Molecular mechanism of protrusion formation during cell-to-cell spread of Listeria

Abstract

The bacterial pathogen Listeria monocytogenes spreads within human tissues using a motility process dependent on the host actin cytoskeleton. Cell-to-cell spread involves the ability of motile bacteria to remodel the host plasma membrane into protrusions, which are internalized by neighboring cells. Recent results indicate that formation of Listeria protrusions in polarized human cells involves bacterial antagonism of a host signaling pathway comprised of the scaffolding protein Tuba and its effectors N-WASP and Cdc42. These three human proteins form a complex that generates tension at apical cell junctions. Listeria relieves this tension and facilitates protrusion formation by secreting a protein called InlC. InlC interacts with a Src Homology 3 (SH3) domain in Tuba, thereby displacing N-WASP from this domain. Interaction of InlC with Tuba is needed for efficient Listeria spread in cultured human cells and infected animals. Recent structural data has elucidated the mechanistic details of InlC/Tuba interaction, revealing that InlC and N-WASP compete for partly overlapping binding surfaces in the Tuba SH3 domain. InlC binds this domain with higher affinity than N-WASP, explaining how InlC is able to disrupt Tuba/N-WASP complexes.

Keywords: InlC; Listeria monocytogenes; SH3 domain; Tuba; cell-to-cell spread; cortical tension; protrusion; structural elucidation.

Figure 1

Bacterial and host factors controlling protrusion formation in polarized epithelial cells. (A) Steps in the intracellular life cycle of Listeria. (1) internalization of bacteria into host cells, (2) escape from phagosomes, (3) bacterial replication in the cytosol, (4) actin based motility, (5) formation of bacterial protrusions, (6) engulfment of protrusions, and (7) dissolution of the double membranous vacuole. The process of cell–cell spread comprises steps 4–7. This minireview focuses on molecular events controlling protrusion formation (indicated with an asterisk). “TJ” denotes tight junctions. (B) Domain structure of the human scaffolding protein Tuba. SH3 domains (light blue) are numbered 1–6. SH3 domains 1–4 bind the GTPase Dynamin, whereas SH3 domain 6 (SH3-6) interacts with N-WASP. SH3-6 is also targeted by the secreted listerial protein InlC. Ligands of SH3-5 are unknown. The Dbl Homology (DH) domain has guanine nucleotide exchange activity for the human GTPase Cdc42. The Bar domain presumably binds lipids of the plasma membrane inner leaflet. Physiological processes controlled by Tuba ligands are listed. (C) Relief of Tuba-mediated inhibition of Listeria spreading by InlC and other bacterial factors: (i) Without InlC or other bacterial proteins, listerial protrusion formation is restrained by [1] Tuba SH3-6 binding to N-WASP to recruit N-WASP to the plasma membrane and/or activate N-WASP, and [2] Tuba-mediated stimulation of Cdc42 to impair protrusions by activating N-WASP and/or other effectors (?). (ii). InlC relieves Tuba-mediated inhibition of bacterial spread by blocking N-WASP binding to Tuba SH3-6. In addition, an unknown bacterial factor “X” promotes protrusion formation by reducing Cdc42 activity. (D) Model of tight junction perturbation by InlC. In uninfected polarized epithelial cells or cells infected with ΔinlC bacteria (left panel), host Tuba, N-WASP and Cdc42 together promote cortical tension presumably via Tuba-mediated activation of Cdc42 and of N-WASP as well as Cdc42-GTP activation of N-WASP and possibly of additional effectors. In cells infected with wild-type Listeria (right panel), cortical tension is diminished by InlC displacing N-WASP from Tuba SH3-6 and by Cdc42 inhibition via an unidentified bacterial factor “X.” Reduced tension allows efficient protrusion formation by Listeria.

Figure 2

Molecular Displacement of human N-WASP from human Tuba SH3-6 by InlC of Listeria monocytogenes: the critical physiological interaction of N-WASP with Tuba is mediated by the recognition of a proline-rich peptide of N-WASP (blue, residues 346–357) by a peptide binding groove on the sixth SH3 domain (SH3-6, cyan) of Tuba. This interaction is disrupted by InlC (shades of red: pink—N-terminal Cap domain, red—central leucine rich repeat or LRR domain, maroon—C-terminal Ig-like domain) secreted by L. monocytogenes during infection, which binds to a partly overlapping surface of Tuba SH3-6. While the latter interaction is not itself very tight (K_D = 5.4 μM), it is sufficiently tight to outcompete the interaction of N-WASP with Tuba SH3-6 which at K_D = 46.5 μM is roughly an order of magnitude weaker but typical of transient or reversible, physiological interactions.