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Immunochemical analysis of plasmid-encoded proteins released by enteropathogenic Yersinia sp. grown in
Abstract:
Enteropathogenic Yersinia sp. releases plasmid-associated proteins of low molecular mass (26-67 kilodaltons) at 37 degrees C. In this study, the optimum conditions for the release of proteins were assessed and the released proteins (RPs) were analyzed for the manner of release, immunochemical characteristics, and the location of the genes necessary for their synthesis. Protein release was strongly enhanced when growth media were markedly depleted of calcium ions by precipitation with oxalate or chelation with EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. RP yields were greatest when Yersinia spp. were in the exponential growth phase. The RPs appeared to be released from the Yersinia spp. by secretion rather than by pinching off of membrane vesicles, because the RPs did not sediment during high-speed centrifugation nor were they contaminated to any significant degree with lipopolysaccharide. Moreover, immunoblot analysis revealed only traces of protein species related to RPs within the outer membranes of plasmid-positive Yersinia spp. grown at 37 degrees C under calcium-restricted conditions. Immunoblot studies also showed that the RPs of Y. enterocolitica serotypes O:3, O:8, and O:9 and the RP of Y. pseudotuberculosis serotype I are highly cross-reactive. Finally, the immunoprecipitates of the products of minicells which harbor Yersinia plasmids were used to demonstrate that at least three proteins immunochemically related to the released fraction were plasmid encoded. These results suggest that at least three of the RPs may be related to or identical with previously described plasmid-encoded Yersinia outer membrane proteins.
Insights
Enteropathogenic Yersinia species release specific proteins (RPs) under calcium-depleted conditions. These released proteins are secreted, not from membrane vesicles, and at least three are plasmid-encoded.
Area of Science:
- Microbiology
- Molecular Biology
- Immunology
Background:
- Enteropathogenic Yersinia species release low molecular mass proteins at 37°C.
- These plasmid-associated proteins play a role in virulence.
Purpose of the Study:
- To optimize conditions for the release of these proteins.
- To analyze the characteristics of released proteins (RPs).
- To determine the genetic location of RPs.
Main Methods:
- Yersinia spp. growth under calcium-restricted conditions (oxalate precipitation, EGTA chelation).
- Analysis of protein release during exponential growth phase.
- High-speed centrifugation and immunoblot analysis to assess release mechanism and protein localization.
- Immunoprecipitation using minicells harboring Yersinia plasmids.
Main Results:
- Protein release significantly enhanced in calcium-depleted media.
- RPs released via secretion, not membrane vesicles, with minimal lipopolysaccharide contamination.
- RPs from Y. enterocolitica (O:3, O:8, O:9) and Y. pseudotuberculosis (serotype I) are cross-reactive.
- At least three RPs are plasmid-encoded, potentially identical to known outer membrane proteins.
Conclusions:
- Calcium restriction is key for Yersinia protein release.
- Released proteins are secreted and plasmid-encoded.
- RPs may represent a subset of plasmid-encoded outer membrane proteins involved in Yersinia pathogenesis.