In Vivo Targets of Pasteurella Multocida Toxin

Many Pasteurella multocida strains are carried as commensals, while some cause disease in animals and humans. Some type D strains cause atrophic rhinitis in pigs, where the causative agent is known to be the Pasteurella multocida toxin (PMT). PMT activates three families of G-proteins—Gq/11, G12/13, and Gi/o—leading to cellular mitogenesis and other sequelae. The effects of PMT on whole animals in vivo have been investigated previously, but only at the level of organ-specific pathogenesis. We report here the first study to screen all the organs targeted by the toxin by using the QE antibody that recognizes only PMT-modified G-proteins. Under our experimental conditions, short-term treatment of PMT is shown to have multiple in vivo targets, demonstrating G-alpha protein modification, stimulation of proliferation markers and expression of active β-catenin in a tissue- and cell-specific manner. This highlights the usefulness of PMT as an important tool for dissecting the specific roles of different G-alpha proteins in vivo.

Cloning, expressing and purification of recombinant C-terminal PMT of Pasteurella multocida

Pasteurella multocida strains produce heat-stable toxin Pasteurella Multocida Toxin (PMT) which is the main virulence factor causing pasteurellosis in pigs. Therefore, PMT protein is considered as a promising candidate to study recombinant vaccine against pasteurellosis in pigs. In this study, we cloned the nucleotide sequence coding C-terminal fragment of PMT (tPMT-780), from acid amine 506 to acid amine 1285, into pRSET-A vector and expressed it on Escherichia coli BL21 bacteria. The SDS-PAGE result showed that tPMT-C780 was well-expressed by IPTG 1mM induction and determined to be insoluble protein, therefore it was purified by denaturation method using Ni-NTA beads. The protein elution samples contained only one protein band similar with the size of tPMT-C780 and positively reacted with PMT-specific antibody. These results suggested that we were successful in expressing and purifying C-terminal protein of PMT toxin, tPMT-C780. This is the primary material for further studies to produce recombinant vaccine against pasteurellosis in pigs.

Involvement of Osteocytes in the Action of Pasteurella multocida Toxin

Pasteurella multocida toxin (PMT) causes progressive atrophic rhinitis with severe turbinate bone degradation in pigs. It has been reported that the toxin deamidates and activates heterotrimeric G proteins, resulting in increased differentiation of osteoclasts and blockade of osteoblast differentiation. So far, the action of PMT on osteocytes, which is the most abundant cell type in bone tissue, is not known. In MLO-Y4 osteocytes, PMT deamidated heterotrimeric G proteins, resulting in loss of osteocyte dendritic processes, stress fiber formation, cell spreading and activation of RhoC but not of RhoA. Moreover, the toxin caused processing of membrane-bound receptor activator of NF-κB ligand (RANKL) to release soluble RANKL and enhanced the secretion of osteoclastogenic TNF-α. In a co-culture model of osteocytes and bone marrow cells, PMT-induced osteoclastogenesis was largely increased as compared to the mono-culture model. The enhancement of osteoclastogenesis observed in the co-culture was blocked by sequestering RANKL with osteoprotegerin and by an antibody against TNF-α indicating involvement of release of the osteoclastogenic factors from osteocytes. Data support the crucial role of osteocytes in bone metabolism and osteoclastogenesis and identify osteocytes as important target cells of PMT in progressive atrophic rhinitis.