Characterization of the heat shock response in Brucella abortus and isolation of the genes encoding the GroE heat shock proteins.

摘要

In an effort to define the heat shock response in the bovine intracellular pathogen Brucella abortus, a rough variant lacking extensive lipopolysaccharide was pulse-labeled with [35S]methionine following exposure to elevated temperatures. The major heat shock proteins observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography migrate at 70, 62, 18, and 10 kDa. The maximum response was observed between 42 and 46 degrees C and within 2 to 3 h of the shif in temperature and varied slightly for the different proteins. Accumulation of the 62-kDa heat shock protein (62-kDa Hsp) was observed to continue for up to 5 h following the shift in temperature. In an effort to better define the heat shock response and its potential relationship with protective immunity, genes encoding the major heat shock proteins were isolated from recombinant libraries constructed from B. abortus S19 and S2308 and sequenced. The 62-kDa Hsp shares more than 60% amino acid homology with members of the GroEL family and is immunoprecipitated with polyclonal antibodies to Escherichia coli GroEL and monoclonal antibodies to mycobacterial Hsp 65. Western blot (immunoblot) analysis with pooled sera from vaccinated and infected cattle revealed that the 62-kDa Hsp is a predominantly recognized antigen. The roles of these gene products during environmental stress and in protective immunity against brucellosis are under investigation.