Ultrastructural localization of the Pasteurella multocida toxin in a toxin-producing strain

Abstract

Toxigenic strains of Pasteurella multocida produce the 147 kDa protein Pasteurella multocida toxin (PMT) which is responsible for the osteoclastic bone resorption in progressive atrophic rhinitis in pigs and induces such resorption in all experimental animals tested so far. In the present study we have carried out immunocytochemistry on formaldehyde-and glutaraldehyde-fixed ultracryocut P. multocida using a pool of monoclonal antibodies against different epitopes on PMT as the first layer and affinity purified rabbit anti-mouse IgG as the second layer. Goat anti-rabbit IgG conjugated with 5 nm gold particles was used as marker. The gold particles were silverenhanced prior to examination in the transmission electron microscope. Whole bacteria were also immunostained after fixation and critical point drying and examined by scanning transmission electron microscopy. The results showed that PMT was located in the cytoplasm of P. multocida. PMT could not be detected on intact, undamaged P. multocida by scanning electron microscopy. Neither pili nor flagella could be detected on the surface of the negatively stained P. multocida strains investigated. PMT has a series of characteristics encompassed in the definition of an exotoxin. However, that PMT was not secreted by living intact P. multocida is unexpected for an exotoxin.

The Pasteurella multocida toxin is encoded within a lysogenic bacteriophage

“…Previous studies have shown that PMT is not secreted from in vitro grown cells (Nakai et al ., 1985; iDali et al ., 1991). Thus, electron microscopy demonstrated that the toxin was cytoplasmic in P. multocida , and that no toxin could be detected on intact undamaged P. multocida (iDali et al ., 1991). Furthermore, the toxin has no apparent signal sequence.…”

“…The toxin is a modular protein, with domains for cell‐binding/internalization at the N‐terminus and for enzymatic activity at the C‐terminus (Busch et al ., 2001; Pullinger et al ., 2001), and therefore has the characteristics of a secreted exotoxin. However, it is not secreted from the bacteria during in vitro culture, and ultrastructural studies of toxigenic P. multocida demonstrated that PMT was localized in the bacterial cytoplasm (Nakai et al ., 1985; iDali et al ., 1991).…”

Phenotypic and pathobiological properties of Corynebacterium aquaticum isolated from diseased striped bass

“…On the other hand, secretion of some exotoxins is dependent on the phase of growth of the bacterium and its cultural conditions (Wretling & Heden 1933, O'Reilly & Day 1983, Idali et al 1991) while other toxins remain in the cytoplasm and are released only after cell lysis. In fact, when the ECP of Corynebacterium aquaticum from striped bass were collected at 24 h (instead of at 48 h), the protein content and proteolytic activity of the ECP were very low (0.2 mg ml-' and 1.2 units ml-' respectively), no cytotoxins were detected, and the ECP lacked toxicity for fish.…”

Pasteurella multocida toxin

“…Ultrastructural studies of toxigenic P. multocida showed that PMT was localized in the cytoplasm of the bacteria [217,218]. Although it is known that PMT is expressed under noninducing conditions, it is possible that phage induction results in PMT release from cells during infection.…”