Mechanisms Of Listeriolysin O-triggered ERK1/2 Phosphorylation During Infection By Listeria Monocytogenes

Listeria monocytogenes is a gram-positive intracellular bacterium.It contains listeriolysin O(LLO),which causes perforation of the host cells membrane and is essential for intracellular replication of Listeria.Previous studies have found that LLO can cause phosphorylation of the mitogen-activated protein kinase(MAPK)family protein ERK1/2 after host cells infection,but the mechanism is unknown.In this study,the molecular mechanism of ERK1/2 phosphorylation was analyzed by Caco-2 cells infected with L.monocytogenes.We infected or incubated Caco-2 cells with Listeria and purified recombinant protein LLO(5n M)to detect ERK1/2 phosphorylation levels.We found that the phosphorylation level of ERK1/2 in Caco-2 cells was significantly increased by L.monocytogenes infection and was LLOdependent.The phosphorylation level of ERK1/2 was positively correlated with the LLO concentration.However,LLO lost the ability to activate ERK1/2 phosphorylation after blocking membrane binding sites with cholesterol.We also found that LLO deletion of the PEST-like sequence did not affect its activation of ERK1/2 phosphorylation,suggesting that LLO activating phosphorylation of ERK1/2 is independent of the active site and is associated with the membrane binding sites.Further studies demonstrated that it did not trigger intracellular ERK1/2 phosphorylation even at high concentrations of LLO(>20 n M)when the key amino acids of LLO membrane-binding sites mutated(LLOT515AL516A).This suggests that LLO entering host cells is a critical step in the activation of ERK1/2 phosphorylation.For this reason,we treated Caco-2 cells with the LLO mutant LLON478AV479 A which controls the entry of cells,and we found that LLO mutant LLON478AV479 A activated ERK1/2 phosphorylation completely depending on the integrity of the cell membrane.Then we used the propidium iodide to track the integrity of the cell membrane consequently.We found that ERK1/2 did not phosphorylate when the concentration of LLON478AV479A(5 n M)was insufficient to disrupt the integrity of the cell membrane.However,ERK1/2 phosphorylation was initiated when the concentration of LLON478AV479A(20 n M)could destroy the integrity of the cell membrane.This suggests that LLO triggers ERK1/2 phosphorylation in the host cell cytoplasm rather than on the cell membrane.In addition,we also found that L.monocytogenes lacking LLO or expressing LLOT515AL516 A lost proliferative capacity in mouse macrophages,but the plaque assay showed that L.monocytogenes expressing LLOT515AL516 A remained cytotoxic and capable of intercellular diffusion.This indicates that the Listeria monocytogenes infected host cells are related to the number of bacteria and the membrane binding ability of LLO.Taken together,this study found that L.monocytogenes-infected host cells are highly dependent on host cell membrane integrity by LLO-induced phosphorylation of ERK1/2.T515 and L516 are the key amino acid sites for LLO to trigger phosphorylation of ERK1/2.The results are important for further clarifying the intrinsic mechanism between Listeria infection and host signaling pathways as well as the pollution prevention and control and public health of gram-positive foodborne intracellular bacteria.