Reduced virulence of the MARTX toxin increases the persistence of outbreak-associated Vibrio vulnificus in host reservoirs

Abstract

Opportunistic bacteria strategically dampen their virulence to allow them to survive and propagate in hosts. However, the molecular mechanisms underlying virulence control are not clearly understood. Here, we found that the opportunistic pathogen Vibrio vulnificus biotype 3, which caused an outbreak of severe wound and intestinal infections associated with farmed tilapia, secretes significantly less virulent multifunctional autoprocessing repeats-in-toxin (MARTX) toxin, which is the most critical virulence factor in other clinical Vibrio strains. The biotype 3 MARTX toxin contains a cysteine protease domain (CPD) evolutionarily retaining a unique autocleavage site and a distinct β-flap region. CPD autoproteolytic activity is attenuated following its autocleavage because of the β-flap region. This β-flap blocks the active site, disabling further autoproteolytic processing and release of the modularly structured effector domains within the toxin. Expression of this altered CPD consequently results in attenuated release of effectors by the toxin and significantly reduces the virulence of V. vulnificus biotype 3 in cells and in mice. Bioinformatic analysis revealed that this virulence mechanism is shared in all biotype 3 strains. Thus, these data provide new insights into the mechanisms by which opportunistic bacteria persist in an environmental reservoir, prolonging the potential to cause outbreaks.

Keywords: MARTX toxin; Vibrio vulnificus; cysteine protease domain; effector; infectious disease; virulence factor.

Figure 1

The CPD of the V. vulnificus biotype 3 MARTX toxin is autocleaved atypically.A, multiple sequence alignment of CPDs of MARTX toxins from various Vibrio species. Completely conserved residues are highlighted in red, sequences for the β-flap region are indicated in dark yellow, X₁-L-X₂ motifs (autocleavage sites) are displayed in green, and autocleavage residues (VLE) of the V. vulnificus BAA87 CPD are indicated in blue. Within the β-flap region, secondary structure elements of the V. cholerae CPD (PDB ID 3EEB) are indicated. Multiple sequence alignment was carried out using MultiAlin, and the figure was generated with ESPript. The following Vibrio strains are shown: V. vulnificus BAA87 (WP_039507922.1), V. vulnificus MO6-24/O (WP_015728045.1), V. vulnificus FORC_009 (WP_060534095.1), V. cholerae N16961 (AAD21057.1), and V. anguillarum (RMZ64238.1). B, autocleavage site analysis of CPD_BAA87. The N-terminal sequence of autocleaved CPD_BAA87 is EVSGQ, as determined by Edman sequencing. The first amino acid (E) of the autocleaved CPD is highlighted in red. C and D, in vitro autocleavage assay. CPD_BAA87, its mutants CPD_AWT/ALA and CPD_VLE/ALA, and CPD_MO6-24/O were incubated with equivalent molar concentrations of InsP₆, resolved by SDS-PAGE, and visualized by Coomassie staining. Autocleaved CPDs are indicated by arrows. The intensity of the cleaved protein on SDS-PAGE was quantified using NIH ImageJ and plotted. The plots are from three independent experiments. CPD, cysteine protease domain; MARTX, multifunctional autoprocessing repeats-in-toxin.

Figure 2

The structure of CPD_BAA87suggests nonfunctionality for processing of effector domains.A, crystal structure of CPD_BAA87, consisting of the core catalytic domain and β-flap. The helices, β-strands, and loop regions are shown in red, yellow, and green, respectively. B, superimposition of the catalytic domain of CPD_BAA87 onto the corresponding domain of CPD_cholerae (white). Residues corresponding to different secondary structures of CPD_BAA87 are shown in color, and residues corresponding to CPD_cholerae are shown in black. The catalytic residues of CPD_BAA87 (H4183 and C4232) and CPD_cholerae (H3519 and C3568) are indicated. C, superimposition of the β-flap of CPD_BAA87 onto the corresponding region of CPD_cholerae (white). Active residues are indicated for each CPD. D, the electrostatic surface potential of CPD_cholerae shows a positively charged pocket for InsP₆ binding (upper left panel). The residues involved in formation of the pocket are indicated. Residues involved in InsP₆ binding are shown as a stick model within CPD_cholerae displayed as a ribbon model (upper right panel). The β-flap_cholerae residues critically involved in formation of the InsP₆-binding pocket are indicated in red (upper panels). The electrostatic surface potential of CPD_BAA87 reveals a highly basic pocket formed by the corresponding residues of CPD_cholerae (lower left panel). The corresponding residues involved in the InsP₆-binding pocket of CPD_BAA87 are displayed, including the residues corresponding to the critical β-flap_cholerae residues for InsP₆ binding, which are shown in red (lower right panel). E, crater-like hydrophobic surface formed by structural movement of the β-hairpin in the β-flap region via interacting forces. Hydrogen bonds between residues are shown as dashed lines. CPD, cysteine protease domain; MARTX, multifunctional autoprocessing repeats-in-toxin.

Figure 3

Autocleaved CPD_BAA87becomes nonfunctional for processing of MARTX toxin effectors.A, the steric configuration of the β-flap (ribbon model in red) blocking the active site of the autocleaved CPD_BAA87. Catalytic residues are shown in red (Cys4232) and blue (His4183) on a surface model of the catalytic domain (in white). The N-terminal flexible region (Glu4068–Ala4090) is shown as a red dashed line, and Trp4091 is shown with a red stick model. B, the steric configuration of the β-flap, showing autocleaved CPD_cholerae, which is functional for processing of MARTX toxin effectors. Catalytic residues are shown in red (Cys3568) and blue (His3519) on the surface model of the catalytic domain (in white). The two structures are displayed in the same orientation. CPD, cysteine protease domain; MARTX, multifunctional autoprocessing repeats-in-toxin.

Figure 4

Evolutionary insights into the disabled effector domain processing of CPD_BAA87.A and B, in vitro autocleavage assay for CPD proteins. CPDs were incubated with InsP₆ for the indicated durations. The intensities of the bands representing the cleaved protein on SDS-PAGE were quantified with NIH ImageJ and plotted. The plots are from three independent experiments. Autocleaved CPDs are indicated by arrows. C, in vitro MARTX effector domain processing assay. The purified effector region of MARTX_BAA87 toxin was co-incubated with the indicated CPDs in the presence of 1 mM InsP₆ for 1 h at 37 °C. Effector products processed by each CPD were analyzed by SDS-PAGE (left panel). The bar graph shows the proportion of the uncleaved effector region relative to the effector region in the absence of CPD (right panel). The intensity of each band representing the uncleaved effector region was quantified with NIH ImageJ, and the mean of individual values were statistically analyzed by a one-way ANOVA with multiple comparisons (∗∗p < 0.005; ∗∗∗∗p < 0.0001; n.s., not significant). CPD, cysteine protease domain; MARTX, multifunctional autoprocessing repeats-in-toxin.

Figure 5

The reduced virulence of the MARTX_BAA87toxin is mediated by CPD_BAA87.A, schematic representation of the V. vulnificus strains used for cell rounding assays and mouse survival experiments. Parental, V. vulnificus MO6-24/O strain; MO6-24/O/MARTX_BAA87, V. vulnificus MO6-24/O expressing MARTX_BAA87 (i.e., in which the MCF and CPD of MARTX_MO6-24/O are substituted with the ExoY, DmX, and CPD of MARTX_BAA87); MO6-24/O/MARTX_BAA87/CPD/β-flap_MO6-24/O, an engineered strain in which the β-flap of CPD_BAA87 is substituted with that of MO6-24/O; MO6-24/O/MARTX_BAA87/CPD_{MO6-24/O-mimic}, an engineered strain in which CPD_BAA87 in MO6-24/O/MARTX_BAA87 is substituted with CPD_MO6-24/O. Mutated residues and the substituted regions in CPD_{MO6-24/O-mimic} are indicated. B, representative images of HeLa cells treated with PBS as a control or the indicated V. vulnificus strains at an multiplicity of infection of 0.1 for 120 min. C, quantification of rounded HeLa cells from images obtained after 60, 90, and 120 min of the treatments described in B. The proportion of rounded HeLa cells relative to the total number of HeLa cells was calculated. Data are shown as the mean ± standard deviation (SD) from three independent images (∗∗∗∗p < 0.0001). D, survival of mice challenged by subcutaneous injection of the indicated V. vulnificus strains (n = 10; pooled data from two independent experiments with five mice per group). Data from the MO6-24/O/MARTX_BAA87 strain and the MO6-24/O/MARTX_BAA87/CPD_{MO6-24/O-mimic} strain were compared by the log-rank test. CPD, cysteine protease domain; MARTX, multifunctional autoprocessing repeats-in-toxin.

Figure 6

The functionality of CPDs correlates with the evolutionary classification of V. vulnificus biotypes.A, proposed model for MARTX_BAA87 processing. Upon entry into host cells, the CPD of MARTX_BAA87 is allosterically activated by binding to InsP₆ and autocleaved (left panel). DmX released from the autocleaved CPD is then allosterically activated via the interaction with ARF and autocleaved (middle panel). The released DmX complexed with ARF disrupts the Golgi structure, and DmX may induce cell shrinking via modification of an unknown target(s) (16, 28). B, phylogenetic tree of CPDs from different V. vulnificus strains. Amino acid sequence analysis revealed that CPDs can be classified into three groups based on their sequences and predicted functional discrepancies, shown as arcs outside of the tree. The biotypes of V. vulnificus strains are indicated with different symbols: biotype 1, red circle; biotype 2, blue circle; biotype 3, green circle; unidentified, magenta diamond. ARF, ADP-ribosylation factor; CPD, cysteine protease domain; DmX, domain X effector; DUF1, domain of unknown function; ExoY, ExoY-like adenylate cyclase domain; MARTX, multifunctional autoprocessing repeats-in-toxin; RID, Rho GTPase-inactivation domain.