Abstract
Listeria monocytogenes is a gram positive, food-borne bacteria that causes gastroenteritis, meningitis and abortion in pregnant women. The ability to enter into the mammalian cells is critical for survival and pathogenesis of L. monocytogenes. L. monocytogenes expresses a surface protein Internalin A (InlA) which specifically interacts with the host protein E-cadherin, a calcium-dependent adhesion molecule important in cell-cell junction formation. InlA/E-cadherin interaction is critical for crossing the intestinal barrier and leads to signalling events that activate actin polymerization within host cells. Localized actin polymerization at the sites of bacterial entry causes membrane remodelling leading to engulfment of bacteria inside vacuoles. A key question is what other host physiological processes are exploited by L. monocytogenes in order to internalize itself through InlA/E-cadherin pathway? Here, I demonstrate that L. monocytogenes stimulates host exocytosis, a process in which intracellular vesicles fuse with plasma membrane, by activating the exocyst complex. Using exocytic probe VAMP3-GFP to detect exocytosis and InlA-coated beads as a model for L. monocytogenes, I found that InlA-coated beads stimulate localized exocytosis at the sites of entry. Exocytosis is critically regulated by N-ethylmaleimide sensitive factor (NSF), a protein that recycles SNAREs after SNARE-mediated vesicular fusion with plasma membrane. Using TAT-NSF fusion peptide to inhibit the function of NSF, I found that the entry of InlA-coated beads as well as exocytosis was significantly reduced indicating that NSF plays important role in InlA-mediated entry. Exocytosis is also regulated by the exocyst complex, a conserved octameric complex comprised of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84 subunits. RNAi-mediated depletion of the exocyst subunits Sec6, Exo70 and Exo84 significantly reduced the entry of InlA-coated beads indicating their role in entry. I also assessed the role of exocyst regulator RalA using RNAi and found that depletion of RalA significantly reduces entry. My data shows that reduction in entry of InlA-coated beads after inhibiting NSF or depleting Sec6, Exo70, Exo84 and RalA is not due to reduced surface levels of E-cadherin indicating that exocytosis is stimulated downstream of E-cadherin activation. Collectively, these results indicate that L. monocytogenes subverts exocytosis along with the exocyst complex and its regulator RalA to facilitate entry into mammalian cells via InlA-mediated pathway.
The normal function of E-cadherin on epithelial cells is to interact homophilically to form adherens junctions. Initially, two participating cells explore their environment by forming lamellipodia or membrane ruffles prior to forming initial contact sites where E-cadherin interactions occur in puncta, structures that comprise of clusters of homophilic E-cadherin dimers. The formation of exploratory structures early during adherens junction formation is mediated by actin polymerization at the cell periphery that provides force that bring two plasma membranes together. The initial contact sites expand as more E-cadherin homophilic interactions develop, leading to maturation of the adherens junction. An important question is whether processes apart from actin polymerization contribute to the formation of adherens junctions. My preliminary results using latex beads coated with the extracellular domain of E-cadherin show that homophilic interaction between E-cadherin molecules can stimulate exocytosis. These findings raise the interesting possibility that membrane delivery through exocytosis might contribute to adherens junction formation.