The multifunctional-autoprocessing repeats-in-toxin (MARTXVv) toxin that harbours 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 hydrophilaMARTXAh toxin are shown for the first time to induce cell rounding. Alanine mutagenesis across the C-terminal subdomain of MCFVv identified an Arg-Cys-Asp (RCD) tripeptide motif shown to comprise a cysteine protease catalytic site essential for autoprocessing of MCFVv. The autoprocessing could be recapitulated in vitro by the 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 with other C58 peptidases and is here proposed to represent a new subgroup of autopeptidase found within a number of putative large bacterial toxins.
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