Listeria monocytogenes Exploits Host Caveolin for Cell-to-Cell Spreading

Abstract

Listeria monocytogenes moves from one cell to another using actin-rich membrane protrusions that propel the bacterium toward neighboring cells. Despite cholesterol being required for this transfer process, the precise host internalization mechanism remains elusive. Here, we show that caveolin endocytosis is key to this event as bacterial cell-to-cell transfer is severely impaired when cells are depleted of caveolin-1. Only a subset of additional caveolar components (cavin-2 and EHD2) are present at sites of bacterial transfer, and although clathrin and the clathrin-associated proteins Eps15 and AP2 are absent from the bacterial invaginations, efficient L. monocytogenes spreading requires the clathrin-interacting protein epsin-1. We also directly demonstrated that isolated L. monocytogenes membrane protrusions can trigger the recruitment of caveolar proteins in a neighboring cell. The engulfment of these bacterial and cytoskeletal structures through a caveolin-based mechanism demonstrates that the classical nanometer-scale theoretical size limit for this internalization pathway is exceeded by these bacterial pathogens. IMPORTANCE Listeria monocytogenes moves from one cell to another as it disseminates within tissues. This bacterial transfer process depends on the host actin cytoskeleton as the bacterium forms motile actin-rich membranous protrusions that propel the bacteria into neighboring cells, thus forming corresponding membrane invaginations. Here, we examine these membrane invaginations and demonstrate that caveolin-1–based endocytosis is crucial for efficient bacterial cell-to-cell spreading. We show that only a subset of caveolin-associated proteins (cavin-2 and EHD2) are involved in this process. Despite the absence of clathrin at the invaginations, the classical clathrin-associated protein epsin-1 is also required for efficient bacterial spreading. Using isolated L. monocytogenes protrusions added onto naive host cells, we demonstrate that actin-based propulsion is dispensable for caveolin-1 endocytosis as the presence of the protrusion/invagination interaction alone triggers caveolin-1 recruitment in the recipient cells. Finally, we provide a model of how this caveolin-1–based internalization event can exceed the theoretical size limit for this endocytic pathway.

Distribution of PDLIM1 at actin‐rich structures generated by invasive and adherent bacterial pathogens

“…monocytogenes is an invasive pathogen that uses surface bound (and secreted) effector proteins to exploit an array of host endocytic/exocytoic (Dowd et al, 2020; Gianfelice et al, 2015; Veiga et al, 2007; Veiga & Cossart, 2005), cytoskeletal (Theriot, Rosenblatt, Portnoy, Goldschmidt‐Clermont, & Mitchison, 1994; Chakraborty et al, 1995; Welch, Iwamatsu, & Mitchison, 1997; Welch, Rosenblatt, Skoble, Portnoy, & Mitchison, 1998; Dhanda et al, 2018; see also Lambrechts, Gevaert, Cossart, Vandekerckhove, & Van Troys, 2008 for a review) and cytosolic (Dhanda, Lulic, Vogl, et al, 2019; Faralla et al, 2018; Gouin et al, 2019; Rajabian et al, 2009; Walker, Chua, & Guttman, 2018) proteins over the course of their infectious cycle. A crucial target of these microbes is the host actin cytoskeleton as they rearrange it into five distinct actin‐rich structures: (a) clathrin‐mediated endocytic cups for their initial invasion (Veiga & Cossart, 2005; Veiga et al, 2007; see also Pizarro‐Cerdá, Kühbacher, & Cossart, 2012 for a review), (b) actin clouds, (c) comet/rocket tails to move intracellularly (Tilney & Portnoy, 1989; see also Lambrechts et al, 2008 for a review), (d) membrane protrusions to spread between cells (Tilney & Portnoy, 1989; see also Lamason & Welch, 2017; Weddle & Agaisse, 2018 for reviews), and (e) membrane invaginations that engulf the corresponding membrane protrusions in adjacent cells (Dhanda et al, 2020).…”

“…Here the bacterium engages with the host cell periphery and uses the propulsive force of the actin tail to push against the host cell plasma membrane as it forms a finger‐like membrane protrusion (Tilney & Portnoy, 1989; see also Lamason & Welch, 2017; Weddle & Agaisse, 2018 for reviews). The membrane protrusion pushes into a corresponding membrane invagination in the neighboring cell that is concentrated with caveolin endocytic components (Dhanda et al, 2020; Sanderlin et al, 2019). Continuous cell‐to‐cell spreading ultimately enables the bacteria to colonize organs and breach tissue‐based barriers within the host (Bakardjiev, Theriot, & Portnoy, 2006; Brundage et al, 1993; Faralla et al, 2018; Le Monnier et al, 2007).…”

Host Lipid Rafts as the Gates for Listeria monocytogenes Infection: A Mini-Review

“…L. monocytogenes cell-to-cell spreading is not limited to transfer to phagocytic cells ( 61 ). Dhanda et al further investigated the membrane invagination in neighboring non-phagocytic cells ( Figure 1C , right) ( 66 ). While caveolin-based and lipid raft-dependent endocytosis is supposed to be a process that internalizes extracellular material into bulb-shaped caveolae no larger than 100 nm, L. monocytogenes membrane protrusions triggered the recruitment of caveolar proteins and PS in a neighboring cell ( 66 ).…”

“…Dhanda et al further investigated the membrane invagination in neighboring non-phagocytic cells ( Figure 1C , right) ( 66 ). While caveolin-based and lipid raft-dependent endocytosis is supposed to be a process that internalizes extracellular material into bulb-shaped caveolae no larger than 100 nm, L. monocytogenes membrane protrusions triggered the recruitment of caveolar proteins and PS in a neighboring cell ( 66 ). Knock-down of caveolin-1 reduced invagination length in the neighboring cells and L. monocytogenes cell-to-cell spreading without detectable effect on the length of the actin comet tail and protrusion in initial infected cells ( 66 ).…”

“…While caveolin-based and lipid raft-dependent endocytosis is supposed to be a process that internalizes extracellular material into bulb-shaped caveolae no larger than 100 nm, L. monocytogenes membrane protrusions triggered the recruitment of caveolar proteins and PS in a neighboring cell ( 66 ). Knock-down of caveolin-1 reduced invagination length in the neighboring cells and L. monocytogenes cell-to-cell spreading without detectable effect on the length of the actin comet tail and protrusion in initial infected cells ( 66 ). This suggests that caveolin can mediate engulfment of large materials, such as L. monocytogenes -containing membrane protrusions, which was not supposed to be achieved based on the size of caveolae.…”