Summary Pasteurella multocida toxin (PMT) is an AB toxin that causes pleiotropic effects in targeted host cells. The N-terminus of PMT (PMT-N) is believed to harbor the membrane receptor binding and translocation domains responsible for mediating cellular entry and delivery of the C-terminal catalytic domain into the host cytosol. Previous studies have implicated gangliosides as the host receptors for PMT binding. To gain further insight into the binding interactions involved in PMT binding to cell membranes, we explored the role of various membrane components in PMT binding, utilizing four different approaches: TLC-overlay binding experiments with 125I-labeled PMT, PMT-N or PMT-C; pull-down experiments using reconstituted membrane liposomes with full-length PMT, surface plasmon resonance (SPR) analysis of PMT-N binding to reconstituted membrane liposomes, and SPR analysis of PMT-N binding to HEK-293T cell membranes without or with sphingomyelinase, phospholipase D, or trypsin treatment. Results revealed that, in our experimental system, full-length PMT and PMT-N did not bind to gangliosides, including GM1, GM2 or GM3, but instead bound to membrane phospholipids, primarily the abundant sphingophospholipid sphingomyelin (SM) or phosphatidylcholine (PC) with other lipid components. Collectively, these studies demonstrate the importance of SM for PMT binding to membranes and suggest the involvement of a protein co-receptor.
The zoonotic pathogen 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 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 of three distinct modular subdomains (C1-C2-C3), where C1 contains a membrane localization domain (MLD), C2 has as-of-yet undefined function, and C3 catalyzes 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 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 into the cytosol non-native GFP cargo, 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 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 1105-1278. 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.
An immunosuppressive tumor microenvironment has hampered the efficacy of immunotherapy in prostate cancer. However, radiation-induced immunological effects can partly mediate anti-tumor effects by promoting a pro-inflammatory environment potentially responsive to immunotherapy. Herein, we examined the immunomodulatory properties of a radiopharmaceutical therapy (RPT) with NM600 radiolabeled with either a beta or alpha emitter in two prostate cancer models. 225Ac-NM600, but not 177Lu-NM600, promoted significant anti-tumor effects and improved overall survival. Immunomodulatory effects were dose, radionuclide, and tumor type-dependent. 225Ac-NM600 elicited an array of immunomodulatory effects such as increased CD8/Treg ratio, activation of effector and memory T cells, abrogation of infiltrating suppressor cells (e.g., Tregs and MDSCs), and increased levels of Th1 cytokine and pro-inflammatory chemokines. Importantly, we demonstrate the need to carefully characterize the immune responses elicited by RPT both pre-clinically and clinically to maximize tumor control and avoid potential counterproductive immunosuppressive effects.
Correction for Clemons et al., "Cytosolic delivery of multidomain cargos by the N terminus of Pasteurella multocida toxin." Infect Immun 86:e00596-18.
Pasteurella multocida toxin (PMT), the major virulence factor responsible for zoonotic atrophic rhinitis, is a protein deamidase that activates the alpha subunit of heterotrimeric G proteins. Initial activation of G alpha-q-coupled phospholipase C-beta-1 signaling by PMT is followed by uncoupling of G alpha-q-dependent signaling, causing downregulation of downstream calcium and mitogenic signaling pathways. Here, we show that PMT decreases endogenous and exogenously expressed G alpha-q protein content in host cell plasma membranes and in detergent resistant membrane (DRM) fractions. This membrane depletion of G alpha-q protein was dependent upon the catalytic activity of PMT. Results indicate that PMT-modified G alpha-q redistributes within the host cell membrane from the DRM fraction into the soluble membrane and cytosolic fractions. In contrast, PMT had no affect on G alpha-s or G beta protein levels, which are not substrate targets of PMT. PMT also had no affect on G alpha-11 levels, even though G alpha-11 can serve as a substrate for deamidation by PMT, suggesting that membrane depletion of PMT-modified G-alpha-q has specificity.
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