Fig 1.
N. cinerea co-localises with components of cortical plaques on epithelial cells.
(A) Epithelial cells were infected for 3 h with N. cinerea expressing GFP and stained for CD44, ezrin or actin. Bacteria co-localised with each protein (white arrows); magnified areas in the panels on the right show the honeycomb-like arrangement of each protein. (B) Non-infected A549 cells were immunostained for CD44, ezrin or actin and analysed by microscopy. Magnified areas shown in the panels on the right do not show a honeycomb-like arrangement. Scale bars, 10 μm. (C) Frequency of co-localisation of each protein in honeycomb-like arrangement at the site of attachment was determined by scoring 50 microcolonies. Data shown represent the mean ± SD of three independent experiments; NS, not significant. (D) Epithelial cells were infected for 3 h with N. cinerea and double fluorescence labelling was performed. Actin (red) and CD44 (green) in the top panels; or ezrin (red) and CD44 (green) in the bottom panels. Scale bars correspond to 10 μm. (E) XZ sections of cells dual labelled for actin (red) and CD44 (green), or CD44 (green) and ezrin (red). Bacteria and nuclei were stained with DAPI (white). Arrows indicate cellular protrusions enriched with actin-CD44 or ezrin-CD44.
Fig 2.
N. cinerea requires a cholesterol-rich plasma membrane for an efficient host-cell colonisation.
(A) Epithelial cells were treated with MβCD or MβCD and cholesterol (MβCD+Chol) then infected for 3 h. Adhesion of N. cinerea was reduced in the presence of MβCD and restored to wild-type levels with addition of cholesterol. (B) Frequency of CD44 co-localisation in honeycomb-like structures with adherent N. cinerea in the presence of MβCD or MβCD+Chol was compared to non-treated cells (NT). (C) XZ-section images of microcolonies attached to host cells were acquired by confocal microscopy. CD44 was stained with anti-CD44 (red) and cell-associated bacteria are in green. (D) CD44 localisation (red) in cells treated with MβCD or MβCD+Chol and infected for 3 h with N. cinerea expressing GFP (green) at an MOI of 100. DNA was stained with DAPI (shown blue in merge). Scale bar, 10 μm. Data shown are the mean ± SD of three independent experiments: NS, not significant; *p <0.05; ***p<0.0005 using one-way ANOVA test for multiple comparison.
Fig 3.
Tfp are not required for colocalisation of N. cinerea with CD44 honeycomb-like structures.
(A) Epithelial cells were infected with wild-type N. cinerea 346T (Wt) or N. cinerea 346TΔpilE1/2 both expressing GFP (green in merge) at an MOI of 100. At 3 hpi, cells were stained for CD44 (red in merge). Scale bar, 10 μm. (B) Quantification of CD44 colocalisation with bacterial colonies. Results represent the mean ± SD of three independent experiments. NS, not significant using unpaired two-tailed Student’s t-test.
Fig 4.
N. cinerea Tfp are required for microcolony formation and fusion.
(A) Epithelial cells were infected with N. cinerea wild-type (Wt) expressing sfCherry for 16 h. Images were captured at 10 min intervals; time points 2, 4 and 8 h are shown. (B) Microcolony size was quantified by measuring their surface area. Each line corresponds to a single microcolony tracked over time. Data shown are for eight microcolonies from one representative experiment of three independent experiments. (C) Epithelial cells infected with N. cinerea 346TΔpilE1/2 expressing GFP. Images were captured at 10 min intervals, and images from 2, 4 and 8 h post infection are shown. (D) Epithelial cells co-infected with wild-type N. cinerea (Wt, red in merge) and the pilE mutant (ΔpilE1/2, green in merge). Images are representative of three independent experiments performed in triplicate. In each case merged panels show both phase-contrast and fluorescence channels (GFP and Texas Red). Scale bars, 75 μm.
Fig 5.
N. cinerea and N. meningitidis form mixed microcolonies on epithelial cells.
(A) Representative images of epithelial cells co-infected with N. cinerea (N. cinerea-sfGFP) and N. meningitidis for 3 h. Lower panels are enlarged images of boxed area in upper panels. Nuclei and bacterial DNA were stained with DAPI (red). Each DAPI stained spot corresponds to a single bacterium (left-hand and merge panels). White arrows indicate N. meningitidis (DAPI positive, GFP negative) within a mixed microcolony. CD44 is shown in grey (merge panels). Scale bar, 10 μm. (B) Frequency of mixed or single species microcolonies quantified by confocal microscopy. Data shown are from at least 150 microcolonies in three independent experiments; the number of mixed microcolonies was expressed as a percentage of all microcolonies. NS, not significant; *, p<0.05; **, p< 0.005 (one-way ANOVA). (C) XZ optical section analysis of a mixed microcolony containing N. meningitidis (Nm 8013, red) and N. cinerea (Nc 346T, green) on epithelial cells. White arrows show N. meningitidis in close contact with N. cinerea on the cell surface.
Fig 6.
Scanning electron microscopy of mixed Neisseria microcolonies on epithelial cells.
SEM analysis of A549 cells infected for 6 h with N. cinerea 346T (A), N. meningitidis 8013 (B) or cells co-infected with both species (C). White arrows indicate N. cinerea, and red arrows highlight N. meningitidis. Scale bars, 1 μm.
Fig 7.
N. cinerea reduces association of N. meningitidis with epithelial cells.
(A) Cells were infected with N. cinerea (Nc346T) for 4.5 h prior to infection with N. meningitidis (Nm8013). The number of cell associated bacteria of each species was determined 1.5 h later. Results are the mean ± SD of three independent experiments carried out in triplicate. NS, not significant; ***p<0.0005 (unpaired two-tailed Student’s t-test). (B and C) Epithelial cells were infected with N. meningitidis (Nm 8013) alone or with N. cinerea (Nc 346T). The number of cell associated bacteria (CFU/mL) was determined at time points as indicated. Filled shapes show the number of CFU/well in single infections, while empty shapes show the number of CFU/well in co-infections. Each data point represents a single well from three independent experiments conducted in triplicate. NS, not significant; *, p<0.05 (unpaired two-tailed Student’s t-test). (D) Epithelial cells were infected with N. meningitidis (Nm 8013) alone or co-infected with N. cinerea (Nc 346T) at a ratio of 1:100 (Nm 8013 to Nc 346T) for 20 h. (E) Single and mixed cultures of N. meningitidis (8013) and N. cinerea (346T) were grown in the absence of cells for 6 hrs, and the number of bacteria was determined by selective plating. Results are the mean +SD of three independent experiments carried out in triplicate. NS, not significant. (F) Epithelial cells were infected with N. meningitidis (Nm 8013) alone or co-infected with E. coli (BL21 pET21b) at an MOI of 50 for each strain. Cell associated N. meningitidis and E. coli (CFU/well) was determined at 6 hpi. Filled circles show Nm8013 (red) and E. coli (blue) in single infections; filled squares show Nm8013 (red) and E. coli (blue) in co-infection. Results are the mean ± SD of 9 replicates from three independent experiments. NS, not significant; *p<0.05; **p<0.005 (unpaired two-tailed Student’s t-test). (G) Epithelial cells were infected with N. meningitidis (Nm 8013) alone or co-infected with wild-type N. cinerea (Nc346T) or a mutant lacking ACP (NcΔacp). At 6 hpi, cell associated N. meningitidis was quantified and presented as CFU per well. Filled circles show Nm8013 bacterial numbers in single infections; empty circles or empty triangles show levels of Nm8013 in co-infection. Results are the mean ± SD of at least three independent experiments carried out in duplicate. NS, not significant; *, p<0.01; **, p<0.001 (one-way ANOVA test for multiple comparison).
Fig 8.
N. cinerea restricts N. meningitidis microcolony motility and expansion.
(A) Representative time-lapse images of epithelial cells infected with N. meningitidis pNCC101::sfCherry alone (red, Nm8013), or with N. cinerea 346T expressing GFP (green, Nc346T) or N. cinerea 346TΔpilE1/2 expressing GFP (green, NcΔpilE1/2). Time points 3, 4 and 6 hpi are shown. Scale bar, 50 μm. (B) The frequency of microcolonies containing Nc346T and Nm8013 assessed by time-lapse microscopy. Most microcolonies harbour both species. Data shows the mean + SD of at least 700 Nm8013 microcolonies from three independent experiments. NS, not significant; ***, p<0.0005 (unpaired two-tailed Student’s t-test). (C) The size of N. meningitidis aggregates in single and mixed infections was quantified by measuring the area of meningococcal microcolonies. Each point shows the mean ± SD (dotted lines) of 36 microcolonies from three independent experiments. NS, not significant; *, p<0.01; **, p<0.001; ***, p<0.0005 (one-way ANOVA test). (D) Total distance travelled by N. meningitidis microcolonies in presence or absence of N. cinerea. Cumulative microcolony displacement was calculated based on the total distance covered per microcolony between 3 and 6 hpi. Data shown represent the mean + SD of 36 microcolonies tracked from three independent experiments. NS, not significant; ***, p<0.0005 (one-way ANOVA test for multiple comparisons).