Induced autoprocessing of the cytopathic Makes caterpillars floppy-like effector domain of the Vibrio vulnificus MARTX toxin.
The multifunctional-autoprocessing repeats-in-toxin (MARTX(Vv)) 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 (MCF(Vv)) is previously unstudied. Using transient expression and protein delivery, MCF(Vv) and MCF(Ah) from the Aeromonas 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 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.
Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, 60611, USA.
Shivangi
S
Agarwal
Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, 60611, USA.
Shivani
S
Agarwal
Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, 60611, USA.
Marco
M
Biancucci
Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL, 60611, USA.
Karla J F
KJ
Satchell
D016980:Aeromonas hydrophila / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D001329:Autolysis; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D020134:Catalytic Domain; D002460:Cell Line; D048430:Cell Shape / Q000187:drug effects; D016000:Cluster Analysis; D057056:Cysteine Proteases / Q000737:chemistry / Q000235:genetics; D004252:DNA Mutational Analysis; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D017386:Sequence Homology, Amino Acid
D001427:Bacterial Toxins; D057056:Cysteine Proteases