InlP enhances transcytosis through MDCK polarized monolayers.

(A) Intracellular growth curves of wild type 10403S L. monocytogenes in MDCK (black solid line) or MDCK AF-6-/- (black dashed line) cells and of ΔinlP L. monocytogenes in MDCK (gray solid line) or MDCK AF-6-/- (gray dashed line) cells. Unpolarized monolayers of MDCK cell lines were infected with 10403S at MOI of 150:1. Gentamicin was added at 50 μg/ml to kill extracellular bacteria and maintained in media thereafter. Each growth curve represents the means and standard deviations of colony forming units (CFU) over time from three separate experiments performed in triplicates. (B) Afadin limits initial L. monocytogenes invasion. Flow cytometry quantifying the number of L. monocytogenes-containing MDCK and MDCK AF-6-/- cells 5 hours post infection. Data were normalized to 1 for wild-type MDCK cells infected with ΔactA L. monocytogenes for each experiment, and pooled from two independent experiments (each experiment is depicted by different symbols). (C) Amount of transcytosis by wild-type, ΔinlP, and ΔactA L. monocytogenes through wild-type MDCK monolayers. Data were normalized to 1 for MDCK cells infected with wild-type L. monocytogenes for each experiment, and pooled from four independent experiments. Each experiment is depicted by different symbols. (D) Amount of transcytosis by wild-type, ΔinlP, and ΔactA L. monocytogenes through MDCK AF-6-/- monolayers. Data were normalized to 1 for AF-6-/- MDCK cells infected with wild-type L. monocytogenes for each experiment, and pooled from three independent experiments. Each experiment is depicted by different symbols. (E) Amount of transcytosis by wild-type, ΔinlP, and ΔLRR5 L. monocytogenes through MDCK monolayers. Data were normalized to 1 for MDCK cells infected with wild-type L. monocytogenes for each experiment, and pooled from three independent experiments. Each experiment is depicted by different symbols.