CHARACTERIZATION OF A 17-KILODALTON ANTIGEN OF BARTONELLA-HENSELAE REACTIVE WITH SERA FROM PATIENTS WITH CAT-SCRATCH DISEASE

A library of Bartonella (Rochalimaea) henselae DNA was constructed in the cloning vector lambda ZAPII and screened for expression of antigenic proteins by using a pool of sera from patients who had been diagnosed with cat scratch disease (CSD) and had antibodies to Bartonella spp., as determined by indirect fluorescent-antibody (IFA) assay. Ten immunoreactive phages were subcloned as recombinant plasmids by in vivo excision. All 10 recombinants expressed a protein of approximately 17 kDa when they were examined by immunoblot with the pool of human sera. Restriction endonuclease digestion of each recombinant plasmid indicated seven profiles, suggesting that cloning bias was not the reason for repeated isolation of clones expressing the 17-kDa antigen. The gene coding for the 17-kDa antigen was sequenced and shown to code for an open reading frame of 148 amino acids with a predicted molecular mass of 16,893 Da. The amino terminus of the deduced amino acid sequence was hydrophobic in nature and similar in size and composition to signal peptides found in gram-negative bacteria. The remainder of the deduced amino acid sequence was more hydrophilic and may represent surface-exposed epitopes. Further subcloning of the 17-kDa antigen as a biotinylated fusion protein in the expression vector PinPoint Xa-2 resulted in a 30-kDa protein that was highly reactive on immunoblots with individual serum samples from patients with CSD. The agreement between reactivity with the 30-kDa fusion protein on immunoblot analysis and the results obtained by IFA assay was 92% for IFA-positive sera and 88% for IFA-negative sera. The recombinant-expressed 17-kDa protein should be of value as an antigen for serologic diagnosis of CSD and Bartonella infections and warrants further study in attempts to develop a subunit vaccine to prevent long-term Bartonella infection in cats and the potential for further spread of these organisms to humans.