Identification of Vibrio cholerae type III secretion system effector proteins

Abstract

AM-19226 is a pathogenic O39 serogroup Vibrio cholerae strain that lacks the typical virulence factors for colonization (toxin-coregulated pilus [TCP]) and toxin production (cholera toxin [CT]) and instead encodes a type III secretion system (T3SS). The mechanism of pathogenesis is unknown, and few effector proteins have been identified. We therefore undertook a survey of the open reading frames (ORFs) within the ∼49.7-kb T3SS genomic island to identify potential effector proteins. We identified 15 ORFs for their ability to inhibit growth when expressed in yeast and then used a β-lactamase (TEM1) fusion reporter system to demonstrate that 11 proteins were bona fide effectors translocated into HeLa cells in vitro in a T3SS-dependent manner. One effector, which we named VopX (A33_1663), is conserved only in V. cholerae and Vibrio parahaemolyticus T3SS-positive strains and has not been previously studied. A vopX deletion reduces the ability of strain AM-19226 to colonize in vivo, and the bile-induced expression of a vopX-lacZ transcriptional fusion in vitro is regulated by the T3SS-encoded transcriptional regulators VttR(A) and VttR(B). An RLM1 yeast deletion strain rescued the growth inhibition induced by VopX expression, suggesting that VopX interacts with components of the cell wall integrity mitogen-activated protein kinase (MAPK) pathway. The collective results show that the V. cholerae T3SS encodes multiple effector proteins, one of which likely has novel activities that contribute to disease via interference with eukaryotic signaling pathways.

FIG. 1.

(A) Schematic representation of the T3SS island found in strain AM-19226. The region shown is ∼49.7 kb. Genes encoding T3SS apparatus proteins are shown as dark gray arrows, two transcriptional regulators (vttR_A and vttR_B) and a putative transcriptional regulator are shown as black arrows, the gene encoding the VopF effector protein (A33_1696) is checkered, non-T3SS-related genes encoding proteins presumably involved in virulence and the P4 integrase are shown in light gray, and genes selected for and tested in yeast are shown as white arrows. (B) Yeast growth inhibition assay. Yeast strains carrying AM-19226 T3SS ORFs (designated by the A33 locus number) cloned in pBG1805 were grown in SCD-Ura to saturation, serially diluted 10-fold, and then spotted onto SCD-Ura or SCGal-Ura plates. The strain harboring pBG1805 served as the negative control (showing no inhibition of yeast growth), whereas the strain carrying A33_1696, encoding the VopF protein, served as the positive control (showing inhibition of yeast growth). Experiments were repeated three times with similar results.

FIG. 2.

Yeast growth inhibition screen in medium containing caspofungin. Strains carrying either vector alone (pBG1805), pBG1805 expressing a dubious yeast ORF (YAL069W), or pBG1805-based plasmids encoding V. cholerae strain AM-19226 putative effector proteins were grown in noninducing selective synthetic medium containing dextrose. Ten-fold serial dilutions of yeast strains were spotted onto selective medium containing 2% galactose and 30 to 80 ng/ml of caspofungin. The yeast strain carrying the bck1-Δ mutation is sensitive to caspofungin and serves as a control for caspofungin activity. Experiments were repeated three times with similar results.

FIG. 3.

Identification of the bona fide effector proteins. Confocal microscopic images showing the translocation of Bla fusion proteins by the T3SS into HeLa cells. HeLa cells were cocultured with AM-19226 strains expressing different Bla fusion proteins and then observed for fluorescence following excitation at 409 nm. Emission due to FRET can be viewed as green fluorescence at 520 nm, whereas disruption of FRET by Bla fusion protein activity results in emission at 447 nm (blue fluorescence). The white bars represent 50 μm. One representative field is shown for each strain, and each AM-19226 protein was tested for translocation at least twice. (A) Results of experiments conducted using the wild-type T3SS AM-19226 strain (competent for translocation). (B) Representative examples of assays conducted using the T3SS-ΔvcsN strain (translocation deficient).

FIG. 4.

Growth curves and mouse competition assay. (A) OD₆₀₀ recorded for the VopX-positive (solid line; n = 5) and VopX-negative (dashed line; n = 5) strains in a 96-well plate after 1:200 dilutions of overnight cultures in LB. (B) Competition assays in CD-1 infant mice were performed using a lacZ deletion derivative of strain AM-19226 (MD996) and ΔvopX strain AAC477. The results of a single experiment are shown, where each symbol represents the competitive index (CI) from a single animal (n = 8). The bar indicates the mean CI. The experiment was repeated with similar results.

FIG. 5.

Genetic approach in yeast for identification of VopX functional target. (A) The components of the cell wall integrity pathway in yeast. (B) Deletion of the RLM1 gene suppresses yeast growth inhibition by VopX. Strains with the wild-type CWI or a bck1-Δ, slt2-Δ, or rlm1-Δ deletion carrying pBG1805, pBG1805-YAL069W, pBG1805-VopF, or pBG1805-VopX were plated onto SCD-Ura or SCGal-Ura. (C) Complementation of the RLM1 gene in the rlm1-Δ strain. Strains with the wild-type CWI or the rlm1-Δ deletion and carrying pBG1805 and pYCplac111-based plasmids were plated onto SCD-Ura-Leu or SCGal-Ura-Leu (lacking uracil and leucine).

FIG. 6.

vopX-lacZ reporter fusion expression is modulated by ToxR homologs in the presence of bile. β-Galactosidase activity levels were measured in strains carrying a single-copy chromosomal lacZ gene fused to putative vopX promoter sequences. Strains carrying the vcsRTCNS2 reporter fusion and the promoterless reporter were used as control strains. Wild-type background strains expressing the reporter fusion were grown overnight in LB medium without (light gray bars) or with 0.4% bile (checkered bars). Reporter fusion strains with the following deletions were simultaneously assayed in four additional genetic backgrounds after growth in LB plus 0.4% bile overnight: ΔvttR_A (dark gray bars), ΔvttR_B (vertical striped bars), ΔvttR_A ΔvttR_B (narrow hatched bars), and ΔtoxR (wide hatched bars). The experiment was repeated, with similar results, and the data shown represent the results of a single experiment using three individual colonies of each strain. Units, μmol o-nitrophenyl-β-d-galactopyranoside hydrolyzed/min/optical density at 600 nm.