ActA is required for crossing of the fetoplacental barrier by Listeria monocytogenes

Abstract

The facultative intracellular bacterial pathogen Listeria monocytogenes induces severe fetal infection during pregnancy. Little is known about the molecular mechanisms allowing the maternofetal transmission of bacteria. In this work, we studied fetoplacental invasion by infecting mice with various mutants lacking virulence factors involved in the intracellular life cycle of L. monocytogenes. We found that the placenta was highly susceptible to bacteria, including avirulent bacteria, such as an L. monocytogenes mutant with an hly deletion (DeltaLLO) and a nonpathogenic species, Listeria innocua, suggesting that permissive trophoblastic cells, trapping bacteria, provide a protective niche for bacterial survival. The DeltaLLO mutant, which is unable to escape the phagosomal compartment of infected cells, failed to grow in the trophoblast tissue and to invade the fetus. Mutant bacteria with inlA and inlB deletion (DeltaInlAB) grew in the placenta and fetus as well as did the wild-type virulent stain (EGDwt), indicating that in the murine model, internalins A and B are not involved in fetoplacental invasion by L. monocytogenes. Pregnant mice were then infected with an actA deletion (DeltaActA) strain, a virulence-attenuated mutant that is unable to polymerize actin and to spread from cell to cell. With the DeltaActA mutant, fetal infection occurs, but with a significant delay and restriction, and it requires a placental bacterial load 2 log units higher than that for the wild-type virulent strain. Definitive evidence for the role of ActA was provided by showing that a actA-complemented DeltaActA mutant was restored in its capacity to invade fetuses. ActA-mediated cell-to-cell spreading plays a major role in the vertical transmission of L. monocytogenes to the fetus in the murine model.

FIG. 1.

Infection of pregnant mice with L. innocua. Pregnant BALB/c mice were injected i.v. with 5 × 10⁷ bacteria by day 14 of gestation. Animals were monitored by quantifying bacterial growth at intervals. (A) Bacterial growth in spleen and blood of pregnant mice. The results shown are means ± standard errors from groups of five mice (each experiment was repeated twice) and are expressed as the log₁₀ bacteria (CFU) per organ or log₁₀ bacteria per ml of blood. (B) The kinetics of bacterial growth are represented in the left panels by dot plots corresponding to individual bacterial counts for each placenta (top panel) and each fetus (lower panel) for the indicated times. In the right panels, means ± standard errors for each time are given.

FIG. 2.

Infection of pregnant mice with ΔInlAB and ΔLLO mutants. Pregnant BALB/c mice were injected i.v. at day 14 of gestation with 5 × 10⁵ of wild-type L. monocytogenes (EGD) and its isogenic hly or inlAB deletion mutants. Animals were monitored by quantifying bacterial growth at intervals. (A) Bacterial growth in organs (spleen, liver and brain) and in blood of pregnant mice for EGDwt and ΔInlAB strains. Results shown are means ± standard errors of the means from groups of three to five mice and are expressed as the log₁₀ bacteria (CFU) per organ or log₁₀ bacteria per ml of blood. (B) The kinetics of bacterial growth are represented on the left panels by dot plots corresponding to individual bacterial counts for each placenta (top panel) and each fetus (lower panel) for the indicated times and strains. In the right panels, means ± standard errors for each time are given for wild-type, ΔInlAB, and ΔLLO strains. The numbers reported near each value correspond to the percentage of infected placenta among all analyzed placentas. When observed, fetal losses are symbolized by a cross. P values reported in each graph correspond to the comparison between EGDwt and the indicated mutant. NS, not statistically different.

FIG. 3.

Role of ActA in the crossing of the fetoplacental barrier. Pregnant BALB/c mice were injected intravenously at day 14 of gestation with 5 × 10⁵ L. monocytogenes actA deletion mutant cells (ΔActA) or the ΔActA mutant complemented with actA (ΔActA+actA). As compared to the isogenic mutants, bacterial growth for the EGDwt strain is indicated by the dotted lines. Animals were monitored by quantifying bacterial growth at the indicated times. (A) Bacterial growth in organs (spleen, liver, and brain) and in blood of pregnant mice infected with ΔActA (left panels) and actA-complemented ΔActA+actA (right panels) strains. Results shown are means ± standard errors from groups of three to five mice and are expressed as the log₁₀ bacteria (CFU) per organ or log₁₀ bacteria per ml of blood. (B) The kinetics of bacterial growth are represented on the left panels by dot plots corresponding to individual bacterial counts for each placenta (top panel) and each fetus (lower panel) for the indicated times and strains. In the right panels, the means ± standard errors for each time and strain are given for ΔActA and the actA-complemented ΔAct mutant and P values for the comparison between the two strains are also reported. The numbers reported near each value correspond to the percentage of infected placenta among all analyzed placentas. All data are representative of three independent experiments. P values reported in each graph correspond to the comparison between EGDwt and the indicated mutant. NS, not statistically different.

FIG. 4.

Histological examination of placental infectious foci obtained with wild-type L. monocytognes and its ΔInlAB and ΔActA isogenic mutants. Gram-Weigert staining at different magnifications (top and bottom panels) were performed on placental sections 72 h after intravenous infection with 2 × 10⁵ wild-type L. monocytogenes cells or with mutants with the inlAB locus (ΔInlAB) or actA (ΔActA) deleted. Arrows indicate the important spreading to adjacent and distant cells. Arrowheads symbolized the well-delimited peripheral zones from foci induced by infection due to the ΔActA mutant.

FIG. 5.

Model of crossing of the fetoplacental barrier by L. monocytogenes. Two layers of syncytiotrophoblastic cells (ScT) and one layer of endothelial cells (EC) surrounding a fetal vessel (FV) constitute the murine fetoplacental barrier. L. monocytogenes (LM) infects the syncytiotrophoblastic layer and crosses the fetoplacental barrier by ActA-dependent cell-to-cell spreading.