LAP-Hsp60 complex modulates epithelial tight junction barrier

Abstract

During infection, Listeria adhesion protein (LAP), a housekeeping enzyme, acts as a tight junction modulator (TJM) through interaction with Hsp60 to facilitate Listeria monocytogenes translocation across the intestinal epithelial barrier. Here, we used purified LAP as a potential TJM to overcome the limiting and variable effects observed by other agents in the class. We structurally determined the LAP interaction alone and in complex with Hsp60 utilizing cryo-EM and computational analysis. LAP structure resolved at 2.83 Å, forms multimeric interlocking dimers and tetramers, and the N-domain interacts with Hsp60, while the C-domain bridges bacterial surface receptor InlB. The structural studies complement LAP-mediated cyclic peptide drugs (vancomycin and desmopressin) absorption across the intestinal barrier in a mouse model without inducing inflammation or adverse effects on the TJ architecture. This study demonstrates that the LAP-Hsp60 complex is the basis for downstream utilization of LAP or peptide mimetics as promising TJM for improved peroral biologics delivery.

Keywords: Hsp60; LAP; cryo-EM; mouse; peptide drug delivery; structure-function; tight junction modulator.

Fig. 1 |. The cryo-EM structure of tetrameric LAP.

a. The cryo-EM map of the LAP tetramer allows unambiguous assignment of the side chains. Each monomer is in a different color, and each domain is indicated. b. The tetrameric structure of LAP consists of interlocking subunits flanked on each end by C-domains. c. The tetramer consists of two dimers arranged in a pseudo-2-fold symmetry that assemble along the C-domain of LAP. d. Each dimer consists of extended interlocking monomers (green and pink), each consisting of an N-domain, an extended linker region, and a C-domain. e. Each tetramer structure consists of two LAP dimers (green/pink; blue/violet) that align well with one another with RMSD of 0.1 Å.

Fig. 2 |. The structure of the LAP-Hsp60 complex using cryo-EM and computational modeling.

a. The location of the top 10 solutions from the computational docking of the N-domain of LAP to Hsp60 (gray) are shown as spheres representing the center of mass of each solution; green agrees with the location found in our cryo-EM, red is in other locations. Orthogonal views are shown. b. The models are shown for each of the six solutions that agree with the cryo-EM and share similar binding modes, given that Hsp60 is a heptamer. c. Only the top solution is now shown of the N-domain of LAP (orange) with Hsp60 (gray). d. An enlarged view of only the N-domain of LAP (orange) with a monomer of the primary interaction molecule from Hsp60 (gray), depicting shape complementarity. e. The electrostatic properties of the N-domain of LAP show a strongly positive region (blue) along the region that interacts with Hsp60. f. The electrostatic properties of the Hsp60 assembly show strongly negative regions (red) at the interface of each of the Hsp60 monomers, where the N-domain of LAP binds (yellow dashed circle). g. An enlarged view of the interacting region of N-domain LAP (orange) with Hsp60 (gray). h. An orthogonal view of panel G looking at the binding interface of the N-domain of LAP consisting of a buried surface area of ~1000 Ų, with the interacting residues highlighted into the three primary interacting groups including (i) D101-E108 (gray), (ii) S430-Q438 (green), and (iii) K391-I402 (cyan).

Fig. 3 |. The cryo-EM structure of dimeric LAP and a structural model for the InlB-LAP-Hsp60 complex.

a. A separate 3D class showed the dimeric structure of LAP, flanked on each end by C-domains; no density was observed for the N-domain for these flanking domains. b. AlphaFold was used to model the GW1–3 domains of lnlB with the LAP dimer (ribbon). The locations of InlB for the top 50 results are shown as spheres, with the green spheres clustered in proximity to the predicted interaction site indicated in the panel. c. Orthogonal views of the AlphaFold models are shown in cartoon representation for select InlB models indicated in green from panel B; the numbers indicate the rank of each model from AlphaFold. d. The electrostatic properties of InlB and the LAP dimer are shown with the indicated charge complementary surfaces. The dimeric interface in LAP across the C-domains is indicated by the yellow dashed oval, with the putative interacting interface displayed by the dashed black box. e. A model for the InlB-LAP-Hsp60 interaction complex is shown based on cryo-EM studies, analysis of surface electrostatics, and computational modeling.

Fig. 4 |. LAP increases paracellular permeability in cultured cell models.

a. Overview of paracellular permeability study in transwell setup. b. LAP dose-dependent effect on FITC- 4 kDa dextran (FD4) permeability through Caco-2 cell monolayers. c-d. Apparent permeability coefficient (${\mathrm{P}}_{\mathrm{a}\mathrm{p}\mathrm{p}}$) measurement of LAP-mediated FD4 through Caco-2 (c) and MDCK cell line (d). e. Comparison of ${\mathrm{P}}_{\mathrm{a}\mathrm{p}\mathrm{p}}$ value of FD4 after treatment with LAP, EDTA, and Na-Caprate. f-g. LAP and subdomain protein (N1, N2, C1, and C2)-mediated TEER and FD4 permeability through Caco-2 monolayers in 1 h. h-i. Effect of fasted simulated stomach (FaSSGF) and small intestinal (FaSSIF) fluid on LAP mediated FD4 permeability. j-k. Peptides from the N-terminal domain of LAP that interact with Hsp60 (j) and their effect on FD4 flux through Caco-2 monolayers in 1 h (k). Peptides 1, 2 and 3 were combined. Peptide 4 is located outside the Hsp60 binding domain. All error bars represent SEM (n = 3–6). ****p < 0.0001; **p < 0.01; *p < 0.05; ns, no significance.

Fig. 5 |. LAP facilitates paracellular permeability across the intestinal barrier in mice

a. Overview of paracellular permeability study in C57BL/6 mice. b-c. LAP-Dose-dependent response for 4-kDa FITC-dextran (FD4) permeability in urine (b) and serum (c) of mice. d-e. Time-dependent response for FD4 permeability in urine (d) and serum (e) of mice. f. Gross pathology of the intestine 4 h post gavage g. Green fluorescence emission from feces (4-h-post gavage from mice treated with FD4 (arrow) but not from LAP+FD4. h. Hematoxylin and Eosin (H&E)-stained histopathological images of mouse ileal sections. i-k. ELISA analysis of inflammation markers, Lipocalin-2 (i) CRP (j) and IL-6 (k). All error bars represent SEM (n=3–6). ****p < 0.0001; ***p < 0.001; **p < 0.01; ns, no significance.

Fig. 6 |. LAP-mediated intestinal epithelial barrier opening is transient

a. Overview of mice experiment to demonstrate TJ recovery. b-c. FD4 permeability in serum (b) and urine (c) of mice after 8 h of LAP treatment. d. Appearance of mouse intestine after 8 h of LAP treatment. e. Confocal images of ileal tight junction protein occludin, claudin-1, β-actin and E-cadherin after LAP treatment. f-h. Intracellular commensal bacterial counts in the intestine after aerobic (f) and anaerobic (g) incubation, in blood MLN, spleen and liver (h) after 4 h of LAP or subdomain proteins treatment. All error bars represent SEM (n = 3–6). ****p < 0.0001; ***p < 0.001; **p < 0.01; ns, no significance.

Fig. 7 |. LAP facilitates protein (peptide) drug passage across the intestinal epithelium in mice.

a-b. Agar diffusion assay showing antimicrobial activity of vancomycin mixed with LAP and subdomain proteins against Lm and Sa (a). Graphical representation of zones of inhibition (b). c-d. LAP and subdomain-mediated delivery of vancomycin in serum (c) and urine (d) 4 h post-gavage e-f. LAP and subdomain-mediated delivery of desmopressin in plasm (e) and urine (f) 4 h post gavage. g-i. Gross pathology (g), Histopathology image (h), and scores (i) j. Mass-Spec analysis of LAP in mouse serum following gavage with LAP+vancomycin. All error bars represent SEM (n = 3–8). ****p < 0.0001; ***p < 0.001; **p < 0.01; ns, no significance.