The MCF Toxin of the Extracellular Pathogen Vibrio vulnificus is Activated by and Targets Host GTPases

Abstract

Vibrio vulnificus (Vv) is a Gram‐negative bacterial pathogen typically found in warm coastal waters. This pathogen causes severe life‐threatening gastrointestinal and wound infections, most commonly from the consumption of contaminated seafood or an exposed wound. Infections progress quickly to sepsis, and death; approximately 90% of patients are hospitalized and >50% die, often as fast as 48 hours after symptom onset. Of concern, the incidence of Vv is increasing, in part due to its expanding geographical distribution caused by climate change.Bacterial pathogens cause debilitating and lethal infections that are often difficult to treat. Understanding how to manage and control the infections they cause is immensely important for improving quality of life and preventing death. The toxins secreted by extracellular bacteria are frequently the primary virulence factors required to either establish colonization, promote maintenance, growth, and/or eventually dissemination. As such bacterial toxins are extremely important in pathogenesis, and they affect and manipulate many host cellular processes.The primary Vv virulence factor associated with sepsis and subsequent death is the composite MARTX toxin. The MARTX toxin is secreted as a single large polypeptide composed of glycine‐rich repeats at both the N‐ and C‐termini between which there are up to five variable effector domains and a cysteine protease domain (CPD). The repeat regions form a pore in the plasma membrane and the effectors and CPD are translocated across. Within the cell, the CPD auto‐cleaves at multiple sites to release each of the arrayed effectors individually into the host cytoplasm. The MARTX toxin thus serves as an effector delivery platform for translocation of multiple toxic virulence factors inside cells as a single bolus.The Makes Caterpillars Floppy‐like (MCF) effector domain is among the most commonly present Vv MARTX effector domains. MCF is unique from most other MARTX domains in that it utilizes its own autoproteolytic activity to activate itself once inside the host cell. MCF autoprocessing requires stimulation by host ARFs, small GTPases that cycle between GDP‐ or GTP‐bound forms and localize to specific subcellular locations. Once auto‐processed, MCF binds phosphatidylinositol‐5‐phosphate (PtdIns5P), a lipid enriched on Golgi membranes, and its exposed N‐terminal glycine is acetylated. Combined, this results in the generation of “active” MCF (aMCF). aMCF induces the intrinsic apoptotic pathway, causes cell rounding, and nuclear fragmentation. Furthermore, aMCF causes vesiculation of the Golgi apparatus, and destroys the mitochondria.Remarkably, while ARF stimulated autoprocessing results in aMCF, ARFs are not the direct target leading to the cytotoxic effects. The biochemical link between the activation of MCF and its cytotoxicity remains unknown. We explore how MCF activation leads to its disruption of host endocytic trafficking proteins. We examine how these proteins may be targeted and modified by MCF. Furthermore, we assess how the disruption of these host proteins ultimately leads to the dissolution of the endomembrane signaling network and the cytotoxicity we observe. This study begins to elucidate the biochemical activity of this important domain and gives insight into how it may promote disease progression. This work will be critical for advancing the understanding how these MARTX toxins function during Vv pathogenesis.