The identification and characterization of a fibronectin-binding protein of Borrelia hermsii expressed in the blood during experimental relapsing fever

Only a small number of proteins that are expressed by the tick-borne relapsing fever (RF) agent B. hermsii during vertebrate host infection have been characterized, and the emphasis has been on the distinctive variable antigens of this species. The overall goal of this study was to identify and characterize additional B. hermsii proteins expressed during vertebrate host infection. In Chapter 2 I describe the identification and characterization of a non-variable protein of B. hermsii that was among the cell-free antigens present in the blood of infected mice. This protein was identified by mass spectrometry and then comparison of the deduced peptides to a database of open reading frames of the B. hermsii genome. The protein was identified as open reading frame (ORF) BHA007 near the left end of the 174 kb linear plasmid in B. hermsii. Orthologous ORFs were found on the linear plasmids of other RF species but not in Lyme disease Borrelia species. The cellular location of the protein was assessed by detergent fractionation and in situ protease susceptibility. For further in vitro studies the gene was synthesized, expressed in Escherichia coli, and the recombinant protein was purified. In Chapter 3 I investigated the characteristics of expression and subcellular localization of BHA007. This included analysis of in vivo expression that was carried out by mass spectrometry and RNA-seq analysis of spirochetes recovered from the blood as well as by immunoassay analysis with sera from infected animals. It was present in the proteome of spirochetes in the blood, and infected mice developed antibody to it during infection. In Chapter 4 I investigated a possible function for BHA007 during infection. Binding of selected host proteins was assessed by a competitive binding assay with labeled and unlabeled proteins. Recombinant BHA007 bound both human and bovine fibronectin with an affinity of ~40 nM, but only weakly or not at all other host proteins. The results indicate that BHA007 and orthologs of other RF species is a surface-exposed membrane protein that is expressed by spirochetes in the blood of infected animals and has fibronectin-binding activity. The protein has potential to be used for immunodiagnostic assays and perhaps as a vaccine.