitidis-infected cells at each time point. *, P < 0.05.

Meningococcal infection decreases the number of Detroit 562 cells in the S phase. Detroit 562 cells were either left uninfected or were infected with MC58 or α711 (MOI of 100) for a 24-h time period. Cell proliferation analysis was performed using a Click-iT EdU Alexa Fluor 488 flow cytometry assay kit and FxCycle Far Red stain. During the final 2 h of infection, Detroit 562 cells were treated with 20 μM EdU, harvested, and then analyzed by flow cytometry. EdU was stained with Alexa Fluor 488 azide according to the manufacturer's protocol, followed by staining with 1 μM FxCycle Far Red stain for DNA content analysis. The cells were then analyzed by flow cytometry using either 488-nm excitation for the EdU Alexa Fluor 488 dye or 633-nm excitation for the FxCycle Far Red stain. (A, upper panel) The results of a representative experiment are shown. The complete population (total) and the EdU-positive (EdU) populations are indicated. (Lower panel) The results of a statistical analyses of the percentages of the cell population incorporating EdU in uninfected control cells, MC58-infected cells, or α711-infected cells from three independent experiments are shown. The data are shown as means ± the standard deviations (*, P < 0.05). (B, upper panel) The results of a representative experiment are shown, and cells in the G₁, S, and G₂/M phases are gated in boxes. (Lower panel) The results of a statistical analyses of the percentages of the cell population in the S phase in uninfected control cells, MC58-infected cells, or α711-infected cells from three independent experiments are shown. The data are shown as means ± the standard deviations (*, P < 0.05).

G₁-phase arrest is induced by viable bacteria but not by heat-killed bacteria. Detroit 562 cells either were infected with viable N. meningitidis strain MC58, 8013/clone12, α711, or α4 (MOI 100) or were exposed to heat-killed (hk) bacteria. Uninfected cells served as a control. Cells were harvested for PI staining at 24 h p.i. and analyzed by flow cytometry. (A) The results of representative experiments are shown. (B) The results of statistical analyses of the percentages of the cell population in the G₁, S, and G₂/M phases are shown. The data are shown as means ± the SD obtained from three independent experiments (*, P < 0.05).

Meningococcal supernatants do not have any effect on the cell cycle distribution in epithelial cells. N. meningitidis strain MC58, 8013/clone 12, α711, or α4 was grown to the exponential phase and harvested by centrifugation, and supernatants were collected, filter sterilized, and transferred to VivaSpin columns for protein concentration. Detroit 562 cells either were left uninfected or were infected with viable MC58 (as a positive control) or were exposed to 25- or 100-μl portions of concentrated meningococcal supernatants. Cells were harvested for PI staining at 24 h p.i. and analyzed by flow cytometry. (A) The results of representative experiments are shown. (B) The graphs show the statistical analyses of the percentages of the cell population in the G₁, S, and G₂/M phases. The data are shown as means ± the SD obtained from three independent experiments (*, P < 0.05).

Pathogenic N. meningitidis strains and carrier isolates influence cyclin D1 and cyclin E protein levels. (A) Detroit 562 cells were either left uninfected (control) or infected with N. meningitidis MC58, 8013/clone 12, α711, or α4 (MOI 100) for 24 h. Cell lysates of control and N. meningitidis-infected cells were collected and analyzed for cyclin D1 and cyclin E protein expression by immunoblot analysis. β-Actin was used to normalize protein loading. The figure shows a representative Western blot with lanes from different areas of the same blot. (B) Bar diagram showing the fold increase in the normalized mRNA expression of cyclin D1 and cyclin E in Detroit 562 cells infected with MC58 or α711 in comparison to uninfected control cells. Real-time PCR data were analyzed according to the comparative ΔC_T method by first normalizing the sample values (cyclin D1 and cyclin E) to the reference gene values (GAPDH) in infected and uninfected control cells, respectively, and then calculating the relative change in expression (as a fold increase) in infected compared to uninfected cells. Experiments were performed three times, and error bars represent the standard errors of the mean (ns, not significant).

Pathogenic N. meningitidis strains and carrier isolates increase p21^WAF1/CIP1 protein levels in Detroit 562 cells and induce redistribution of p21^WAF1/CIP1 and p27^CIP1. (A) Cells were either left uninfected (control) or infected with N. meningitidis MC58, 8013/clone12, α711, or α4 (MOI 100) for a 24-h time period and were analyzed for p21^WAF1/CIP1 protein expression by Western blot analysis. Band intensities were quantified by densitometric analysis as described for cyclin D1 and cyclin E using ImageJ and normalized to β-actin. The figure shows a representative Western blot with lanes from different areas of the same blot. (B and C) Subcellular distribution of p21^WAF1/CIP1 (B) and p27^CIP1 (C) in N. meningitidis-infected Detroit 562 cells assessed by immunofluorescence microscopy. Colors in panel B: green, p21^WAF1/CIP1 protein stained with specific antibody and secondary antibody conjugated with Alexa Fluor 488; blue, nuclei stained with DAPI; light green, colocalization. Colors in panel C: red, p27^CIP1 stained with specific antibody and secondary antibody conjugated with Cy3; blue, nuclei stained with DAPI.