Pasteurella multocida Toxin as a Transporter of Non-Cell-Permeating Proteins

Abstract

bThe protein toxin Pasteurella multocida toxin (PMT) is the causative agent of atrophic rhinitis in pigs, leading to atrophy of the nasal turbinate bones by affecting osteoblasts and osteoclasts. The mechanism of PMT-induced intoxication is a deamidation of ␣-subunits of heterotrimeric G proteins, including G␣ q , G␣ 13 , and G␣ i , thereby causing persistent activation of the G proteins. Here we utilized PMT as a transporter of the non-cell-permeating A domain of diphtheria toxin (DTa). Fusion proteins of PMT and DTa ADP-ribosylated elongation factor 2, the natural target of diphtheria toxin, leading to cell toxicity. PMT-DTa effects were competed by PMT, indicating binding to the same cell surface receptor. Fluorescently labeled PMT-DTa and PMT colocalized with specific markers of early and late endosomes. Bafilomycin A, which inhibits vacuolar H ؉ -ATPase, blocked PMT-DTainduced intoxication of HEK-293 cells. By constructing various PMT-DTa chimeras, we identified a minimal region of PMT necessary for uptake of DTa. The data suggest that PMT is able to transport cargo proteins into eukaryotic cells by utilizing the PMT-specific uptake route.

Modular domain swapping among the bacterial cytotoxic necrotizing factor (CNF) family for efficient cargo delivery into mammalian cells

“…For example, full-length botulinum neurotoxin serotype D (BoNT/D) has been fused with several heterologous cargos for delivery of enzymatically active cargo (14). In the case of a fusion of Pasteurella multocida toxin (PMT) with the catalytic activity domain of diphtheria toxin (DTa), including the native PMT cargo enhanced the cellular DTa-mediated activity of PMT-DTa 10-fold over PMTΔC-DTa without the PMT cargo (15). Identifying key molecular determinants of inter-domain compatibility will enable the design of stable toxin-based delivery platforms and will improve the efficacy of cytosolic cargo delivery.…”

Pasteurella multocida toxin

“…Both mechanisms are important for trafficking from early to late endosomes. A more recent study using confocal colocalization experiments with markers of intracellular compartments and fluorescently marked PMT supports this DT-like trafficking model of PMT, where the toxin is internalized to early and late endosomes and, from there, translocates to the cytoplasm [260]. PMT colocalized with Rab5 and early endosome antigen 1 (EEA1), which both are markers for early endosomes.…”

“…This process appears to involve a low pH-dependent event, since the addition of weak lysosomotrophic bases, such as methylamine, ammonium chloride, or chloroquine, blocked PMT action by buffering the intra-endosomal acidification [180]. Additionally, PMT signaling could be inhibited by the treatment of cells with bafilomycin A1, a specific inhibitor of the vacuolar H + -ATPase (v-ATPase) that physiologically acidifies endosomes [259][260][261]. Involvement of a low pH-dependent, membrane translocation event in PMT action was further supported by another study in which PMT intracellular activity could be induced by a low pH pulse after PMT was bound to cells at 4°C, even in the presence of the endocytosis inhibitor bafilomycin A1 [261].…”

“…Interestingly, the mutation of Asp-401, which is nearby, but outside the helix-loop-helix motif, completely inhibited the action of PMT. The importance of this motif in the translocation process was shown in another study by the construction of various cytotoxic PMT-DT chimeras [260]. A series of N-terminal fragments of PMT were fused to the catalytic domain of DT, and the effects of cellular intoxication were measured.…”

Crystal structure of full-length cytotoxic necrotizing factor CNFYreveals molecular building blocks for intoxication

“…Notably, translocation and delivery of the β-lactamase TEM into the host cell cytoplasm was achieved when the D1-2 fragment was extended by the third domain (D3) comprising the imperfect β-barrel domain ( Fig 4D), demonstrating that this subunit is an essential part of the translocation apparatus. Interestingly, the N-terminus of Pasteurella multocida exotoxin PMT possesses high homology to the first three domains of CNF Y (Fig 3C) and a hybrid toxin consisting of this N-terminal fragment (residues 1-505) of PMT and the ADP-ribosylating domain of DT was able to intoxicate cells (Bergmann et al, 2013), indicating a conserved translocation module consisting of domains D1-3.…”