Cytosolic delivery of multidomain cargos by the N terminus of Pasteurella multocida toxin

Nathan C. Clemons, Yuka Bannai, Elizabeth E. Haywood, Yiting Xu, James D. Buschbach, Mengfei Ho, Brenda A. Wilson

Research output: Contribution to journalArticlepeer-review

Abstract

The zoonotic pathogen Pasteurella multocida produces a 146-kDa modular toxin (PMT) that enters host cells and manipulates intracellular signaling through action on its Gα protein targets. The N terminus of PMT (PMT-N) mediates cellular uptake through receptor-mediated endocytosis, followed by the delivery of the C-terminal catalytic domain from acidic endosomes into the cytosol. The putative native cargo of PMT consists of a 710-residue polypeptide with three distinct modular subdomains (C1-C2-C3), where C1 contains a membrane localization domain (MLD), C2 has an as-yet-undefined function, and C3 catalyzes the deamidation of a specific active-site glutamine residue in Gα protein targets. However, whether the three cargo subdomains are delivered intact or undergo further proteolytic processing during or after translocation from the late endosome is unclear. Here, we demonstrate that PMT-N mediates the delivery of its native C-terminal cargo as a single polypeptide, corresponding to C1-C2-C3, including the MLD, with no evidence of cleavage between subdomains. We show that PMT-N also delivers nonnative green fluorescent protein (GFP) cargo into the cytosol, further supporting that the receptor-binding and translocation functions reside within PMT-N. Our findings further show that PMT-N can deliver C1-C2 alone but that the presence of C1-C2 is important for the cytosolic delivery of the catalytic C3 subdomain by PMT-N. In addition, we further refine the minimum C3 domain required for intracellular activity as comprising residues 1105 to 1278. These findings reinforce that PMT-N serves as the cytosolic delivery vehicle for C-terminal cargo and demonstrate that its native cargo is delivered intact as C1-C2-C3.

Original languageEnglish (US)
Article numbere00248-18
JournalInfection and immunity
Volume86
Issue number8
DOIs
StatePublished - Aug 1 2018

Keywords

  • AB-type toxin
  • Atrophic rhinitis
  • Cargo delivery domain
  • Cytotoxicity
  • Deaminase toxin
  • Dermonecrotic toxin
  • Membrane translocation
  • Modular toxin
  • Pasteurellosis
  • Receptor-mediated endocytosis

ASJC Scopus subject areas

  • Parasitology
  • Microbiology
  • Immunology
  • Infectious Diseases

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