CELLULAR AND MOLECULAR MECHANISM OF LISTERIA ADHESION PROTEIN-MEDIATED BACTERIAL CROSSING OF THE INTESTINAL BARRIER
The crossing of host barriers (intestinal, blood-brain, and placental) is a critical step for systemic infections caused by entero-invasive pathogens. In the intestine, the epithelial cells are the first line of defense against enteric pathogens. Listeria monocytogenes is a facultative-intracellular foodborne pathogen that first crosses the intestinal barrier to cause a systemic infection. However, the underlying mechanism is not well understood.
We demonstrate that Listeria adhesion protein (LAP) promotes the translocation of L. monocytogenes across the intestinal barrier in mouse models (A/J and C57BL/6). Relative to the wild-type (WT; serotype 4b) or the isogenic bacterial invasion protein Internalin A mutant (ΔinlA) strain, the lap─ strain showed significant defect in translocation across the intestinal barrier and colonization of the mesenteric-lymph nodes, liver and spleen in the early phase of infection (24 h and 48 h). LAP induces intestinal epithelial barrier dysfunction for increased translocation as evidenced by increased permeability to 4-kDa FITC-dextran (FD4), a marker of paracellular permeability, in the serum and urine of WT and ΔinlA- infected mice and across Caco-2 cell barrier, but not the lap─ mutant strain. Microscopic examination confirmed localization of the WT and ΔinlA strains in the tight junction, a crucial barrier of intestinal paracellular permeability, in the mouse ileal tissue but the lap─ strain remained confined in the lumen. LAP also upregulates TNF-α and IL-6 in intestinal epithelia of mice and in Caco-2 cells for increased permeability.
Investigation of the underlying molecular mechanisms of LAP-mediated increase in intestinal permeability by using lap─ mutant strain, purified LAP and shRNA-mediated Hsp60 suppression, we demonstrate that LAP interacts with its host receptor, Hsp60, and activates the canonical NF-κB signaling, which in turn facilitates myosin light-chain kinase (MLCK)-mediated opening of the epithelial barrier via the cellular redistribution of major epithelial junctional proteins claudin-1, occludin, and E-cadherin. Pharmacological inhibition of NF-κB or MLCK in cells or genetic ablation of MLCK in mice (C57BL/6) prevents mislocalization of epithelial junctional proteins, intestinal permeability and L. monocytogenes translocation across the intestinal barrier.
Furthermore, LAP also promotes L. monocytogenes translocation across the intestinal barrier and systemic dissemination in a Mongolian gerbil that are permissive to the bacterial invasion proteins; InlA-and InlB-mediated pathways; similar to that in humans. We show a direct LAP-dependent and InlA-independent pathway for L. monocytogenes paracellular translocation across the intestinal epithelial cells that do not express luminally accessible E-cadherin. Additionally, we show a functional InlA/E-cadherin interaction pathway that aids L. monocytogenes translocation by targeting cells with luminally accessible E-cadherin such as cells at the site of epithelial cell extrusion, epithelial folds and mucus-expelling goblet cells. Thus, L. monocytogenes uses LAP to exploit epithelial innate defense in the early phase of infection to cross the intestinal epithelial barrier, independent of other invasion proteins.
This work fills a critical gap in our understanding of L. monocytogenes pathogenesis and sheds light to the complex interplay between host-pathogen interactions for bacterial crossing of the crucial intestinal barrier.