Figure 4

Confocal microscopy analysis of the mannosyl/bovine serum albumin-fluorescein isothiocyanate (man/BSA-FITC) colocalization with Streptococcus pneumoniae capsule in Schwann cells (SC). (A) Optical section of infected Schwann cells cultured for 48 h, immunolabeled for anti-pneumococcal antiserum (red) and reacted with Man/BSA-FITC (green). Active CTLDs of MR in infected SCs were observed after receptor-ligand binding assays with Man/BSA-FITC (red, yellow and white dashed squares in A). Higher-magnification views of the red, yellow and white boxes in A show details of S. pneumoniae adhered to the cellular surface (B) or internalized by SC in C and D. Internalized bacteria can be seen throughout the cytoplasm of the SCs (thin arrows in C and D), some of which lack the polysaccharide capsule (thick arrow in D). (E) Optical section at the maximum nuclei diameter of A with the orthogonal plane images cut at the yellow and red lines, and projected in the upper and right columns, respectively. Orthogonal projections show colocalization of both markers (arrows). The nuclei of SCs and/or bacterial DNA (blue dots) are stained with DAPI. The DAPI counterstaining shows the bacterial DNA surrounded by intense labeling of the pneumococcus capsule that reacted with the anti-pneumococcal antiserum (B - D). These results are representative of five separate experiments. Scale bar = 30 μm in (A); 1.5 μm in (B); 2 μm in (C - D); 18 μm in (E).