The bacterial virulence factor InlC perturbs apical cell junctions and promotes cell-to-cell spread of Listeria

Abstract

Introductory Paragraph Several pathogenic bacteria, including Listeria monocytogenes, use an F-actin motility process to spread between mammalian cells1. Actin ‘comet tails’ propel Listeria through the cytoplasm, resulting in bacteria-containing membrane protrusions that are internalized by neighboring cells. The mechanism by which Listeria overcomes cortical tension to generate protrusions is unknown. Here, we identify bacterial and host proteins that directly regulate protrusions. We show that efficient spreading between polarized epithelial cells requires the secreted Listeria virulence protein InlC. We next identify the mammalian adaptor protein Tuba as a ligand of InlC. InlC binds to a C-terminal SH3 domain in Tuba, which normally engages the human actin regulatory protein N-WASP2. InlC promotes protrusion formation by inhibiting Tuba and N-WASP, most likely by impairing binding of N-WASP to the Tuba SH3 domain. Tuba and N-WASP are known to control the structure of apical junctions in epithelial cells3. We demonstrate that, by inhibiting Tuba and N-WASP, InlC makes taut apical junctions become slack. Experiments with myosin II inhibitors indicate that InlC-mediated perturbation of junctions accounts for the role of this bacterial protein in protrusion formation. Collectively, our results suggest that InlC promotes bacterial dissemination by relieving cortical tension, thereby enhancing the ability of motile bacteria to deform the plasma membrane into protrusions.

Impact of the Listeria monocytogenes Protein InlC on Infection in Mice

“…Such tissues include enterocytes lining the intestinal lumen, hepatocytes, and the brain endothelium (2). In polarized enterocytes, the secreted bacterial protein InlC acts after ActA-mediated comet tail formation to enhance the production of L. monocytogenes protrusions (6). InlC stimulates protrusion formation by antagonizing the function of a human signaling protein called Tuba.…”

“…Tuba is a large scaffolding protein that contains several functional domains, including a carboxyl-terminal Src homology 3 (SH3) domain that interacts with the human actin regulatory protein N-WASP (7). Genetic and biochemical data indicate that InlC inhibits host Tuba by binding to this SH3 domain, thereby disrupting Tuba/N-WASP complexes (6). How InlC-mediated inhibition of host Tuba and N-WASP leads to enhanced protrusion formation is not fully understood.…”

“…How InlC-mediated inhibition of host Tuba and N-WASP leads to enhanced protrusion formation is not fully understood. Based on the effects of L. monocytogenes infection on the morphology of apical junctions in enterocytes, it was hypothesized that InlC might dissipate cortical tension, thereby reducing the force required to deform the plasma membrane into protrusions (6).…”

“…Binding of eukaryotic SH3 domain ligands is mediated by short peptide sequences of the consensus PxxP (where x is any amino acid) or, less commonly, other sequences, such as PxxDY or RxxK (9). Interestingly, interaction of InlC with the Tuba SH3 domain involves an RxxK sequence in InlC (6). Replacement of the lysine residue in this sequence with an alanine results in an InlC protein (InlC.K173A) that folds normally but is partly defective in binding to the Tuba SH3 domain (6).…”

“…Interestingly, interaction of InlC with the Tuba SH3 domain involves an RxxK sequence in InlC (6). Replacement of the lysine residue in this sequence with an alanine results in an InlC protein (InlC.K173A) that folds normally but is partly defective in binding to the Tuba SH3 domain (6). The InlC.K173A protein binds to the SH3 domain of human Tuba at about 1/6 the affinity of wild-type InlC.…”