Edwardsiella piscicida Enters Nonphagocytic Cells via a Macropinocytosis-Involved Hybrid Mechanism

Edwardsiella piscicida is an important pathogen that infects a wide range of hosts from fish to human. Recent studies demonstrated that E. piscicida can invade and survive within multiple non-phagocytic cells, but the internalization mechanism remains poorly understood. Here, we employed HeLa cells as a non-phagocytic cell model to investigate the endocytic strategy used by the pathogenic E. piscicida isolate EIB202. Using a combination of optical and electron microscopy, we observed obvious membrane ruffles and F-actin rearrangements in HeLa cells after EIB202 infection. We also revealed that EIB202 internalization significantly depended on the activity of Na + /H + exchangers and multiple intracellular signaling events related to macropinocytosis, suggesting that E. piscicida utilizes the host macropinocytosis pathway to enter HeLa cells. Further, using inhibitory drugs and shRNAs to block specific endocytic pathways, we found that a caveolin-dependent but not clathrin-dependent pathway is involved in E. piscicida entry, and that its entry requires dynamin and membrane cholesterol. Together, these data suggest that E. piscicida enters non-phagocytic cells via macropinocytosis and caveolin-dependent endocytosis involving cholesterol and dynamin, improving the understanding of how E. piscicida interacts with non-phagocytic cells. IMPORTANCE Bacterial internalization is the first step in breaking through the host cell defense. Therefore, studying the mechanism of bacterial internalization improves the understanding of the pathogenic mechanism of bacteria. In this study, the internalization process on non-phagocytic cells by Edwardsiella piscicida was evaluated. Our results showed that E. piscicida can be internalized into non-phagocytic cells via macropinocytosis and caveolin-mediated endocytosis, and that cholesterol and dynamin are involved in this process. These results reveal a new method for inhibiting Edwardsiella piscicida infection, providing a foundation for further studies of bacterial pathogenicity.