Dataset Information

Cytosolic Delivery of Multidomain Cargos by the N Terminus of Pasteurella multocida Toxin.

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 G? 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 G? 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.

SUBMITTER: Clemons NC

PROVIDER: S-EPMC6056849 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Cytosolic Delivery of Multidomain Cargos by the N Terminus of Pasteurella multocida Toxin.

Clemons Nathan C NC Bannai Yuka Y Haywood Elizabeth E EE Xu Yiting Y Buschbach James D JD Ho Mengfei M Wilson Brenda A BA

Infection and immunity 20180723 8

The zoonotic pathogen <i>Pasteurella multocida</i> 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 ( ...[more]

PMID: 29784857

Similar Datasets

Project description:Bone remodeling is a continuous process to retain the structural integrity and function of the skeleton. A tight coupling is maintained between osteoclast-mediated resorption of old or damaged bones and osteoblast-mediated formation of new bones for bone homeostasis. While osteoblasts differentiate from mesenchymal stem cells, osteoclasts are hematopoietic in origin and derived from myeloid precursor cells. Osteoclast differentiation is driven by two cytokines, cytokine receptor activator of NF-κB ligand (RANKL), and macrophage colony-stimulating factor. Imbalances in the activity of osteoblasts and osteoclasts result in the development of bone disorders. Bacterially caused porcine atrophic rhinitis is characterized by a loss of nasal ventral conche bones and a distortion of the snout. While Bordetella bronchiseptica strains cause mild and reversible symptoms, infection of pigs with toxigenic Pasteurella multocida strains causes a severe and irreversible decay. The responsible virulence factor Pasteurella multocida toxin (PMT) contains a deamidase activity in its catalytical domain that constitutively activates specific heterotrimeric G proteins to induce downstream signaling cascades. While osteoblasts are inhibited by the toxin, osteoclasts are activated, thus skewing bone remodeling toward excessive bone degradation. Still, the mechanism by which PMT interferes with bone homeostasis, and the reason for this unusual target tissue is not yet well understood. Here, we show that PMT has the potential to differentiate bone marrow-derived macrophages into functional osteoclasts. This toxin-mediated differentiation process is independent of RANKL, a cytokine believed to be indispensable for triggering osteoclastogenesis, as addition of osteoprotegerin to PMT-treated macrophages does not show any effect on PMT-induced osteoclast formation. Although RANKL is not a prerequisite, toxin-primed macrophages show enhanced responsiveness to low concentrations of RANKL, suggesting that the PMT-generated microenvironment offers conditions where low concentrations of RANKL lead to an increase in the number of osteoclasts resulting in increased resorption. PMT-mediated release of the osteoclastogenic cytokines such as IL-6 and TNF-α, but not IL-1, supports the differentiation process. Although the production of cytokines and the subsequent activation of signaling cascades are necessary for PMT-mediated differentiation into osteoclasts, they are not sufficient and PMT-induced activation of G protein signaling is essential for efficient osteoclastogenesis.

Dataset Information

Cytosolic Delivery of Multidomain Cargos by the N Terminus of Pasteurella multocida Toxin.

Publications

Cytosolic Delivery of Multidomain Cargos by the N Terminus of Pasteurella multocida Toxin.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets