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Extracellular neuraminidase production by a Pasteurella multocida A:3 strain associated with bovine pneumonia
D J White1, W L Jolley, C W Purdy
1Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
Abstract:
The properties of an extracellular neuraminidase produced by a Pasteurella multocida A:3 strain that was isolated in a case of bovine pneumonia were examined during growth in a defined medium. This enzyme (isolated from concentrated culture supernatants of P. multocida A:3) was active against N-acetylneuramin lactose, human alpha-1-acid glycoprotein, fetuin, colominic acid, and bovine submaxillary mucin. Enzyme elaboration was correlated with the growth of the organism in a defined medium, with maximum quantities produced in the stationary phase. The enzyme was purified by a combination of ammonium sulfate fractionation, ion exchange on DEAE-Sephacel, and gel filtration on Sephadex G-200. The purified neuraminidase possessed a specific activity of 9.36 mumol of sialic acid released per min per mg of protein against fetuin. The enzyme possessed a pH optimum of 6.0 and a Km of 0.03 mg/ml. The P. multocida A:3 neuraminidase had a molecular weight of approximately 500,000 as estimated by gel filtration. The enzyme was stable at 4 and 37 degrees C for 3 h. Approximately 75% of the neuraminidase activity was lost within 30 min at 50 degrees C. Greater than 90% of the enzyme activity was destroyed within 10 min at temperatures of > or = 65 degrees C. The P. multocida neuraminidase does not appear to be serologically related to the Pasteurella haemolytica A1 neuraminidase since antiserum prepared against the purified P. haemolytica enzyme did not neutralize the P. multocida enzyme.
Insights
This study characterized the neuraminidase enzyme from Pasteurella multocida A:3, finding it active against various substrates and stable under specific conditions. The enzyme is not serologically related to Pasteurella haemolytica A1 neuraminidase.
Area of Science:
- Microbiology
- Enzymology
- Veterinary Science
Background:
- Bovine pneumonia can be associated with Pasteurella multocida strains.
- Extracellular enzymes produced by bacteria play roles in pathogenesis.
- Neuraminidases are enzymes that cleave sialic acids, important in host-pathogen interactions.
Purpose of the Study:
- To characterize the extracellular neuraminidase produced by a Pasteurella multocida A:3 strain.
- To investigate the enzyme's activity, purification, and stability.
- To compare the Pasteurella multocida A:3 neuraminidase with other related enzymes.
Main Methods:
- Cultivation of Pasteurella multocida A:3 in defined medium.
- Purification of neuraminidase using ammonium sulfate fractionation, ion exchange, and gel filtration.
- Enzyme activity assays using various substrates (fetuin, N-acetylneuramin lactose, etc.).
- Determination of enzyme kinetics (pH optimum, Km), molecular weight, and thermal stability.
Main Results:
- The Pasteurella multocida A:3 neuraminidase was active against multiple substrates, including fetuin.
- Maximum enzyme production occurred during the stationary growth phase.
- Purified enzyme had a specific activity of 9.36 U/mg against fetuin, with a pH optimum of 6.0 and Km of 0.03 mg/ml.
- The enzyme's molecular weight was estimated at 500,000 Da.
- The enzyme exhibited stability at 4°C and 37°C but was rapidly inactivated at temperatures above 50°C.
- Antiserum against Pasteurella haemolytica A1 neuraminidase did not neutralize the Pasteurella multocida A:3 enzyme, indicating no serological relation.
Conclusions:
- Pasteurella multocida A:3 produces a potent extracellular neuraminidase.
- The enzyme's properties suggest a potential role in the pathogenesis of bovine pneumonia.
- The Pasteurella multocida A:3 neuraminidase is distinct from the Pasteurella haemolytica A1 neuraminidase.