Vibrio cholerae strains with mutations in an atypical type I secretion system accumulate RTX toxin intracellularly

Abstract

This study shows that the Vibrio cholerae RTX toxin is secreted by a four-component type I secretion system (TISS) encoded by rtxB, rtxD, rtxE, and tolC. ATP-binding site mutations in both RtxB and RtxE blocked secretion, demonstrating that this atypical TISS requires two transport ATPases that may function as a heterodimer.

FIG. 1.

(A) The gene clusters of V. cholerae rtx and E. coli hly vary significantly. (B) rtxB and rtxD overlap by 46 nt in different reading frames, as shown by amino acid sequences of RtxB and RtxD. (C) Genetic organization of rtxB::Km, ΔrtxB, ΔrtxD, and ΔrtxE strains. In order to accommodate the 46-nt overlap of rtxB and rtxD, the ATG of the rtxD gene was moved to the position of the ATG of rtxB downstream of the promoter for the creation of strain BBV21. For ΔrtxD, the stop codon of rtxD was moved to the position of the stop codon of rtxB to create strain BBV19. For the creation of the ΔrtxE strain, the stop codon of rtxE was moved to the position of the stop codon of rtxD to create strain CW149. (D) Locations of point mutations introduced to create strains RtxBK496A, RtxDΔLRER, and RtxEK522A. The Shine-Dalgarno sequence of rtxE is underlined. The figure was generated in MacVector 7.0. (Oxford Biosystems) with sequences from E. coli (accession numbers M10133 and M12863 [10, 11]) and V. cholerae (accession number NC_002505 [16]). CHP, conserved hypothetical protein; *, stop codon.

FIG. 2.

RTX toxin is not secreted by TISS mutant BBV16. (A) Thirty-seven micrograms of concentrated supernatant fluids or (B) 119 μg of cell lysate from strains KFV43 (wild type [wt]), KFV80 (ΔrtxACBD), and BBV16 (rtxB::Km) was separated on a sodium dodecyl sulfate-6% polyacrylamide gel, and RTX toxin was detected by Western blotting with polyclonal anti-RtxA antibody. Asterisks mark breakdown products of the RTX toxin in concentrated supernatant fluids and possible full-length toxin in cell lysates. Numbers indicate molecular masses in kilodaltons.

FIG. 3.

Active RTX toxin is detected in cell lysates of a polar TISS mutant, BBV16. HEp-2 cells were treated with equal total protein concentrations of (A and C) concentrated supernatant fluids or (B and D) cell lysate from strains KFV43 (wild type [wt]), KFV80 (ΔrtxACBD), and BBV16 (rtxB::Km) as described in the text. Cells were (A and B) photographed for rounding or (C and D) collected for detection of actin cross-linking by Western blot analysis; 20 mM Tris-1 mM EDTA (pH 8.0) with 10% glycerol was used as a mock treatment. Monomer (M), dimer (D), trimer (T), and quatramer (Q) forms of actin are indicated at the right of the gels. Recovery of actin cross-linking activity in cell lysates of KFV43 varied between experiments. Numbers indicate molecular masses in kilodaltons.

FIG. 4.

TISS point mutants accumulate toxin intracellularly. HEp-2 cells were treated with (A) supernatant fluids or (B) clarified lysates prepared from strains KFV43 (wild type [wt]), CW128 (ΔrtxA), KFV80 (ΔrtxACBD), BBV3 (RtxBK496A), BBV2 (RtxEK522A), and BBV1(RtxDΔLRER). Luria broth and 20 mM Tris-1 mM EDTA (pH 8.0) were used as mock controls for supernatant- and cell lysate-treated cells, respectively. Recovery of actin cross-linking activity in cell lysates of KFV43 varied between experiments. Numbers indicate molecular masses in kilodaltons.

FIG. 5.

Model of RTX toxin TISS.

FIG. 6.

rtx gene clusters in five species share a similar genomic structure that includes the atypical TISS. The figure was generated in MacVector 7.0 (Oxford Biosystems) with sequences from V. cholerae (accession number NC_002505 [16]), Vibrio vulnificus (accession number NC_005140 [6]), Photorhabdus luminescens (accession number NC_005126 [9]), and Environmental Sargasso Sea sequence assembly CH006444 (29). The Xenorhabdus boviennii rtx cluster was downloaded from the publicly available database at www.xenorhabdus.org provided by B. Goldman of The Monsanto Company and collaborators. Frameshift (FS), insertion sequence (IS), conserved hypothetical protein (CHP).