Magnesium ion disrupts LAP surface re‐association of Listeria monocytogenes by dissociation of InlB

Listeria monocytogenes crosses intestinal epithelial barrier for the systemic spread, which is facilitated in part, by Listeria adhesion protein (LAP). LAP, a housekeeping enzyme called acetaldehyde alcohol dehydrogenase, is present in both pathogenic and nonpathogenic Listeria . LAP is secreted with the aid of SecA2 translocon, and its’ re‐association on the surface of pathogenic Listeria i.e., L. monocytogenes is critical for its function. However, the cell wall component(s) involved in LAP interaction is unknown, which was investigated. Using L. monocytogenes F4244 (WT), an isogenic lap − strain and purified recombinant LAP in the Caco‐2 cell adhesion assay and Western blot, we examined LAP re‐association on lap − cells. Extraneous added LAP restored adhesion capacity of lap − , but not of L. innocua to Caco‐2 cells, suggesting the presence of LAP‐interacting receptor(s) only on pathogenic bacteria. To locate LAP interaction partners, co‐immunoprecipitation of WT cell wall fraction using anti‐LAP mAb was analyzed by SDS‐PAGE. Selected protein bands were subjected to MALDI‐TOF protein identification showing involvement of internalin B (InlB). Next, we investigated the effect of MgCl 2 pretreatment on LAP re‐association on the cell surface, cell‐free supernatant, and consequent bacterial adhesion. Mg 2+ is a wall teichoic acid (WTA) charge neutralizing agent that reduced levels of InlB binding on the WT cell wall, resulting in surface disassociation of LAP. However, only partial surface reduction of LAP was observed in Δ inlB strain suggesting more than one ligand is involved. In conclusion, our results indicate that both InlB and WTA may participate in LAP re‐association on L. monocytogenes cell surface, which is essential for its pathogenic activities. Support or Funding Information The technical assistance of Uma Aryal, Valerie Ryan, and Aaron Gall and funding from USDA‐ARS project number 59‐8072‐6‐001 through the Center for Food Safety Engineering at Purdue University are acknowledged.LAP Surface Ligand Fishing Expedition As the experiment scheme shown (A), several Co‐immunoprecipated candidates were visualized by Sliver staining. Red arrow point to a potential receptor around 70 kDa (B). Abundance of each identified protein was graphed according to relative protein abundance (C). Internalin B is the first protein present only in pathogenic strain.MgCl 2 Treatment Affects LAP Dissociation and Bacterial Adhesion WB protein analysis in Supernatant and Cell Wall Fraction (A), growth curve (B) and adhesion (C) of LmWT grow with additional MgCl 2 in TSBYE.