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. 2000 Oct 16;19(20):5315-23.
doi: 10.1093/emboj/19.20.5315.

In vivo covalent cross-linking of cellular actin by the Vibrio cholerae RTX toxin

K J Fullner  1 , J J Mekalanos
Affiliations

In vivo covalent cross-linking of cellular actin by the Vibrio cholerae RTX toxin

K J Fullner et al. EMBO J. .

Abstract

Enteric pathogens often export toxins that elicit diarrhea as a part of the etiology of disease, including toxins that affect cytoskeletal structure. Recently, we discovered that the intestinal pathogen Vibrio cholerae elicits rounding of epithelial cells that is dependent upon a gene we designated rtxA. Here we investigate the association of rtxA with the cell-rounding effect. We find that V. cholerae exports a large toxin, RTX (repeats-in-toxin) toxin, to culture supernatant fluids and that this toxin is responsible for cell rounding. Furthermore, we find that cell rounding is not due to necrosis, suggesting that RTX toxin is not a typical member of the RTX family of pore-forming toxins. Rather, RTX toxin causes depolymerization of actin stress fibers and covalent cross-linking of cellular actin into dimers, trimers and higher multimers. This RTX toxin-specific cross-linking occurs in cells previously rounded with cytochalasin D, indicating that G-actin is the toxin target. Although several models explain our observations, our simultaneous detection of actin cross-linking and depolymerization points toward a novel mechanism of action for RTX toxin, distinguishing it from all other known toxins.

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Figures

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Fig. 1. Vibrio cholerae induces rounding of HEp-2 cells dependent upon rtxA. (A) Schematic representation of the CTXΦ/rtxA locus of the large chromosome of V.cholerae. Only genes relevant to this study are designated and the diagram is not drawn to scale. Bars indicate regions deleted from Bah1P and Bah2P. (B) Cell rounding after 75 min incubation of HEp-2 cells with PBS-washed bacteria. An equal volume of PBS was used as the mock infection control.
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Fig. 2. Rounded cells have not undergone necrosis. Lactate dehydrogenase (LDH) (A) and Cr51 (B) release to the medium were measured after 3 h incubation of HEp-2 cells with bacteria. The percentage release was determined by the activity in the sample divided by the activity from cells lysed with Triton X-100. Values shown are the mean of triplicates in a single assay with the standard deviation. (C and D) HEp-2 cells incubated with Bah1P (C) or Bah2P (D) for 2 h were stained with SYTO10 (membrane permeant, green) and DEAD-RED (membrane impermeant, red) to assay membrane integrity. Photographs are representative of the field of cells. A count of >100 cells showed no significant difference in the ratio of red/green cells in Bah1P- and Bah2P-inoculated cells. Mock-infected control cells appeared similar to Bah2P shown in (D).
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Fig. 3. Depolymerization of actin stress fibers depends on rtxA. A total of 105 HEp-2 cells were inoculated with PBS (A and E), 107 Bah1P (B, F and G), 107 Bah2P (C and H) or 2.5 µM cytochalasin D (D, I and J). Inoculated cells were incubated at 37°C in 5% CO2 for either 1 (A–D) or 2 h (E–J), after which cells were stained with fluorescent phalloidin (green). (G and J) Cells counterstained with propidium iodide (red) to facilitate location of cells in (F) and (I), respectively.
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Fig. 4. The RTX toxin does not modify the Rho GTPase similarly to C.difficile toxin A (CdA). (A) A PSI-BLAST search (Altschul et al., 1997) comparing RtxA with the DDBJ/EMBL/GenBank database detected a region of limited sequence similarity to CdA as shown (score = 46.9, E = 0.002). (B) To check for a CdA-like modification of Rho in Bah1P-infected cells, Rho protein in 50 µg of cell extract was ADP-ribosylated with exoenzyme C3 in vitro. Prior modification of Rho by CdA protects cells from further modification by C3 as shown in lane 8.
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Fig. 5. In vivo cross-linking of actin depends on rtxA. Western blot detection of (A) actin or (B) tubulin in cell extracts prepared from HEp-2 cells after co-incubation with PBS (mock), PBS-washed bacterial cultures (as indicated), 2.5 µM cytochalasin D (CytD) or 5 nM C.difficile toxin A (CdA) for the time intervals indicated at the top.
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Fig. 6. The 84 kDa actin species is a dimer of actin. The Coomassie Blue-stained SDS–polyacrylamide gel on the left shows actin species following elution from a DNase I affinity column in G-buffer containing 50% formamide. The sequences of 21 independent peptides derived from trypsin fragments of dimer form are shown on the right alongside the corresponding protein that the peptide matches most closely.
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Fig. 7. Detection of RtxA in concentrated culture supernatant fluids by western blotting. A 2.6 µg aliquot of concentrated culture supernatant fluids from KFV43 (RTX+) and CW128 (RTX–) was loaded in each lane. RtxA was detected using pre-immune rabbit serum (left) or serum raised against RtxA138 (right). Arrows on the right denote the location of RtxA-specific bands. Those marked with an asterisk denote the three major breakdown products.
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Fig. 8. Antibody neutralization of RtxA inhibits actin cross-linking. A 30 µg aliquot of IgG1 from the indicated rabbit sera was added to 4 µg of concentrated supernatant preparations of KFV43 (RTX+) or CW128 (RTX–). Bovine serum albumin (BSA) was substituted for either antibody or supernatant preparation in equal concentration for control samples. The mixtures were incubated on ice for 30 min and then were added to 5 × 105 HEp-2 adherent cells covered with RPMI 1640 medium without additions. Cells were incubated at 37°C in 5% CO2 for 75 min prior to western blot detection of actin.
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Fig. 9. Cytochalasin D does not block actin cross-linking. Cells were incubated with 2.5 µM cytochalasin D for 1 h, at which time cells were visually rounded. A 4 µg aliquot of concentrated supernatant preparations of KFV43 (RTX+) or CW128 (RTX–) was then added. Cells were incubated at 37°C in 5% CO2 for 2 h prior to western blot detection of actin.
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Fig. 10. Proposed models for the action of the RTX toxin. The secreted toxin (R) could enter the cells [1] gaining direct access to the cytoplasmic G-actin [2]. The RTX toxin could thus be the enzyme that then covalently cross-links G-actin, disrupting the equilibrium between F- and G-actin, leading to the depolymerization of stress fibers. Alternatively, RTX could activate an endogenous cross-linking protein (CP), which then carries out the cross-linking reaction [3]. Finally, RTX may function either from within or from outside the cell to activate signal pathways to activate endogenous proteins for both depolymerization and cross-linking [4].
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