Identification and characterization of the Pasteurella multocida toxin translocation domain

Abstract

SummaryThe Pasteurella multocida toxin (PMT) is a potent mitogen which enters the cytosol of eukaryotic cells via a low pH membrane translocation event. In common with the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), the core of the PMT translocation domain is composed of two predicted hydrophobic helices (H1 -residues 402-423, H2 -437-457) linked by a hydrophilic loop (PMT-TL -424-436). The peptide loop contains three acidic residues (D425, D431 and E434), which may play a role equivalent to D373, D379 and E382/383 in CNF1. To test this hypothesis, a series of point mutants was generated in which acidic residues were mutated into the permanently charged positive residue lysine. Individual mutation of D425, D431 and E434 each caused a four-to sixfold reduction in toxin activity. Interestingly, mutation of D401 located immediately outside the predicted helixloop-helix motif completely abolished toxin activity. Individual mutations did not affect cell binding nor greatly altered toxin structure, but did prevent translocation of the surface-bound proteins into the cytosol after a low pH pulse. Moreover, we demonstrate using an in vitro assay that PMT undergoes a pHdependent membrane insertion.

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

“…Translocation of PMT-DTa into the cytosol depends on acidification of the endosome, as revealed by inhibition of EF-2 ADP-ribosylation by PMT-DTa upon treatment with an inhibitor of vacuolar H ϩ -ATPase (47). This is in congruence to previous findings that the biological effect of PMT depends on acidification during uptake (11,21). To follow intracellular trafficking, PMTDTa was labeled site specifically at the N terminus with a fluorescent epitope.…”

“…The receptor binding and translocation domains are located in the N terminus (aa 1 to 574). Whereas the receptor binding domain has not been characterized in detail, two amphipathic helices, covering residues 402 to 457, are suggested to be involved in membrane insertion and translocation (21). So far, the cell surface receptor of PMT is not known (22).…”

Crystal structures reveal a thiol protease-like catalytic triad in the C-terminal region ofPasteurella multocidatoxin

“…Thus, we consider that C-PMT represents the intracellularly active moiety of the toxin. This does not conflict with the fact that the membrane translocation domain is reportedly composed of regions spanning residues 402-457 (14). Furthermore, the receptor-binding domain could be located on the N-terminal side of the translocation domain.…”

“…Previous reports have shown that PMT binds to an unknown receptor on target cells, is internalized by endocytosis, and escapes from the acidic endosome into the cytoplasm. The N-terminal region of the toxin is believed to mediate these procedures (14,17). We hypothesize about the subsequent action of PMT as follows: Although it is unknown whether the Nterminal region is clipped off when the toxin molecule enters the cytoplasm, the C-terminal region, at least, corresponding to C-PMT, is transferred to the vicinity of the plasma membrane with the aid of the C1 domain.…”

“…However, the molecular mechanism of the action of PMT remains unknown. The toxin is at least believed to bind a putative receptor on target cells, to be internalized by endocytosis, and to enter the cytoplasm from the endosome (7,14), implying that the toxin possesses an enzymatic region to modify as-yet-unknown intracellular target molecules. PMT consists of a single polypeptide chain of 1,285 aa.…”