Construction Of Vaccine Vector Based On LLO Attenuated Listeria Monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen that escapes from a phagosome and grows within the cytosol of host cells.This bacterium secretes a pore-forming cholesterol-ependent cytolysin,listeriolysin O?LLO?,a major virulence factor that enables the bacteria to cross the phagosomal membrane and is also involved in activation of cellular processes.L.monocytogenes has evolved multiple sophisticated mechanisms to regulate the activity of LLO in the phagosome and minimize activity in host cytosol.The results of previous studies showed that LLO lost the hemolysis activity after the deletion of amino acids 472-483,so whether Listeria monocytogenes vaccine could be constructed based on the phenomenon.In order to further study the novel functions on immunity of L.monocytogenes,we used LLO as the research object.Here in this study,we identified Asn₄₇₈ and Val₄₇₉ at the C-terminal of LLO as novel critical residues that were required for haemolytic activity of LLO and bacterial virulence.The double mutant LLO_N478AV479A showed a more remarkable decrease in the haemolytic activity both at acidic and neutral pHs compared with the wild-type LLO.L.monocytogenes synthesizing LLO_N478A,or LLO_V479A in culture supernatants had the strong capability to lyse erythrocytes,similar to the bacteria expressing wild-type LLO.The bacteria secreting the mutated LLO_N478AV479A has barely detectable haemolytic activity,but host cell cytotoxicity exhibited,escaped,intracellularly grew and cell-to-cell spread with the same efficiency as the strain expressing wild-type LLO,however,resulted in a highly-attenuated in virulence in the mouse listeriosis model.These data strongly suggest that these two residues at the C-terminal are key sites that are required for haemolytic activity of LLO and essential for Listeria pathogenicity in mice.The finding that the nearly non-haemolytic mutant expressing LLO_N478AV479A478AV479A capable of growing intracellularly indicates that mutagenesis of a virulence determinant can attenuate virulence and provides a novel approach to the development of live vaccine strains for immunotherapy.In order to explore the relationship between Listeria infection and activation of MAPK signaling pathway,we used Listeria EGD-e and Caco-2 epithelial cells as reference strains and infected subjects,the study found that the bacteria can phosphorylate ERK1/2 and p38 signaling molecules to activate ERK1/2 and p38 signaling pathway.Infection with the?hly strain lacking the LLO failed to phosphorylate ERK1/2 and p38.We here for the first time demonstrate that LLO is responsible for activation of ERK1/2 and p38 signaling pathway via phosphorylation on ERK1/2and p38 during L.monocytogenes infection.The finding will elucidate the complex mechanism of MAPK signaling pathway during L.monocytogenes infection.