Distinct Roles of the Repeat-Containing Regions and Effector Domains of the Vibrio vulnificus Multifunctional-Autoprocessing Repeats-in-Toxin (MARTX) Toxin

Abstract

Vibrio vulnificus is a seafood-borne pathogen that destroys the intestinal epithelium, leading to rapid bacterial dissemination and death. The most important virulence factor is the multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin comprised of effector domains in the center region flanked by long repeat-containing regions which are well conserved among MARTX toxins and predicted to translocate effector domains. Here, we examined the role of the repeat-containing regions using a modified V. vulnificus MARTX (MARTXVv) toxin generated by replacing all the internal effector domains with β-lactamase (Bla). Bla activity was detected in secretions from the bacterium and also in the cytosol of intoxicated epithelial cells. The modified MARTXVv toxin without effector domains retained its necrotic activity but lost its cell-rounding activity. Further, deletion of the carboxyl-terminal repeat-containing region blocked toxin secretion from the bacterium. Deletion of the amino-terminal repeat-containing region had no effect on secretion but completely abolished translocation and necrosis. Neither secretion nor translocation was affected by enzymatically inactivating the cysteine protease domain of the toxin. These data demonstrate that the amino-terminal and carboxyl-terminal repeat-containing regions of the MARTXVv toxin are necessary and sufficient for the delivery of effector domains and epithelial cell lysis in vitro but that effector domains are required for other cytopathic functions. Furthermore, Ca2+-dependent secretion of the modified MARTXVv toxin suggests that nonclassical RTX-like repeats found in the carboxyl-terminal repeat-containing region are functionally similar to classical RTX repeats found in other RTX proteins.

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

“…Ampicillin resistance of V. vulnificus strains was determined using a disk diffusion assay as previously described (18) using AM10 antibiotic disks (Thermo Scientific). Bla translocation into HeLa cells was determined using CCF2-AM as a reporter for flow cytometry conducted as previously described (18). Lysis of cells exposed to V. vulnificus at a multiplicity of infection (MOI) of 10 was quantified by lactate dehydrogenase (LDH) release as previously described (18) using a CytoTox 96 nonradioactive cytotoxicity assay kit (Promega, Madison, WI) according to the manufacturer's protocol.…”

“…The plasmid was modified further by site-directed mutagenesis using primers 3 and 4 to introduce the codon for C3351A substitution, and the resulting plasmid, pSA9, was transformed to S17pir. The plasmids were then transferred by conjugation from S17-1pir to V. vulnificus strain BS1407, a derivative of CMCP6 which has an unmarked deletion to remove the vvhA hemolysin gene and in which all rtxA1 gene sequences for the effector domains are replaced with a promoterless betalactamase gene (bla) sequence (18). Conjugation was followed by selection for double homologous recombinants using sucrose counterselection as previously described (31).…”

“…The 1,131 nucleotides (nt) of rtxA1 corresponding to MCF Vv (nt 9607 to 10737) was amplified from V. vulnificus CMCP6 genomic DNA using primers 1 and 2. The amplified fragment was digested with SalI, ligated into plasmid pHGJ5 (18), and transformed into Escherichia coli SM10pir cells to generate pSA8. The plasmid was modified further by site-directed mutagenesis using primers 3 and 4 to introduce the codon for C3351A substitution, and the resulting plasmid, pSA9, was transformed to S17pir.…”

“…Only when the bacteria produce the MARTX Vv toxin are there a significant reduction in mitochondrial membrane potential, release of cytochrome c, and processing of caspase 3, despite the presence of intact vvhA and vvpM genes in these bacteria. This ability of V. vulnificus to induce mitochondrial damage during coculture was shown further to depend upon the presence of Ca 2ϩ in the medium (17), which was recently shown to be essential for secretion of the MARTX Vv toxin from V. vulnificus (18), recapitulating the linkage of the MARTX Vv toxin to mitochondrial damage.…”

“…The repeat regions form a pore at the eukaryotic plasma membrane that is proposed to translocate up to five distinct effector domains across the eukaryotic plasma membrane (18,19). These effector domains are then released into the cytosol by induction of the autoprocessing cysteine protease domain (CPD) that is stimulated by the small molecule inositol hexakisphosphate (18,20). It has been shown that the repeat regions are sufficient for pore formation and necrosis but that the effector domains are required for the cytopathic activities of the cell, including cell rounding (18).…”