A tripartite cytolytic toxin formed by Vibrio cholerae proteins with flagellum-facilitated secretion

Significance Vibrio cholerae, responsible for outbreaks of cholera disease, is a highly motile organism by virtue of a single flagellum. We describe how the flagellum facilitates the secretion of three V. cholerae proteins encoded by a hitherto-unrecognized genomic island. The proteins MakA/B/E can form a tripartite toxin that lyses erythrocytes and is cytotoxic to cultured human cells. A structural basis for the cytolytic activity of the Mak proteins was obtained by X-ray crystallography. Flagellum-facilitated secretion ensuring spatially coordinated delivery of Mak proteins revealed a role for the V. cholerae flagellum considered of particular significance for the bacterial environmental persistence. Our findings will pave the way for the development of diagnostics and therapeutic strategies against pathogenic Vibrionaceae.

d-galactopyranoside (IPTG) followed by growth for 5 h at 25 °C. Cells were harvested by centrifugation, and the pellets were stored at -80 °C until further use. The cell pellets were resuspended in 50 mM Tris-HCl pH 7.6, 0.3 M NaCl, and 10 mM imidazole (lysis buffer) supplemented with 1% triton-X100 and sonicated on ice. The lysate was centrifuged at 63,000 x g for 20 min. The resulting supernatant was passed over a column packed with His60 Niresin (Takara). The column was washed with lysis buffer containing 30 mM imidazole after which the protein was eluted with the same buffer containing 0.3 M imidazole. Next, the histidine tag was removed by incubation with 1% (w/w) TEV protease overnight at 4 °C. After All crystals were soaked for 30 seconds in mother liquor solution supplemented with 20% (v/v) ethylene glycol or 20% (v/v) PEG 400 before they were flash cooled in liquid nitrogen and stored until data collection.
MakB native diffraction data were collected on a Pilatus3 2M detector at beamline ID23-2 and single anomalous diffraction (SAD) data at beamline ID23-1 on a Pilatus 6M F detector at the European Synchrotron Radiation Facility, Grenoble, France (ESRF). Native MakE data were collected at the ESRF beamline ID23-2 on a Pilatus3 2M detector. SAD data were collected at beamline BioMAX, MAX IV in Lund, Sweden. Diffraction images were processed with XDS (5) and scaled with Aimless (6) from the CCP4 program suite (7). The structure of SeMet-labelled MakB was solved with SAD-phasing using AutoRickshaw (8). Density modification and automatic model building were performed using AutoRickshaw and ArpWarp (9) and refined using phenix.refine (10). The SAD MakE data were carefully reprocessed using XDS (5), after which the scaled data were used as input in the CRANK2 pipeline (11). A search for ten Se atoms resulted in an initial model that was further built in COOT (12).
The crystals of the native MakE protein grew in space groups different from the SeMet-labelled proteins, and hence the structure was obtained by molecular replacement using Molrep (13,14) with the SeMet structure as search model. The native structures were refined using phenix.refine (10) and built using rounds of manual building in COOT (12). For the refinement of MakE, translational-libration-screw refinement was used, treating each molecule as an individual TLS group (15).

SDS-PAGE and Immunoblot analysis.
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis were performed according to standard procedures (19). Briefly, bacterial whole cells and culture supernatants were separated by centrifugation at 10,000 × g for 15 min. Cell pellets were suspended in an appropriate volume of 1× SDS buffer and boiled to obtain whole cell lysates. Culture supernatants were filtered through a 0.45 μm PVDF syringe filter (Millipore, USA) and subsequently precipitated with 10% (w/v) trichloroacetic acid (TCA). The samples were centrifuged at 15,000 × g for 15 min at 4 °C to pellet the TCA-precipitated proteins, which were then resuspended in 1× SDS buffer and boiled for 5 min. Protein samples were resolved by SDS-PAGE and processed for immunoblotting. HRP-conjugated goat anti-rabbit IgG (Agrisera AB, Sweden) was used as a secondary antibody. Immunoblot detection was done using Clarity Western ECL substrate (BioRad). Prestained protein molecular weight standards (SM0679, Fermentas) were used to determine the protein molecular weights.

Hemolysis assay
For tests of hemolytic activity, Mak proteins (250 nM) were mixed with a suspension of human erythrocytes (2% of whole blood) in Phosphate-Buffered Saline (PBS) and incubated 120 min at 37 °C. The cytolytic E. coli protein ClyA (250 nM) and Triton X-100 were used as positive controls and PBS as the negative control. After centrifugation, the supernatants were monitored for released haemoglobin, by measurement of absorbance at 545 nm, as an indicator of red blood cell lysis. ClyA was obtained form from E. coli through the procedures for overproduction and purification described earlier (20,21).

Cell lines and cell culture
Human colon cancer cells (Caco-2; RRID:CVCL_0025 and HCT8; RRID:CVCL_2478) were obtained from American Type Culture Collection (ATCC) and maintained in RPMI-1640 or DMEM media supplemented with non-essential amino acids (1:100), sodium pyruvate (1 mM), penicillin (20 Units), streptomycin (20 µg/mL) and 10% fetal bovine serum (FBS) at 37 °C, 5% CO2. All experiments were performed with mycoplasma-free cells.  Identical residues are highlighted in red. Secondary structure elements, based on the MakA structure, are indicated above the sequences. The residues in MakA and MakE predicted to be transmembrane helices are indicated with green bars below the sequence.  Strains represented more than once in the table have undergone repeated sequencing of their genomes and were reported independently. Identification of the mak gene cluster was therefore indicated for each report using the corresponding accession number.

2.
Abbreviations: Chr. 1 = chromosome 1; Chr. 2 = chromosome 2; Chr. = genome composed of only one chromosome. 3. → indicates that the gene cluster is oriented in the clockwise direction relative to the replication origin and ← indicates that the gene cluster is oriented in the counterclockwise direction.