The Shigella Virulence Factor IcsA Relieves N-WASP Autoinhibition by Displacing the Verprolin Homology/Cofilin/Acidic (VCA) Domain

Abstract

Shigella flexneri is a bacterial pathogen that invades cells of the gastrointestinal tract, causing severe dysentery. Shigella mediates intracellular motility and spreading via actin comet tail formation. This process is dependent on the surface-exposed, membrane-embedded virulence factor IcsA, which recruits the host actin regulator N-WASP. Although it is clear that Shigella requires N-WASP for this process, the molecular details of this interaction and the mechanism of N-WASP activation remain poorly understood. Here, we show that co-expression of full-length IcsA and the Shigella membrane protease IcsP yields highly pure IcsA passenger domain (residues 53–758). We show that IcsA is monomeric and describe the solution structure of the passenger domain obtained by small-angle X-ray scattering (SAXS) analysis. The SAXS-derived models suggest that IcsA has an elongated shape but, unlike most other autotransporter proteins, possesses a central kink revealing a distinctly curved structure. Pull-down experiments show direct binding of the IcsA passenger domain to both the WASP homology 1 (WH1) domain and the GTPase binding domain (GBD) of N-WASP and no binding to the verprolin homology/cofilin/acidic (VCA) region. Using fluorescence polarization experiments, we demonstrate that IcsA binding to the GBD region displaces the VCA peptide and that this effect is synergistically enhanced upon IcsA binding to the WH1 region. Additionally, domain mapping of the IcsA interaction interface reveals that different regions of IcsA bind to the WH1 and GBD domains of N-WASP. Taken together, our data support a model where IcsA and N-WASP form a tight complex releasing the N-WASP VCA domain to recruit the host cell machinery for actin tail formation.

The Chlamydia trachomatis secreted effector TmeA hijacks the N-WASP-ARP2/3 actin remodeling axis to facilitate cellular invasion

“…Using bioinformatics, we identified a GBD ligand motif in TmeA, a conserved motif found in WASP and N-WASP, which allows for autoinhibition of the NPF [34]. Effector proteins from pathogenic bacteria such as enterohemorrhagic E. coli (EHEC) EspFu and Shigella flexneri IcsA [23,24] similarly possess this motif to exploit N-WASP regulation. Delivery of EspFu into the host cell results in WASP recruitment and pedestal formation to facilitate bacterial attachment to the surface of the host cell and promotes subsequent effector translocation [22,35].…”

“…Using molecular mimicry, the Escherichia coli T3SS effector protein EspFu circumvents the need for Cdc42 by directly perturbing N-WASP autoinhibition to promote actin assembly, which facilitates translocation of additional T3SS effectors into the host cell [20][21][22]. The Shigella flexneri virulence factor IcsA similarly binds to the GBD to relieve N-WASP autoinhibition, allowing recruitment of the actin machinery necessary for actinbased motility and cell to cell spread [23,24]. While pathogenic manipulation of N-WASP has been suggested to be necessary for bacterial invasion by Salmonella enterica serovar Typhimurium [25] and C. trachomatis [26], how intracellular pathogens hijack N-WASP function to promote invasion is ill-defined.…”

“…activates N-WASP allowing for direct recruitment of the ARP2/3 complex [23]. Both EspFu and IcsA bind with high affinity to the GBD of N-WASP, releasing the WCA region to bind to the ARP2/3 complex [20,21,23].…”

GBPs Inhibit Motility of Shigella flexneri but Are Targeted for Degradation by the Bacterial Ubiquitin Ligase IpaH9.8

“…Mutations were generated by PCR and verified by sequencing. For complementation IpaH9.8 and FLAG::IcsA ( Mauricio et al., 2017 ) were amplified by PCR and cloned into pFPV25.1.…”

Regulatory Hierarchies Controlling Virulence Gene Expression in Shigella flexneri and Vibrio cholerae

“…The released S. flexneri bacteria invade nearby epithelial cells through their basolateral surfaces, promoting actin cytoskeleton rearrangements that result in engulfment of the bacteria ( High et al, 1992 ). This produces bacteria-containing vacuoles that are soon ruptured ( Weiner et al, 2016 ) with the released bacteria inducing actin polymerization at one pole of each microbial cell to achieve motility in the host cell cytosol ( Mauricio et al, 2016 ). The bacterium is capable of moving from one cell to the next via clathrin-dependent endocytosis ( Fukumatsu et al, 2012 ), escaping from the double-membrane-enclosed vacuole after each new invasion ( Welch and Way, 2013 ).…”