Figure 3. L. monocytogenes enters the placenta primarily at invasive villus tips.

(A) Consecutive histological sections of a permissively infected explant at 8 h post-inoculation, frozen and stained for L. monocytogenes (green) and DNA (blue). Left panel and inset 1: red = cytokeratin (CK), expressed by cytotrophoblasts (CTB). In middle panel with insets 2 and 3: red = βHCG (HCG), which primarily stains syncytiotrophoblast (SYN). Subsyncytial cytotrophoblasts (sCTB) underlie the syncytiotrophoblast. Where cytotrophoblasts invade from the villus into the decidua, syncytiotrophoblast breaks, exposing basal surfaces (bSYN). Scattered, isolated bacteria are found mainly in proximal extravillous cytotrophoblasts (EVT). Bar = 100 µm. (B) Distribution of infected cell types in explants infected with ΔActA (top) or 10403S wild type L. monocytogenes (bottom). Each graph represents two sections in each of three explants (average of infected cells counted per explant = 135). For SYN and bSYN, a “cell” was considered to be the area around a single nucleus, roughly the size of a cytotrophoblast. Bars are SEM. (C) Projection of a 3D confocal image showing a whole explant permissively infected with GFP-expressing L. monocytogenes and fixed at 8 h. Anchoring villi (AV), which include invading extravillous cytotrophoblasts, and floating villi (FV), which remain covered with syncytiotrophoblast, are indicated. Red = F-actin. Green = L. monocytogenes. Blue = DNA. Left and top: reconstructed Z series. Because of high F-actin levels in extravillous cytotrophoblasts, bacteria appear yellow. Right and bottom: sum of total GFP intensity over 70 µm Z stack for each X/Y position after background subtraction shows the majority of bacteria are in anchoring villi, in extravillous cytotrophoblasts. Bar = 100 µm.