Induced autoprocessing of the cytopathic Makes caterpillars floppy-like effector domain of theVibrio vulnificus MARTX toxin

Abstract

Summary The multifunctional-autoprocessing repeats-in-toxin (MARTXVv) toxin that harbors a varied repertoire of effector domains is the primary virulence factor of Vibrio vulnificus. Although ubiquitously present among Biotype I toxin variants, the Makes Caterpillars Floppy-like effector domain (MCFVv) is previously unstudied. Using transient expression and protein delivery, MCFVv and MCFAh from the Aeromonas hydrophila MARTXAh toxin are shown for the first time to induce cell rounding. Alanine mutagenesis across the C-terminal subdomain of MCFVv identified an RCD tripeptide motif shown to comprise a cysteine protease catalytic site essential for autoprocessing of MCFVv. The autoprocessing could be recapitulated in vitro by addition of host cell lysate to recombinant MCFVv, indicating induced autoprocessing by cellular factors. The RCD motif is also essential for cytopathicity, suggesting autoprocessing is essential first to activate the toxin and then to process a cellular target protein resulting in cell rounding. Sequence homology places MCFVv within the C58 cysteine protease family that includes the type III secretion effectors YopT from Yersinia spp. and AvrPphB from Pseudomonas syringae. However, the catalytic site RCD motif is unique compared to other C58 peptidases and is here proposed to represent a new subgroup of autopeptidase found within a number of putative large bacterial toxins.

The Makes Caterpillars Floppy (MCF)-Like Domain of Vibrio vulnificus Induces Mitochondrion-Mediated Apoptosis

“…Six-His-tagged LF N , LF N -MCF Vv , and LF N -MCF C3351A were purified after overexpression in BL21(DE3) by nickel affinity chromatography as previously described (26). Anthrax toxin protective antigen (PA) was purified as previously described (26).…”

“…Six-His-tagged LF N , LF N -MCF Vv , and LF N -MCF C3351A were purified after overexpression in BL21(DE3) by nickel affinity chromatography as previously described (26). Anthrax toxin protective antigen (PA) was purified as previously described (26). Cellular intoxications were conducted by adding 168 nM PA to DMEM over cells followed by addition 72 nM LF N or LF N fusion protein, and cells were incubated at 37°C with 5% CO 2 for 24 h unless otherwise stated.…”

“…Although this domain derived its name from the Photorhabdus Makes Caterpillar Floppy toxins Mcf1 and Mcf2, a similar cytopathic effect was not observed when the aligned region of the large Mcf1 toxin was transfected into cells (26). Indeed, while Mcf1 has been linked to induction of apoptosis, the BH3 domain essential for induction of apoptosis by the large Mcf1 toxin maps outside the region that aligns with MCF Vv (24,27).…”

“…This 376-aminoacid domain shares homology with internal domains of other large bacterial toxins, including Photorhabdus Makes Caterpillar Floppy toxins Mcf1 and Mcf2 and Pseudomonas fluorescens FitD toxin (24,25). MCF Vv has recently been shown to be an autoproteolytic cysteine protease that is activated by an as-yet-unidentified heat-resistant proteinaceous component from host cell lysate (26). This suggests that one function of this domain in other large toxins like Mcf1 and FitD is as an autoproteolytic domain to autoprocess the large toxins during toxin translocation.…”

“…This suggests that one function of this domain in other large toxins like Mcf1 and FitD is as an autoproteolytic domain to autoprocess the large toxins during toxin translocation. In addition to autoproteolysis, MCF Vv was further shown to induce a cytopathic effect in cells typified by rounding of different cell type, and this cytopathicity depended upon a catalytic site composed of ar-ginine-3350, cysteine-3351, and aspartate-3352 residues arranged in tandem (26).…”