MARTX effector cross kingdom activation by Golgi-associated ADP-ribosylation factors

Abstract

Summary Vibrio vulnificus infects humans and causes lethal septicemia. The primary virulence factor is a multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin consisting of conserved repeats-containing regions and various effector domains. Recent genomic analyses for the newly emerged V. vulnificus biotype 3 strain revealed that its MARTX toxin has two previously unknown effector domains. Herein, we characterized one of these domain, Domain X (DmXVv). A structure-based homology search revealed DmXVv belongs to the C58B cysteine peptidase subfamily. When ectopically expressed in cells, DmXVv was autoprocessed and induced cytopathicity including Golgi dispersion. When the catalytic cysteine or the region flanking the scissile bond was mutated, both autoprocessing and cytopathicity were significantly reduced indicating DmXVv cytopathicity is activated by amino-terminal autoprocessing. Consistent with this, host cell protein export was affected by Vibrio cells producing a toxin with wild-type, but not catalytically inactive, DmXVv. DmXVv was found to localize to Golgi and to directly interact with Golgi-associated ADP-ribosylation factors ARF1, ARF3, and ARF4, although ARF binding was not necessary for the subcellular localization. Rather, this interaction was found to induce autoprocessing of DmXVv. These data demonstrate that the V. vulnificus hijacks the host ARF proteins to activate the cytopathic DmXVv effector domain of MARTX toxin.

N‐terminal autoprocessing and acetylation of multifunctional‐autoprocessing repeats‐in‐toxins (MARTX) Makes Caterpillars Floppy‐like effector is stimulated by adenosine diphosphate (ADP)‐Ribosylation Factor 1 in advance of Golgi fragmentation

“…However, DmX is only 22% identical to MCF and lacks the signature RCD/Y motif. Further, the aligned His and Asp catalytic residues shared with the C58 peptidases were found to be essential for DmX, whereas they were dispensable in MCF Kim & Satchell, 2016). DmX was likewise found to autoprocess upon stimulation by ARF1, ARF3, and ARF4; however, all three ARFs bound consistently with DmX in cells, unlike MCF which shows a preference for ARF1.…”

“…DmX was likewise found to autoprocess upon stimulation by ARF1, ARF3, and ARF4; however, all three ARFs bound consistently with DmX in cells, unlike MCF which shows a preference for ARF1. Expression of DmX in cells likewise results in dispersion of the Golgi (Kim & Satchell, 2016). However, unlike MCF, DmX does not have an N-terminus blocked to Edman degradation and is not thought to be post-translationally modified in cells.…”

“…Although most V. vulnificus MARTX toxins have MCF, the MARTX toxin for Biotype 3 strains lack this effector (Ziolo et al, 2014). Expression of DmX in cells likewise results in dispersion of the Golgi (Kim & Satchell, 2016). DmX is similar in size to MCF and likewise shows homology to the C58 cysteine peptidases.…”

“…ARF-Myc isoforms were constructed as in Kim and Satchell (2016). CMCP6 ΔvvhA rtxA1::bla and CMCP6 ΔvvhA rtxA1::mcf-bla V. vulnificus strains were generated as detailed in .…”

The Effector Domain Region of the Vibrio vulnificus MARTX Toxin Confers Biphasic Epithelial Barrier Disruption and Is Essential for Systemic Spread from the Intestine

“…Ideally, this experiment would have been conducted using strains with point mutations in the active sites of each MARTX effector domain so as to generate catalytically inactive effector domains in the context of the MARTX holotoxin. However, the catalytic residues of numerous domains have not as yet been identified, and some catalytic point mutants are known to exert intermediate effects when target-binding activity is retained [29, 33, 42, 43]. Therefore, a library of strains was generated in the ΔvvhA background in which each strain harbors an in-frame deletion in the rtxA1 coding region to eliminate a single effector domain from the otherwise functional toxin (Fig 7A).…”