| Listeria adhesion protein (LAP), an alcohol acetaldehyde dehydrogenase (lmo1634) in Listeria monocytogenes, has dual function: enzymatic activity, and adhesion capacity. Recently, our lab reported that LAP is involved in transepithelial translocation of L. monocytogenes through a paracellular route in Caco-2 cell model. In addition, LAP secretion is facilitated by SecA2 and secreted LAP is re-associated on bacterial cell wall to enhance LAP-mediated infection. However, the importance of secreted LAP and the cellular/molecular mechanisms of LAP-mediated translocation were unknown. In this study, LAP secretion levels were examined in fifty-two clinical isolates of L. monocytogenes. Experiments with three high and three low secreting isolates indicated that LAP-mediated L. monocytogenes adhesion, invasion and translocation in Caco-2 cells are each directly related to secreted LAP levels. There was minimal (<2.0%) sequence heterogeneity in lap genes from high and low LAP secreting isolates and they all expressed similar levels of lap transcripts. Western blot analysis of different cell fractions indicated that low LAP-secreting cells accumulated largely in the intracellular (cytosolic) fractions.;To investigate the mechanism of LAP-mediated transepithelial translocation, we performed human gene chip microarray analysis to determine the proteins that may be involved in tight junction opening and also examined the role of TNF-alpha, NF-kappaB, and myosin light chain kinase (MLCK) in this process. Data showed LAP increased TNF-alpha expression in Caco-2 cells likely through activation of NF-kappaB pathway. Curcumin, a NF-kappaB inhibitor, affected TNF-alpha production and also bacterial translocation. MLCK inhibitor, ML-9, affected DextranFITC permeability and also bacterial translocation. Microarray data indicated that LAP down regulates expression of adherence junction proteins, E-cadherin and beta-catenin, which were further confirmed by qRT-PCR, Western blot and fluorescence microscopy staining. In summary, this study revealed that secreted LAP is a key player in LAP-mediated Listeria infection and LAP activates host signaling pathway to modify tight junction proteins which facilitates L. monocytogenes transepithelial translocation through a paracellular route. |
