Effect of Listeria monocytogenes preexposure to mammalian cells under anaerobioc environment in LAP-mediated pathogenesis in subsequent infection under anaerobic conditions

Listeria monocytogenes, the causative agent of listeriosis is ubiquitous in nature. Consumption of contaminated food with this microorganism may cause 20–30% fatality in high-risk immunocompromised individuals. Listeria’s successful survival to harsh conditions in the gut and strategies for adhesion and invasion to host cells are key factors in initiating listeriosis. It is essential to understand in vivo adhesion and invasion mechanisms of Listeria to host cells. Natural conditions that L. monocytogenes encounters during infection inside the host include anaerobiosis, natural microflora, antimicrobial compounds, mucus, and the intestinal fluid. We have previously shown that Listeria adhesion protein (LAP), a house-keeping enzyme of about 96 kDa is involved in receptor-mediated adhesion and transepithelial translocation through intestinal epithelial cells. Moreover, expression of LAP was shown to be increased when bacteria were grown under anaerobic condition. In this study, we examined whether direct contact and incubation of bacteria with the host cells under anaerobic conditions would enhance the expression of LAP and subsequent adhesion to Caco-2 cells. Furthermore, we evaluated LAP expression in L. monocytogenes after growth in modified simulated intestinal fluid (mSIF) containing 3 mg/ml of pancreatin under anaerobiosis and the corresponding effect on adhesion. Bacterial strains, F4244 (WT), lap−, lap+, Δ secA2, and secA2+, were used in this study. To examine contact-dependent LAP expression, bacteria were added to Caco-2 cells (MOI 100:1) and secreted proteins in supernatant were isolated and immunoprobed with antibodies to LAP, NamA, and InlA. Bacteria were recovered by differential centrifugation and adhesion properties of bacteria were subsequently evaluated in an adhesion assay (MOI 10:1). Protein expression data indicated that secretion and surface-associated LAP increased when L. monocytogenes was grown in contact with Caco-2 cells under anaerobic condition. Increased LAP expression also promoted increased adhesion of L. monocytogenes to Caco-2 cells. To examine the effect of mSIF on LAP expression and LAP-mediated adhesion, WT was grown in either BHI or mSIF aerobically or under anaerobiosis and then evaluated for adhesion to Caco-2 cells (MOI 10:1). Data show that LAP expression was slightly lowered when grown in mSIF under aerobic conditions and it was thought to be due to the proteolysis of secreted LAP by proteolytic enzymes present in mSIF. Though, the LAP expression was slightly increased under anaerobiosis, adhesion was not affected. Overall, the data show, bacterial contact with host cells and growth in mSIF can enhance increased LAP secretion in L. monocytogenes when grown under anaerobic condition thus validating the significance of LAP secretion and interaction during infection