CLONING, CHARACTERIZATION AND CONSTRUCTION OF HTRA AND HTRA-LIKE MUTANTS OF BRUCELLA-ABORTUS AND THEIR SURVIVAL IN BALB C MICE/

A genomic library of Brucella abortus S2308 was screened for expressio n of recombinant proteins recognized by sera from mice and from cattle infected with B. abortus. A positive clone, BA1, expressing a 50 kDa peptide was recognized by both sera. Plasmid pBA1, isolated from BA1, was shown by restriction enzyme digestion to possess a 1.9 kb insert. The nucleotide sequence of the pBA1 insert revealed an open reading fr ame with of 1539 bases with a coding capacity of 513 amino acids and a predicted molecular weight of 50 992. The predicted amino acid sequen ce showed 37% identity to E. coli HtrA, a temperature inducible serine protease. A second B. abortus htrA gene, designated htrA-like, was id entified on a different cloned fragment th at a Iso encoded B. abortus recA. The nucleotide sequence of the htrA-like gene revealed an open reading frame of 1422 nucleotides with a coding capacity of 474 amino acids and a predicted molecular weight of 50 155. The deduced amino ac id sequence of the htrA-like gene showed 42% and 36% identity with B. abortus and E. coil HtrAs respectively. Western blotting of E. coli ly sate containing the htrA-like gene was not recognized by sera from B. abortus-infected cattle or mice. B. abortus htrA but not htrA-like rel ieved the temperature sensitive phenotype and permitted growth of an E . coli htrA mutant at 42 degrees C. B. abortus htrA and htrA-like muta nts were constructed and their survival and growth in BALB/c mice was compared to the parental strain S2308. Splenic levels of htrA or htrA- like mutants were initially lower but after 60 days post-infection bot h were higher than the parental strain. Histologic analysis of hepatic inflammatory responses suggested that an initial intense granuloma fo rmation in the htrA group was the basis for early low splenic titers o f bacteria, but that failure to maintain granulomas, as did mice given the parental strain, resulted in a marked secondary rise in splenic b acterial titers.