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 antigen ic proteins by using a pool of sera from patients who had been diagnos ed with cat scratch disease (CSD) and had antibodies to Bartonella spp ., as determined by indirect fluorescent-antibody (IFA) assay. Ten imm unoreactive phages were subcloned as recombinant plasmids by in vivo e xcision. All 10 recombinants expressed a protein of approximately 17 k Da when they were examined by immunoblot with the pool of human sera. Restriction endonuclease digestion of each recombinant plasmid indicat ed 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 o pen reading frame of 148 amino acids with a predicted molecular mass o f 16,893 Da. The amino terminus of the deduced amino acid sequence was hydrophobic in nature and similar in size and composition to signal p eptides found in gram-negative bacteria. The remainder of the deduced amino acid sequence was more hydrophilic and may represent surface-exp osed epitopes. Further subcloning of the 17-kDa antigen as a biotinyla ted fusion protein in the expression vector PinPoint Xa-2 resulted in a 30-kDa protein that was highly reactive on immunoblots with individu al serum samples from patients with CSD. The agreement between reactiv ity with the 30-kDa fusion protein on immunoblot analysis and the resu lts obtained by IFA assay was 92% for IFA-positive sera and 88% for IF A-negative sera. The recombinant-expressed 17-kDa protein should be of value as an antigen for serologic diagnosis of CSD and Bartonella inf ections and warrants further study in attempts to develop a subunit va ccine to prevent long-term Bartonella infection in cats and the potent ial for further spread of these organisms to humans.