Fig 3. Enhanced GBS invasion of Caco-2 cells after disruption of intercellular junctions.

Five-day-old islets and 21-day-old monolayers of Caco-2 cells were infected with GBS and extracellular bacteria were killed with antibiotics. Prior to infection, cells were treated as indicated to disrupt intercellular junctions. (A) Effects of Ca²⁺ and Mg²⁺ on GBS invasion of 5-day-old islets, as measured by CFU counts in cell lysates. DPBS, Dulbecco’s PBS without Ca²⁺ and Mg²⁺. (B) Effect of EGTA on GBS invasion, as measured by CFU counts in cell lysates. (C) Effect of EGTA treatment of Caco-2 cells on S. enterica var. Typhymurium (strain M20) invasion, as measured by CFU counts in cell lysates. Values shown in A, B and C are means ± SD of three independent experiments conducted in triplicate. *, p<0.05 by Mann-Whitney test. (D) Evidence of numerous invading streptococci (Green) in the parabasal region of a 5-day-old islet treated with EGTA. Orthogonal views from x/z and y/z planes are depicted laterally to show the tridimensional location of bacteria. (E) Bacteria interacting with the basal region of 21-day-old monolayers after EGTA treatment. Yellow labels indicate the filter(s) used and the distance, in μm, of the optical section from the slide plane. On the right is a three-dimensional reconstruction centered in the area indicated by the dashed rectangle. Bacteria (Green) were stained with an anti-GBS rabbit serum, followed by Alexa Fluor 488-conjugated anti-rabbit IgG. Actin (Red) and nuclei (Blue) were stained with Phalloidin-iFluor 555 and DAPI, respectively. Scale bar = 10μm.