Neisseria meningitidis Has Two Independent Modes of Recognizing Its Human Receptor CEACAM1
Figure 4
Non-opaque meningococci bind in a CEACAM1-dependent manner to cells derived from meningococcal target tissues.
A) A549 cells were transduced with the indicated lentiviral vectors encoding an irrelevant shRNA (shControl) or a CEACAM1-targeted shRNA (shCEACAM1) or were left without virus. Transduced cells were analysed by flow cytometry with a CEACAM1/CEA-specific monoclonal antibody (clone 4/3/17). The negative control was stained with the Cy2-coupled secondary antibody only (gray curve). Whole cell lysates (WCL) of the cells were probed by Western blotting with monoclonal CEACAM-antibody (clone D14HD11; upper panel) or with monoclonal anti-tubulin antibody (lower panel). Bands derived from CEA, CEACAM1, or CEACAM6 are indicated on the right side. B) shControl- or shCEACAM1-transduced A549 cells were infected with non-opaque meningococci at an MOI of 30 for 3 h. Cell-associated bacteria were stained with a polyclonal antiserum (green) and cells were visualized by differential interference contrast (transmission). Bars represent 10 µm. C) Cell-associated bacteria were detected as in B) and counted. The line represents the median number of cell-associated bacteria and boxes represent the 25%/75% percentile from 30 cells in each sample. Groups were compared by Wilcoxon's signed rank test, * p<0.05. D) Human brain microvascular endothelial cells (HBMECs) were transduced with a GFP- or a CEACAM1-GFP encoding lentivirus. Transduced cell populations were infected with OpaCEA-expressing (Nme OpaCEA) or non-opaque (Nme Opa-) N. meningitidis at an MOI of 30 for 3 h. Samples were differentially stained with polyclonal antiserum against N. meningitidis to differentiate extracellular (arrowhead) and intracellular (small arrow) bacteria. Transduced cells are detected by their GFP signal. Bars represent 10 µm. E) Samples from D) were enumerated for intracellular meningococci as in C). F) Whole cell lysates (WCL) of virus transduced cells from D) were probed with an anti-GFP antibody to demonstrate expression of CEACAM1-GFP.