Fig 1.
Bacterial effector InlP binds host adaptor protein afadin.
(A-B) InlP-GST fusion protein (InlP) or GST protein alone (-) bound to glutathione-Sepharose resin were incubated overnight with protein extracts from (A) MDCK or MDCK AF-6-/- cells or (B) human placenta. Input (IN) and elution fractions (EF) were analyzed by Western blot with anti-afadin antibodies. Actin: loading control. (C-E) Immunofluorescence of human placental villi stained for afadin (red) and DAPI (blue). In panel C the white bar = 10 μm, the asterisk labels a portion of the stroma, and the arrow points to some of the CTBs. White rectangles in panel C indicate the locations of zoomed insets shown in panels D-E.
Fig 2.
Distinct features of InlP as compared to Lmo2027.
(A) Pairwise sequence alignment of InlP and Lmo2027 with conserved amino acids highlighted in red. The LRRs and calcium binding site on InlP are noted. Arrows signify ß-sheets and coils correspond to α-helices. (B) Loading control of pull down experiments on Lmo2027 by Coomassie blue staining. GST protein alone (-), Lmo2027-GST fusion protein (2027) or InlP-GST fusion protein (InlP) bound to glutathione-Sepharose resin used as bait for pull-down experiments with protein extracts from MDCK cells. The elution fraction from pull-down samples using InlP-GST (InlP) as bait were sequentially diluted 5-fold before SDS analysis; undiluted elution fractions were analyzed from pull-down samples using GST (-) or Lmo2027 (2027). Data shown are Coomassie staining and the most abundant band in each lane represents the bait. (C) GST fusion proteins or GST protein alone (-) bound to glutathione-Sepharose resin were incubated overnight with protein extracts from MDCK cells, and elution fractions were analyzed by Western blot with anti-afadin antibodies. The following GST fusion proteins were used: GST-Lmo2027 (Lmo2027) and GST-InlP (InlP). Undiluted elution fractions were analyzed with the exception of the elution fractions marked by black triangles: elution fractions from the experiments using GST-InlP as bait were sequentially diluted by 5-fold before western blot analysis. Actin: loading control. (D) Crystal structure of InlP with schematic depiction of domain layout below. (E) Crystal structure of Lmo2027 with color-coded domains, like in (D).
Fig 3.
LRR5 stabilized afadin- InlP interaction.
(A) Loading control of InlP-afadin binding pull-down experiments with LRR mutants by Coomassie blue staining. GST protein alone (-), InlPΔLRR5-GST fusion protein (ΔLRR5), InlPΔLRR7-GST fusion protein (ΔLRR7), or InlP-GST (InlP) bound to glutathione-Sepharose resin were used as bait for pull-down experiments with protein extracts from MDCK cell line. Data shown are Coomassie staining, and the most abundant band in each lane represents the bait. (B-C) GST fusion proteins or GST protein alone (-) bound to glutathione-sepharose resin were incubated overnight with protein extracts from MDCK cells, and elution fractions (EF) were analyzed by Western blot with anti-afadin antibodies. The following GST fusion proteins were used: GST-InlP (InlP) and GST-InlP with deletions in LRR5 (ΔLRR5) or LRR7 (ΔLRR7). Actin: loading control. Undiluted elution fractions were analyzed with the exception of the elution fractions marked in panel C that were diluted 5-fold. Actin: loading control. (D) Scatter plot showing the logarithm of the ratio of intensity of InlP-GST bound proteins to the intensity of ΔLRR5-GST bound proteins versus total intensity of InlP-GST bound proteins. The intensities (A.U.) of the proteins were identified through mass spectrometry using InlP-GST or InlPΔLRR5-GST as baits to identify host binding partners in the MDCK cell culture extracts. Blue diamonds show all the proteins apart from afadin, which is indicated as orange square. Using this metric, afadin is 2.7 standard deviations away from the mean for this group.
Fig 4.
Cell-ECM traction stresses decrease in magnitude when afadin is not expressed or when InlP is present.
(A) MDCK, MDCK AF-6-/-, and MDCK cell lines stably transfected with inlP under a doxycycline (DOX) inducible promoter were grown on Transwell filters for three days until formation of polarized epithelia. Inulin-FITC was added to the upper compartment of the Transwell insert. The cell permeability was analyzed at different time points by measuring the fluorescence intensity in the lower compartment. MDCK InlP- and MDCK InlP+: stably transfected cells without or with InlP expression secondary to doxycycline exposure for 24 h prior to the addition of Inulin-FITC. (B) Representative phase images (first row) and maps showing the magnitude of traction stresses of confluent MDCK monolayers adherent to hydrogels coated with collagen I (color indicates stress magnitude in Pa). Columns refer to: MDCK InlP-, MDCK InlP+, MDCK AF-6-/- InlP- and MDCK AF-6-/- InlP+. Scale bar corresponds to 20 μm. (C) Time-averaged strain energy (Nn•μm) calculated for multiple regions within confluent MDCK monolayers. Boxplots refer to N = 11 MDCK InlP-, N = 11 MDCK InlP+, N = 12 MDCK AF-6-/- InlP-, N = 12 MDCK AF-6-/- InlP+ monolayer regions. (D-E) TFM results for single cells adherent to polyacrylamide hydrogels coated with collagen I. (D) Instantaneous maps showing the magnitude of traction stresses (color indicates stress values in Pa). Columns refer to: MDCK InlP-, MDCK InlP+, MDCK AF-6-/- InlP- and MDCK AF-6-/- InlP+. Cell outlines are in white. Scale bar corresponds to 20 μm. (E) Time-averaged strain energy (nNμm) from individual cells. Boxplots refer to N = 15 MDCK InlP-, N = 17 MDCK InlP+, N = 15 MDCK AF-6-/- InlP-, and N = 18 MDCK AF-6-/- InlP+. For boxplots in (C), (E) circles represent outliers, and the boxplots’ notched sections show the 95% confidence interval around the median (Wilcoxon–Mann–Whitney test). One or two asterisks denote statistically significant differences between the medians of two distributions (<0.05 or <0.01, respectively; Wilcoxon rank sum test).
Fig 5.
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.