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

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.

Author Summary The bacterium Vibrio vulnificus causes highly lethal infections in susceptible individuals exposed to contaminated water or seafood. V. vulnificus produces a MARTX toxin, which plays a significant role in bacterial virulence. The large MARTX toxin has numerous functional regions that have been characterized in vitro, but have not been functionally assessed during infection. In this study, we use a mouse model to show that a region of the MARTX toxin–the effector domain repertoire–is required for bacterial virulence. This region of the toxin promotes disruption of the intestinal epithelial barrier and movement of bacteria from the initial site of infection to the liver and spleen. Surprisingly, bacterial movement from the intestine occurs even in the absence of overt intestinal tissue damage, indicating that more subtle actions of the effector domains are responsible for promoting systemic bacterial spread. Together, our results advance the field of MARTX toxin research by functionally characterizing the toxin during infection. Results indicate that cytolytic effects on epithelial cells observed in vitro do not directly correspond to activities that are critical to bacterial pathogenesis in vivo and should encourage the study of diverse MARTX toxin functions.