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. 2007 Oct 23;104(43):17117-22.
doi: 10.1073/pnas.0703196104. Epub 2007 Oct 17.

Arp2/3-independent Assembly of Actin by Vibrio Type III Effector VopL

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Free PMC article

Arp2/3-independent Assembly of Actin by Vibrio Type III Effector VopL

Amy D B Liverman et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Microbial pathogens use a variety of mechanisms to disrupt the actin cytoskeleton during infection. Vibrio parahaemolyticus (V. para) is a Gram-negative bacterium that causes gastroenteritis, and new pandemic strains are emerging throughout the world. Analysis of the V. para genome revealed a type III secretion system effector, VopL, encoding three Wiskott-Aldrich homology 2 domains that are interspersed with three proline-rich motifs. Infection of HeLa cells with V. para induces the formation of long actin fibers in a VopL-dependent manner. Transfection of VopL promotes the assembly of actin stress fibers. In vitro, recombinant VopL potently induces assembly of actin filaments that grow at their barbed ends, independent of eukaryotic factors. Vibrio VopL is predicted to be a bacterial virulence factor that disrupts actin homeostasis during an enteric infection of the host.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
VopL, a WH2- and PRM-containing protein, is secreted in a TTSS2-dependent manner from V. para. (A) Schematic diagram and amino acid sequence of VopL indicating the three PRMs (blue) and the three WH2 domains (red). (B) Secretion of VopL by the V. para strains POR-1, POR-2, and POR-3 detected by analysis of secreted proteins that were TCA-precipitated from culture supernatants and analyzed by immunoblotting with a rabbit anti-VopL antibody. HeLa cells were either mock-infected (C and D) or infected with POR-1 (E and F), POR-2 (G and H), or POR-3 (I and J) at a multiplicity of infection of 25 and analyzed by confocal microscopy using rhodamine–phalloidin to stain actin and Hoechst to stain nuclei and bacteria. (Magnification: C–J, ×100.).
Fig. 2.
Fig. 2.
Transfection of VopL into HeLa cells induces formation of actin stress fibers. (A–H) HeLa cells were transfected with either empty vector (A–C) or pSFFV-VopL-FLAG (D–H). The cells were then analyzed by confocal microscopy using rhodamine–phalloidin to stain for actin (A, D, and G), mouse anti-vincullin with a FITC-conjugated secondary antibody (B, E, G, and H), and anti-VopL with an Alexa Fluor 680-conjugated secondary antibody (F and H). (I–P) HeLa cells were transfected with either empty vector and GFP as a marker of transfection (I–K) or pSFFV-VopL-FLAG (no GFP). The cells were then analyzed by confocal microscopy using rhodamine–phalloidin to stain for actin (I, L, and O), rabbit polyclonal anti-non-muscle myosin IIA (J, M, and P), and mouse anti-FLAG antibody to stain for VopL-FLAG (N and P). (Magnification: ×100.)
Fig. 3.
Fig. 3.
VopL activity in the presence of dominant negative RhoA. Shown are images from confocal microscopy using rhodamine–phalloidin to stain for actin in HeLa cells cotransfected with peGFP-N1 (GFP) and empty vector (A), pSFFV-VopL-Flag (B), pcDNA3-(HA)3-RhoA T19N (dominant negative) (C), and RhoA T19N with VopL-Flag (D). (Magnification: ×100.) (E) Quantitation of transfected cells with increased actin fibers in a double blind study.
Fig. 4.
Fig. 4.
Mutation of the WH2 domains affects VopL activity in HeLa cells. (A) Quantitation of transfected cells with increased actin fibers in a double blind study. (B) An alignment of the WH2 domains (with National Center for Biotechnology Information accession numbers) from human WASP (AF115549), N-WASP (D88460), WAVE1 (D87459), WAVE2 (AB026542), WAVE3 (AB020707), Drosophila melanogaster Spire (AF184975), Chlamydia TARP (YP_328278), V. cholerae VopF (AAZ32252), and V. para VopL (NP_800881). A consensus sequence is shown below the alignment, and the residues in VopL that were mutated to alanines in the WH2 domain mutants are indicated by asterisks below the consensus.
Fig. 5.
Fig. 5.
rVopL enhances actin filament assembly in vitro. (A) A rVopL (0.1–5 nM) or rVopL-WH2×3* mutant was incubated with 4 μM actin (5% pyrene-labeled), and changes in fluorescence were measured over time. (B) Comparison of rVopL (5 nM) and Arp2/3 (5 nM) maximally activated with WASP VCA (500 nM). The proteins were incubated with 4 μM actin (5% pyrene-labeled), and changes in fluorescence were measured over time. “Control” refers to the spontaneous assembly of 4 μM actin (5% pyrene-labeled). (C) VopL-nucleated filaments grow at their barbed ends. Shown are pyrene actin polymerization assays in the presence of actin (0.5 μM 20% pyrene), 5 nM VopL-WH2C (WH2C), and mouse capping protein α1β2 (CP) from 0.2 nM to 10 nM. At 10 nM, CP completely inhibits barbed-end filament growth. (D) VopL has no effect on filament elongation. Shown is elongation of 1 μM phalloidin-stabilized actin filaments with 0.5 μM actin monomer (40% pyrene) in the presence and absence of 3 nM VopL-WH2C (WH2C). (E) VopL binds filament sides. Shown are filament binding assays with GST-VopL-WH2C (GST-WH2C) and 5 μM phalloidin-stabilized actin filaments. In this assay, GST-VopL-WH2C is used so that it can be distinguished from actin by SDS/PAGE. Lane 1, 1 μM GST-VopL-WH2C alone; lanes 2–6, 5 μM actin filaments and 1, 2.5, 5, 7.5, and 10 μM GST-VopL-WH2C, respectively; lane 7, 5 μM actin filaments and 10 μM GST; lane 8, 5 μM actin filaments and 10 μM CP. (F) Coomassie-stained SDS/PAGE gel of rVopL (wt, 1 μg) and rVopL-WH2×3* (mt, 1 μg).
Fig. 6.
Fig. 6.
Bacterial virulence factors that manipulate eukaryotic machinery involved in assembly of actin filaments. The formation of actin filaments can be stimulated by activation of Rho-like GTPases that can directly or indirectly activate nucleation factors, which control the rate-limiting step. Bacterial effectors (red) manipulate the state of GTPases by mimicking GTPase-activating proteins (Salmonella SptP and Yersinia YopE), GTPase exchange factors (Salmonella SopE), or even GTPases themselves (WxxxE). They can sever the activity of G proteins using proteolytic (Yersinia YopT) or kinase activity (Yersinia YpkA). Effectors (Listeria ActA, Rickettsia RickA, and Shigella IscA) can also hijack the Arp2/3 nucleation factor complex. Other effectors (Salmonella SipC and Chlamydia TARP) appear to induce actin assembly, albeit inefficiently, and others induce the formation of actin bundles (Salmonella SipA and SipC). VopL is a bacterial effector that appears to have usurped all of the domains necessary to be an extremely efficient nucleation factor for actin and profilin-bound actin.

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