The potent mitogen Pasteurella multocida toxin is highly resistant to proteolysis but becomes susceptible at lysosomal pH

Abstract

The susceptibility of the potent mitogen Pasteurella multocida toxin (PMT) to various proteases was investigated. PMT at a toxin to protease molar ratio of 1 : 1 was resistant to 8 of the 11 proteases tested after one hour. With longer incubation, PMT remained resistant to 7 proteases, and this correlated with a retention of biological activity, indicating that PMT might not require proteolytic cleavage at least until it bound to a cell receptor. Previous evidence had suggested that PMT is processed in the cell via an endosome or lysosome. We have shown that PMT became susceptible to proteolysis when the pH was lowered to 5 or below. This supports the previous suggestion that PMT is processed via a low pH compartment in the cell.

Localization of Functional Domains of the Mitogenic Toxin of Pasteurella multocida

“…PMT has previously been shown to be highly resistant to most proteases at neutral pH (46). One protease, Asp-N, was found to cleave PMT at pH 7, producing three fragments of ca.…”

“…One protease, Asp-N, was found to cleave PMT at pH 7, producing three fragments of ca. 90, 87, and 55 kDa (46). Amino-terminal sequencing of these fragments showed that the toxin was cleaved between residues 473 and 474, and between 509 and 510.…”

“…Its action is also inhibited by neutralizing antibody or methylamine added early but not late after toxin. PMT undergoes a conformational change at low pH, which affects its protease sensitivity and circular dichroism spectra (46,47). This suggests that PMT may be trafficked and perhaps processed through a low-pH compartment.…”

Pasteurella

“…The toxin is passively released upon bacterial lysis and is believed to be actively taken up by host cells via receptor -mediated endocytosis by binding to a ganglioside cell surface receptor (Pettit et al 1993 ). The N -terminal receptor -binding domain is then proteolytically cleaved under low pH conditions (Smyth et al 1995 ), exposing the translocation domain, which allows uptake into the cytoplasm . Here the catalytic C -terminal portion of the protein activates two heterotrimeric G proteins, G α q and G α 12/13 , which in turn affect a number of host signal transduction pathways ).…”

Molecular Biology of Pasteurella multocida Toxin

“…The translocation depends on a putative translocation T-domain, consisting of two predicted hydrophobic helices (residues 402-423 and 437-457) linked by a peptide loop (residues 424-436). It is proposed that acidification induces a structural change in the toxin, which was previously observed utilising circular dichroism and measuring susceptibility to proteases (Smyth et al 1995(Smyth et al , 1999. This structural change exposes the T-domain, allowing it to insert into the vesicular membrane.…”