Abstract
The facultative intracellular pathogen Listeria monocytogenes uses an actin-based motility process to spread within human tissues. Filamentous actin from the human cell forms a tail behind bacteria, propelling microbes through the cytoplasm. Motile bacteria remodel the host plasma membrane into protrusions that are internalized by neighboring cells. A critical unresolved question is whether generation of protrusions by Listeria involves stimulation of host processes apart from actin polymerization. Here we demonstrate that efficient protrusion formation in polarized epithelial cells involves bacterial subversion of host exocytosis. Confocal microscopy imaging indicated that exocytosis is up-regulated in protrusions of Listeria in a manner that depends on the host exocyst complex. Depletion of components of the exocyst complex by RNA interference inhibited the formation of Listeria protrusions and subsequent cell-to-cell spread of bacteria. Additional genetic studies indicated important roles for the exocyst regulators Rab8 and Rab11 in bacterial protrusion formation and spread. The secreted Listeria virulence factor InlC associated with the exocyst component Exo70 and mediated the recruitment of Exo70 to bacterial protrusions. Depletion of exocyst proteins reduced the length of Listeria protrusions, suggesting that the exocyst complex promotes protrusion elongation. Collectively, these results demonstrate that Listeria exploits host exocytosis to stimulate intercellular spread of bacteria.
Highlights
Several intracellular bacterial pathogens, including Listeria monocytogenes, Shigella flexneri, and Rickettsia spp. use an actin-based motility process to spread from infected human cells to neighboring healthy cells [1, 2]
Using an exocytic probe derived from the recycling endosome (RE)-localized v-SNARE VAMP3, we demonstrate that exocytosis is stimulated in protrusions of Listeria
In order to detect exocytosis in Caco-2 BBE1 cells, we constructed an exocytic probe consisting of the v-SNARE protein VAMP3 fused to pHluorin, a pH-sensitive form of GFP [27, 28]
Summary
Several intracellular bacterial pathogens, including Listeria monocytogenes, Shigella flexneri, and Rickettsia spp. use an actin-based motility process to spread from infected human cells to neighboring healthy cells [1, 2]. Cytoplasmic microbes stimulate the polymerization of host actin filaments on one bacterial pole, resulting in the formation of actin “comet” tails These tails propel bacteria through the cytosol and allow contact with the host plasma membrane. In the case of Listeria, the bacterial surface protein ActA induces the formation of actin filaments by activating the host Arp2/3 complex. Apart from this complex, cofilin and capping proteins play critical roles in actin-based movement by increasing treadmilling of uncapped filaments [3]. Studies indicated that force generated by actin-based motility contributes to the generation of bacterial protrusions [4] It remains unclear whether host processes apart from actin polymerization are exploited by Listeria or other bacteria in order to remodel the plasma membrane to generate protrusions. Further genetic experiments demonstrate important roles for the exocyst complex, Rab, Rab, and VAMP3 in Significance
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