Makes caterpillars floppy-like effector-containing MARTX toxins require host ADP-ribosylation factor (ARF) proteins for systemic pathogenicity

Abstract

Significance MARTX toxins present across multiple bacterial genera are primary virulence factors that facilitate initial colonization, dissemination, and lethality in a wide range of hosts, including humans. Upon entry into host cells, the toxins undergo a processing event to release their disease-related modularly structured effector domains. However, the mechanisms underlying processing and activation of diverse effector domains within the toxins remain unclear. Here, we use biochemical and structural biological approaches, in combination with cellular microbiological experiments, to demonstrate how Makes caterpillars floppy-like effector (MCF) or its homolog-containing MARTX toxins process effector modules and fully activate effectors. MCF-containing toxins target ADP-ribosylation factor proteins ubiquitously expressed in cells to activate and disseminate effectors across subcellular compartments simultaneously, eventually leading to systemic pathogenicity. Upon invading target cells, multifunctional autoprocessing repeats-in-toxin (MARTX) toxins secreted by bacterial pathogens release their disease-related modularly structured effector domains. However, it is unclear how a diverse repertoire of effector domains within these toxins are processed and activated. Here, we report that Makes caterpillars floppy-like effector (MCF)-containing MARTX toxins require ubiquitous ADP-ribosylation factor (ARF) proteins for processing and activation of intermediate effector modules, which localize in different subcellular compartments following limited processing of holo effector modules by the internal cysteine protease. Effector domains structured tandemly with MCF in intermediate modules become disengaged and fully activated by MCF, which aggressively interacts with ARF proteins present at the same location as intermediate modules and is converted allosterically into a catalytically competent protease. MCF-mediated effector processing leads ultimately to severe virulence in mice via an MCF-mediated ARF switching mechanism across subcellular compartments. This work provides insight into how bacteria take advantage of host systems to induce systemic pathogenicity.