Role of Flotillin-1 In <i>Listeria monocytogenes</i> Cell-to-Cell Spreading

Listeria monocytogenes spreads intercellularly by creating actin-rich projections that are endocytosed into recipient cells. Caveolin-mediated endocytosis has been implicated in this process, accounting for about 70% in cell-to-cell spread in cells depleted of caveolin-1. Thus, additional mechanisms may contribute to the remaining spread, and we examined the role of flotillin-based endocytosis. We found that flotillin-1 localized to L. monocytogenes invaginations in recipient cells and depletion of flotillin-1 significantly impaired bacterial transfer. Similarly, preventing endogenous flotillin-1 from membrane association significantly reduced bacterial spread. To evaluate whether there was interplay between flotillin-1- and caveolin-1-mediated endocytosis at L. monocytogenes invagination sites, we measured the area of spread in cells with both caveolin-1 and flotillin-1 knockdown and found a further significant decrease in spread and many cells with complete blockage. This work demonstrates that flotillin-based endocytosis is crucial for cell-to-cell spreading of L. monocytogenes and that this endocytic strategy can internalize large membrane protrusions.