Bacteria invade primarily via InlA binding to E-cadherin on cytotrophoblasts not covered by syncytiotrophoblast.

<p>(<b>A</b>) Immunofluorescence of consecutive histological sections. From left to right: red stains βHCG (HCG, a syncytiotrophoblast marker), E-cadherin (Ecad) and EGFR (stains cytotrophoblasts (CTB) and syncytiotrophoblast (SYN) membrane). E-cadherin does not appear on the apical surface of syncytiotrophoblast but is abundant in cytotrophoblasts. Green = <i>L. monocytogenes.</i> Blue = DNA. Bar = 100 µm. (<b>B</b>) Intracellular invasion of <i>L. monocytogenes</i> strains deficient in InlA (ΔA), InlB (ΔB), or InlA/InlB (ΔAB) at 2 h post-inoculation. Each condition represents at least 4 placentas and 3 explants per placenta. Asterisks and crosses denote statistically similar populations.</p

<i>L. monocytogenes</i> enters the placenta primarily at invasive villus tips.

<p>(<b>A</b>) Consecutive histological sections of a permissively infected explant at 8 h post-inoculation, frozen and stained for <i>L. monocytogenes</i> (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>B</b>) Distribution of infected cell types in explants infected with ΔActA (top) or 10403S wild type <i>L. monocytogenes</i> (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. (<b>C</b>) Projection of a 3D confocal image showing a whole explant permissively infected with GFP-expressing <i>L. monocytogenes</i> 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 = <i>L. monocytogenes</i>. 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.</p

Infection progresses from extravillous cytotrophoblasts to stroma over 72 hours.

<p>(<b>A</b>) Histological section of explant permissively infected by <i>L. monocytogenes</i> at 72 h post-inoculation (p.i.)<b>.</b> Inset numbers correspond with right panels. Anchoring villi (AV) are major loci of infection, while floating villi (FV), which lack extravillous cytotrophoblasts (EVT), remain relatively uncolonized. Syncytiotrophoblast (SYN), indicated by βHCG (HCG, red) is still largely uninfected, with spread moving down the subsyncytial cytotrophoblasts (sCTB, insets 1 – 3) and occasionally crossing into stroma (STR, inset 4). (<b>B</b>) AV infection progresses from EVT toward fetus, while FV infection begins at the villus base. Here, a permissive infection shows dissemination from an anchoring villus (top center) to an FV, where bacteria circumnavigate the sCTB (arrowheads) while leaving SYN uninfected. (<b>C</b>) Infection from U-937 cells at 72 h p.i. shows bacteria concentrated in sCTB and bounded by the basement membrane that underlies them. A few bacteria have spread into STR (arrowheads, inset) where fetal capillaries are found. Red = cytokeratin (CK, stains cytotrophoblasts). (<b>D</b>) Explant permissively infected with ΔActA <i>L. monocytogenes</i>, which cannot spread from cell to cell. EVT are filled with bacteria (inset). Red = βHCG. (<b>A–D</b>) Green = <i>L. monocytogenes</i>, Blue = DAPI. Bar = 100 µm. (<b>E</b>) Percentage of AV and FV infected by <i>L. monocytogenes</i> introduced by cell-to-cell spread (i) or from extracellular media (e). FV infection was sporadic. (<b>F</b>) Dissemination of bacteria introduced by both means in AV. All infected AV contained bacteria in EVT. At later timepoints, infection of subsyncytial cytotrophoblasts and stroma rose. Stromal infection was not observed without sCTB infection. (<b>E–F</b>) Each condition represents two sections separated by at least 30 µm on the Z-axis in each of three placentas infected by each means. Bars = SEM.</p

<i>L. monocytogenes</i> infects anchoring villi by cell-to-cell spread.

<p>(<b>A</b>) Histological sections of explant infected by <i>L. monocytogenes</i>-containing U937 macrophage-like cells loaded with green dye (arrowheads). <i>L. monocytogenes</i> are also stained green. Photos are 24 h post-inocculation (p.i.). Both bacteria and U937 cells localize primarily to the E-cadherin-expressing extravillous cytotrophoblasts (EVT). Red:  = Ecad. Blue = DNA. Bar = 100 µm. (<b>B</b>) Bacteria are excluded from syncytiotrophoblast (SYN) and subsyncytial cytotrophoblasts (sCTB). βHCG (red) is primarily expressed by syncytiotrophoblast. Only anchoring villi (AV) are infected, while floating villi (FV) covered in syncytiotrophoblast remain uninfected. Bar = 100 µm. (<b>C</b>) Localization of <i>L. monocytogenes</i> in explants when introduced in extracellular media (e) or by cell-to-cell spread from the intracellular compartment (i) of U937 cells (in the presence of gentamicin) at 24 h p.i. Each condition represents two sections in each of three explants. Bars = SEM.</p

<i>L. monocytogenes</i> grows variably in placental explants.

<p>(<b>A</b>) Intracellular survival of <i>L. monocytogenes</i> in 86 explants from 18 placentas infected with ∼2×10⁶ 10403S (filled circles) or EGDe (open circles) wild type strains for 30 min. Gentamicin was added at 60<i> </i>min to kill extracellular bacteria and maintained in media thereafter. Infection is highly variable and growth is slower than in most cell lines. Bars = median values. (<b>B</b>) Number of internalized bacteria at 2 h post inoculation (p.i.) correlates with the number of anchoring villi in the explant (n = 30 explants, r² = 0.49). (<b>C</b>) Histological section of explant frozen and sliced at 8 h p.i., then stained for <i>L. monocytogenes</i> (green), DNA (blue), and EGFR (red), which stains trophoblast membranes. Bacteria are found in extravillous cytotrophoblasts (EVT) but not syncytiotrophoblast (SYN). Matrigel (MAT) and stroma (STR) are also indicated. Bar = 100 µm.</p

Comparison of in vivo placental structure to placenta explant model.

<p>(<b>A</b>) Structure and orientation of fetus and placenta in uterus at ∼6 weeks of gestation. Fetal structures are represented in shades of blue and purple while maternal are in shades of red. Maternal structures: MY: myometrium, SA: spiral arteries, DD: decidua (uterine lining during pregnancy), IVS: intervillous space filled with maternal blood. Fetal structures: VT: villous tree, CP: chorionic plate, UC: umbilical cord, AF: amniotic fluid. (<b>B</b>) (Enlargement of boxed area in panel A) Maternal blood surrounds the villous tree composed of anchoring (AV) and floating villi (FV), which are covered by a syncytiotrophoblast (SYN) that is underlaid by subsyncytial cytotrophoblasts (sCTB) and a basement membrane. The subsyncytial CTB layer grows increasingly discontinuous in later trimesters. Gas and nutrient exchange with the maternal blood occurs across the syncytiotrophoblast to supply fetal capillaries in the stroma (STR). At the uterine wall, extravillous cytotrophoblasts (EVT) anchor the villous tree in the decidua. Some invade the decidua and move away from the tip to remodel maternal spiral arteries, with altered gene expression patterns as they move (not shown). Notably, E-cadherin expression decreases as VE-cadherin expression rises in distal (relative to fetus) extravillous cytotrophoblasts. (<b>C</b>) A six-week placental explant anchored in Matrigel. Bar = 1 mm. (<b>D</b>) Cartoon representation of the relevant structures seen in panel C.</p

<i>L. monocytogenes</i> infects villous cytotrophoblasts when syncytiotrophoblast is removed.

<p>(<b>A</b>) Placental explant treated with collagenase-containing solution to degrade the syncytiotrophoblast (SYN). Treatment varies; some areas of syncytiotrophoblast remain (e.g. between arrowheads). All villi anchor to form extravillous cytotrophoblasts (EVT). Bar = 1 mm. (<b>B</b>) Left: histological section of enzymatically-treated villus arm, 8 h postinoculation (p.i.). No syncytiotrophoblast remains, permitting infection of both villous cytotrophoblasts (CTB) and extravillous cytotrophoblasts (EVT). Red = E-cadherin (Ecad). Green = <i>L. monocytogenes</i>. Blue = DAPI. Asterisk = Matrigel. Right: Green channel only, color inverted to show <i>L. monocytogenes</i> (solid black) with background fluorescence (faint grey) to show explant outline. Bar = 100 µm. (<b>C</b>) Distribution of infected cell types in enzymatically-treated explants compared to that in untreated explants at 8 h p.i. Here, sCTB refers to villous trophoblasts, which are subsyncytial in untreated explants but exposed after syncytiotrophoblast removal in enzymatically-treated explants. Each condition represents two sections from each of three explants. For syncytiotrophoblast (SYN) and basally accessible syncytiotrophoblast (bSYN), a “cell” was considered to be the area around a single nucleus, roughly the size of a cytotrophoblast. Bars = SEM.</p

Model of <i>L. monocytogenes</i> mechanisms for breaching the maternal-fetal barrier.

<p><i>L. monocytogenes</i> is subjected to multiple bottlenecks when infecting the placenta. Our data support extravillous cytotrophoblasts (EVT) as the primary portal of entry. First (1), relatively few <i>L. monocytogenes</i> reach the maternal decidua, carried by phagocytes. These can infect extravillous cytotrophoblasts by cell-to-cell spread, or by lysing the leukocyte and subsequently infecting extravillous cytotrophoblasts via InlA-E-cadherin interactions. (2) Extravillous cytotrophoblasts further winnow bacterial numbers by delaying the <i>L. monocytogenes</i> intracellular life cycle. If infection progresses, bacteria spread through subsyncytial cytotrophoblasts. (3) The basement membrane underlying these cells presents a third barrier, which few bacteria cross to invade the fetal stroma (STR). On the other hand, <i>L. monocytogenes</i> in the blood contacts only the syncytiotrophoblast (SYN), which is highly resistant to both internalin-mediated infection and intercellular spread. However, it is possible that sites of syncytial damage provide access to subsyncytial cytotrophoblasts. In vivo, such sites are rapidly covered by fibrinoid clots <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000732#ppat.1000732-Benirschke1" target="_blank">[28]</a> that may present yet another physical barrier to infection.</p

Actin cytoskeleton of murine syncytiotrophoblast (SYN) contributes to its elastic strength.

<p>A. Microrheology with an atomic force microscope was used to measure elastic strength of mouse trophoblast stem cells (TSC) and SYN. Photos depict microscopic cantilever positioned above cultured live cells prior to measurement. <b>B.</b> The elastic modulus (Young's modulus) of SYN is significantly higher than that of TSC (p = 4.7×10⁻⁵ by Student's T-test). Elastic modulus of SYN was measured in the exact same spot prior to and after treatment with Cyto-D for 40–60 min. Disruption of the actin cytoskeleton with Cyto-D significantly decreased the elastic modulus of SYN (p = 0.001 by Student's T-test). Bars represent median values. Graph is based on three independent experiments performed in triplicate. <b>C.</b> Immunofluorescence images of the actin (red) in mSYN show that the characteristic actin meshwork (i–iii) is disrupted by 1 hr treatment with Cyto-D (iv–vi). Nuclei are shown in white. Bars in panels i and iv are 50 um. Panels ii–iii and v–vi are representative close-up images of untreated and Cyto-D treated mSYN, respectively. Panels iii and vi show just the actin channel of ii and v respectively. Aggregation of microfilaments in distinct puncta are observed upon treatment. Bars = 10 um.</p

<i>L. monocytogenes</i> (LM) invades and replicates in murine trophoblast stem cells.

<p>A. Colony forming units (CFU) per coverslip at 2, 5, 8, and 24 hours post-inoculation (p.i.) for the following <i>L. monocytogenes</i> (LM) strains were determined: wild type (wt), InlA-deficient (del-InlA), and murinized InlA (InlA^m). Average CFU per coverslip at 2 hours p.i. for each strain is based on nine independent experiments. Average CFU per coverslip at time points 5, 8, and 24 hours p.i. for each strain are based on three independent experiments. Bars represent standard error. Average CFU at 2 hours for InlA^m is 30-fold higher than for wild type and the p-value denotes statistical significance (p = 0.008 by Student's T-test). There is no difference in invasion for wild type versus del-InlA (p = 0.3 by Student's T-test). <b>B.</b> Immunofluorescence image of mTSC at 2 hours p.i.: LM is shown in green, nuclei in white, actin in red. Arrowheads point to foci of infection, Bar = 50 um.</p