IDENTIFICATION OF AN IMMUNOREACTIVE BRUCELLA-ABORTUS HTRA STRESS-RESPONSE PROTEIN

An ll-kb fragment of Brucella abortus genomic DNA cloned into the BamH I site of pUC9 expressed a 6O-kDa protein in Escherichia coli DH5-alph a. Antibodies reactive with this 60-kDa protein were detected by Weste rn blot (immunoblot) analysis in sera from mice, cattle, and goats exp erimentally infected with B. abortus, in sera from mice experimentally infected with Brucella melitensis, and in serum from a dog experiment ally infected with Brucella canis. Similar results were seen with sera obtained from cattle and dogs with naturally acquired brucellosis. Th e gene encoding the 60-kDa Brucella protein was localized to a 2-kb Ec oRI fragment which was also reactive in Southern blots with genomic DN A from other strains of B. abortus as well as with genomic DNA from B. melitensis and B. canis. Nucleotide sequence analysis of the cloned E coRI fragment revealed an open reading frame encoding a protein with a predicted molecular mass of 51,847 Da and an isoelectric paint of 5.1 5. Comparison of the deduced amino acid sequence of the immunoreactive Brucella protein with the SWISS-PROT protein sequence data base revea led that it shares > 40% amino acid sequence identity with the E. coli and Salmonella typhimurium HtrA stress response proteins. Computer-as sisted analysis of this amino acid sequence also predicted that the pu tative Brucella HtrA homolog contains an export signal sequence and a serine protease active site, two structural features characteristic of previously described HtrA proteins. A potential O-E type heat shock p romoter sequence was detected upstream of the cloned Brucella htrA gen e, and Northern (RNA) blot analysis demonstrated that exposure of B. a bortus 2308 to heat shock conditions resulted in a transient elevation of htrA transcription. These results strongly suggest that the immuno reactive 60-kDa Brucella protein is a member of the HtrA class of stre ss response proteins.