Identification of a domain within the multifunctional Vibrio cholerae RTX toxin that covalently cross-links actin

Abstract

The Gram-negative pathogen Vibrio cholerae causes diarrheal disease through the export of enterotoxins. The V. cholerae RTX toxin was previously identified and characterized by its ability to round human laryngeal epithelial (HEp-2) cells. Further investigation determined that cell rounding is caused by the depolymerization of actin stress fibers, through the unique mechanism of covalent actin cross-linking. In this study, we identify a domain within the full-length RTX toxin that is capable of mediating the cross-linking reaction when transiently expressed within eukaryotic cells. A structure/function analysis of the actin cross-linking domain (ACD) reveals that a 412-aa, or a 47.8-kDa, region is essential for cross-linking activity. When this domain is deleted from the full-length toxin gene, actin cross-linking, but not cell rounding, is eliminated, indicating that this toxin carries multiple dissociable activities. The ACD shares 59% amino acid identity with a hypothetical protein from V. cholerae, VC1416, and transient expression of the C-terminal domain of VC1416 also results in actin cross-linking in eukaryotic cells. The presence of this second ACD linked to an Rhs-like element suggests that V. cholerae acquired the domain by horizontal gene transfer and the ACD was inserted into the RTX toxin by gene duplication through the evolution of V. cholerae.

Fig. 1.

Comparative analysis of the primary amino acid sequences of VcRtxA and VvRtxA reveals that the two toxins are similar. Both toxins possess identical N-terminal novel repeats (NR) and C-terminal GD-rich repeats (GD) depicted by hatch marks. The domains unique to each toxin are depicted as shaded boxes.

Fig. 2.

Construction and expression of ACD-EGFP fusions. (A) Schematic representation of the ACD-EGFP full-length and truncated constructs used in this study. The predicted molecular weights of each fusion protein are stated to the right of the construct. Numbers at bottom correspond to the amino acids of the translated product according to the annotation of Lin et al. (4) (GenBank accession no. AF119150). (B) Expression of the full-length and truncation ACD proteins was detected in transiently transfected COS-7 cells. Twenty-four hours after transfection, cell lysates were subjected to SDS/PAGE and Western blotting with anti-GFP antibody. The last lane has been overexposed to detect the expression of CΔ129-EGFP.

Fig. 3.

Functional analysis of ACD-EGFP fusions. COS-7 cells transiently expressing EGFP (A), ACD-EGFP (B), ACD NΔ30-EGFP (C), ACD CΔ129-EGFP (D), ACD CΔ45-EGFP (E), and VC1416-EGFP (F) were observed by using fluorescence microscopy. Images are an overlay of the phase contrast image with the fluorescence micrograph measured at 550-575 nm of a single field viewed at ×400 magnification. (G) COS-7 cells transiently expressing EGFP (lane 1), ACD-EGFP (lane 2), or truncated forms of ACD fused to EGFP (lanes 3-9) were harvested 24 h after transfection. Collected cells were resuspended in SDS buffer, boiled, and subjected to SDS/PAGE and Western blotting with an anti-actin antibody. Arrows mark monomer (M), dimer (D), trimer (T), and quatramer (Q) forms of actin.

Fig. 4.

HEp-2 cells were incubated with PBS or V. cholerae strains with an intact rtxA gene (KFV119), a null mutation in rtxA (KFV92), or an rtxA gene with an in-frame deletion of the ACD (CCO5). (A) Cells were harvested after 90 min of incubation, and actin cross-linking was measured by Western blotting with an anti-actin antibody. Lines at right mark monomer (M), dimer (D), trimer (T), and quatramer (Q) forms of actin. (B) Phase contrast images were acquired after 3 h of incubation at ×200 magnification.

Fig. 5.

VC1416 contains a second copy of ACD. (A) COS-7 cells transiently expressing EGFP, ACD-EGFP, and VC1416-EGFP were measured for actin cross-linking by Western blotting with an anti-actin antibody. Lines at right mark monomer (M), dimer (D), trimer (T), and quatramer (Q) forms of actin. Unt represents untransfected cells. (B) Expression of EGFP, ACD-EGFP, and VC1416-EGFP in COS-7 cells was detected by Western blotting with an anti-GFP antibody.