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

Abstract

Vibrio vulnificus causes highly lethal bacterial infections in which the Multifunctional Autoprocessing Repeats-in-Toxins (MARTX) toxin product of the rtxA1 gene is a key virulence factor. MARTX toxins are secreted proteins up to 5208 amino acids in size. Conserved MARTX N- and C-terminal repeat regions work in concert to form pores in eukaryotic cell membranes, through which the toxin’s central region of modular effector domains is translocated. Upon inositol hexakisphosphate-induced activation of the of the MARTX cysteine protease domain (CPD) in the eukaryotic cytosol, effector domains are released from the holotoxin by autoproteolytic activity. We previously reported that the native MARTX toxin effector domain repertoire is dispensable for epithelial cellular necrosis in vitro, but essential for cell rounding and apoptosis prior to necrotic cell death. Here we use an intragastric mouse model to demonstrate that the effector domain region is required for bacterial virulence during intragastric infection. The MARTX effector domain region is essential for bacterial dissemination from the intestine, but dissemination occurs in the absence of overt intestinal tissue pathology. We employ an in vitro model of V. vulnificus interaction with polarized colonic epithelial cells to show that the MARTX effector domain region induces rapid intestinal barrier dysfunction and increased paracellular permeability prior to onset of cell lysis. Together, these results negate the inherent assumption that observations of necrosis in vitro directly predict bacterial virulence, and indicate a paradigm shift in our conceptual understanding of MARTX toxin function during intestinal infection. Results implicate the MARTX effector domain region in mediating early bacterial dissemination from the intestine to distal organs–a key step in V. vulnificus foodborne pathogenesis–even before onset of overt intestinal pathology.

The Vibrio cholerae MARTX toxin simultaneously induces actin collapse while silencing the inflammatory response to cytoskeletal damage

“…The effector domains, and not the MARTXVc pore, were identified to modulate innate immune signaling in IECs. These data support a previously established model in which the MARTX pore functions primarily as an effector delivery platform and not a direct virulence mechanism (43). How each delivered effector contributes to pathogenesis is still debated.…”

Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis

“…2, B-F). These results led us to conclude that VvPlpA, which is secreted in a T2SS-dependent manner, is an essential virulence factor contributing to V. vulnificus pathogenesis along with other toxins, such as cytolysin/hemolysin and MARTX toxin (5,40,41). Indeed, the expression of neither the cytolysin/hemolysin gene, vvhA, nor the MARTX toxin gene, rtxA1, was affected by plpA mutation in V. vulnificus (data not shown).…”

Vibrio vulnificus MARTX cytotoxin causes inactivation of phagocytosis-related signaling molecules in macrophages

“…The Rho inactivation domain (RID) implicated in causing host cell rounding [ 19 , 20 ] is present in most of the MARTXs of biotype 1 V. vulnificus [ 21 , 22 ]. Other domains, like the actin-crosslinking domain (ACD), Pseudomonas aeruginosa ExoY-like adenylate cyclase (ExoY), cysteine protease domain (CPD), alpha-beta hydrolase (ABH), the Makes caterpillars floppy-like (MCF) and Ras/Rap1-specific endopeptidases (RRSPs) domains are found in the MARTXs of V. cholerae or V. vulnificus [ 21 , 22 ].…”

“…Other domains, like the actin-crosslinking domain (ACD), Pseudomonas aeruginosa ExoY-like adenylate cyclase (ExoY), cysteine protease domain (CPD), alpha-beta hydrolase (ABH), the Makes caterpillars floppy-like (MCF) and Ras/Rap1-specific endopeptidases (RRSPs) domains are found in the MARTXs of V. cholerae or V. vulnificus [ 21 , 22 ]. These effector domains have been demonstrated to exert various effects in the host cells [ 19 , 20 , 23 – 28 ]. The DUF1 (domain of unknown function 1) domain has also been recently shown to interact with prohibitin 1 in HeLa cells to induce cytotoxicity [ 29 ].…”