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The Proteases of American Foulbrood Scales
1School of Pure and Applied Biology, Cardiff University of Wales, Cardiff, CF1 3TL, United Kingdom
Abstract:
The gross protease activity of pathological samples of American foulbrood-infected cadavers from several UK sources was studied. In all cases the bulk of the activity is caused by neutral protease(s) (optimum pH ca. 6.8) that are inhibited by chelating agents such as EDTA and 1,10 phenanthroline (indicating metalloproteases) but not by inhibitors of other classes of proteolytic enzymes. The proteases, which derive from the infectious agent of AFB, Paenibacillus larvae, were unusual in being insensitive to phosphoramidon and in not degrading FAGLA, the artificial substrate specific for most Bacillus metalloproteases. The enzymes in AFB ropes and scales had temperature optima of 60-65°C and were inactivated quickly on incubation at 80°C. Activity at moderate temperatures (37°C) was great on general substrates such as casein, gelatin, and hide powder azure, slight on elastin-Congo red, and nonexistent on collagen. In SDS-polyacrylamide gels the enzymes from the various sources all had molecular weights about 24 kDa. The proteases could be detected only zymographically after brief washing to remove SDS. On silver-stained gels no bands corresponding to the enzymes' activities could be detected. On native polyacrylamide gels enzyme activity was resolved zymographically as at least three metalloprotease bands with samples from different sources showing a variety of patterns.
Insights
Protease activity in American foulbrood (AFB) cadavers is mainly from metalloproteases produced by Paenibacillus larvae. These unique enzymes show specific characteristics, aiding in understanding AFB pathogenesis.
Area of Science:
- Veterinary Microbiology
- Enzymology
- Insect Pathology
Background:
- American foulbrood (AFB) is a devastating bacterial disease affecting honeybee larvae, caused by Paenibacillus larvae.
- Understanding the virulence factors of Paenibacillus larvae, such as proteases, is crucial for developing control strategies.
Purpose of the Study:
- To characterize the gross protease activity present in pathological samples of American foulbrood-infected honeybee cadavers.
- To identify the nature of the proteases responsible for the observed activity and their origin from Paenibacillus larvae.
Main Methods:
- Analysis of protease activity in infected cadavers from various UK sources.
- Enzyme inhibition assays using chelating agents (EDTA, 1,10 phenanthroline) and specific inhibitors.
- Zymography on SDS-polyacrylamide and native polyacrylamide gels to determine molecular weight and identify protease bands.
- Substrate specificity testing (casein, gelatin, hide powder azure, elastin-Congo red, collagen).
- Thermal stability analysis.
Main Results:
- The predominant protease activity was neutral (optimum pH ~6.8) and identified as metalloproteases, inhibited by chelating agents.
- These metalloproteases are derived from Paenibacillus larvae and exhibit unusual resistance to phosphoramidon and inability to degrade FAGLA.
- Enzymes had temperature optima of 60-65°C, were inactivated at 80°C, and showed high activity on general substrates at 37°C.
- Molecular weight was determined to be approximately 24 kDa via SDS-PAGE, with zymography revealing at least three metalloprotease bands on native gels.
Conclusions:
- The study characterizes unique metalloproteases from Paenibacillus larvae contributing to American foulbrood pathogenesis.
- These findings provide insights into the enzymatic mechanisms of AFB and potential targets for future interventions.
- The identified proteases display distinct properties differentiating them from typical Bacillus metalloproteases.