Research Article: Endothelial Cells Use a Formin-Dependent Phagocytosis-Like Process to Internalize the Bacterium Listeria monocytogenes

Date Published: May 6, 2016

Publisher: Public Library of Science

Author(s): Michelle Rengarajan, Arnold Hayer, Julie A. Theriot, Mary O’Riordan.

http://doi.org/10.1371/journal.ppat.1005603

Abstract

Vascular endothelial cells act as gatekeepers that protect underlying tissue from blood-borne toxins and pathogens. Nevertheless, endothelial cells are able to internalize large fibrin clots and apoptotic debris from the bloodstream, although the precise mechanism of such phagocytosis-like uptake is unknown. We show that cultured primary human endothelial cells (HUVEC) internalize both pathogenic and non-pathogenic Listeria bacteria comparably, in a phagocytosis-like process. In contrast with previously studied host cell types, including intestinal epithelial cells and hepatocytes, we find that endothelial internalization of Listeria is independent of all known pathogenic bacterial surface proteins. Consequently, we exploited the internalization and intracellular replication of L. monocytogenes to identify distinct host cell factors that regulate phagocytosis-like uptake in HUVEC. Using siRNA screening and subsequent genetic and pharmacologic perturbations, we determined that endothelial infectivity was modulated by cytoskeletal proteins that normally modulate global architectural changes, including phosphoinositide-3-kinase, focal adhesions, and the small GTPase Rho. We found that Rho kinase (ROCK) is acutely necessary for adhesion of Listeria to endothelial cells, whereas the actin-nucleating formins FHOD1 and FMNL3 specifically regulate internalization of bacteria as well as inert beads, demonstrating that formins regulate endothelial phagocytosis-like uptake independent of the specific cargo. Finally, we found that neither ROCK nor formins were required for macrophage phagocytosis of L. monocytogenes, suggesting that endothelial cells have distinct requirements for bacterial internalization from those of classical professional phagocytes. Our results identify a novel pathway for L. monocytogenes uptake by human host cells, indicating that this wily pathogen can invade a variety of tissues by using a surprisingly diverse suite of distinct uptake mechanisms that operate differentially in different host cell types.

Partial Text

Vascular endothelial cells prevent free movement of material from the bloodstream into underlying tissues by tight regulation of cellular internalization pathways and robust cell-cell junctions. Nonetheless, in a process termed “angiophagy”, endothelial cells lining small-diameter capillaries in the brain, heart, lung, and kidney have been shown to internalize large fibrin or cholesterol clots that are subsequently released into the underlying parenchyma[1,2]. Furthermore, light and electron microscopy studies have established that liver endothelial cells can internalize apoptotic particles and latex beads in situ[3,4]. While this phenomenon is well documented, its molecular regulators have not been elucidated, making it difficult to establish a bona fide role for angiophagy in vivo. Additionally, it is unclear whether an endothelial phagocytosis-like process could be exploited by pathogens to access underlying tissue.

 

Source:

http://doi.org/10.1371/journal.ppat.1005603

 

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