Induced autoprocessing of the cytopathic Makes caterpillars floppy‐like effector domain of the V ibrio vulnificus MARTX toxin

Summary The multifunctional‐autoprocessing repeats‐in‐toxin ( MARTX Vv ) toxin that harbours a varied repertoire of effector domains is the primary virulence factor of V ibrio vulnificus . Although ubiquitously present among B iotype I toxin variants, the ‘Makes caterpillars floppy‐like’ effector domain ( MCF Vv ) is previously unstudied. Using transient expression and protein delivery, MCF Vv and MCF Ah from the A eromonas hydrophila   MARTX Ah toxin are shown for the first time to induce cell rounding. Alanine mutagenesis across the C ‐terminal subdomain of MCF Vv identified an Arg‐Cys‐Asp (RCD) tripeptide motif shown to comprise a cysteine protease catalytic site essential for autoprocessing of MCF Vv . The autoprocessing could be recapitulated in vitro by the addition of host cell lysate to recombinant MCF Vv , 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 MCF Vv within the C 58 cysteine protease family that includes the type III secretion effectors YopT from Y ersinia spp. and A vr P ph B from P seudomonas syringae . However, the catalytic site RCD motif is unique compared with other C 58 peptidases and is here proposed to represent a new subgroup of autopeptidase found within a number of putative large bacterial toxins.