Tick-borne bacterial pathogens of humans cause significant morbidity and mortality throughout the United States and abroad. Lyme disease, caused by Borrelia burgdorferi, is the most prevalent arthropod-borne disease of humans in the United States and many other countries throughout Europe and Asia. Tick-borne relapsing fever, caused by Borrelia hermsii, is endemic in scattered foci throughout many regions of higher elevation in the western United States. [unreadable] [unreadable] We recently completed our genome sequencing efforts with two relapsing fever spirochetes, B. hermsii and B. turicatae, as part of our comparative studies of Lyme disease and relapsing fever spirochetes (manuscript in preparation). The isolates chosen for this effort included B. hermsii DAH that originated from a human case of relapsing fever in Washington, and B. turicatae 91E135 that originated from an Ornithodoros turicata tick in Texas.[unreadable] [unreadable] These spirochetes have the most complex genomes known among the prokaryotes, in that they possess numerous linear and circular plasmids and a linear chromosome slightly less than 1 megabase in size. The low passage, infectious B. burgdorferi B31 has 21 plasmids (12 linear and 9 circular). Our data show the two relapsing fever spirochetes contain 12 - 13 plasmids, including both linear and circular forms. Nearly all of the plasmid-associated genes in B. hermsii and B. turicatae encode either variable major proteins, borrelia direct repeat proteins, or hypothetical proteins that do not match anything in the databases. The linear chromosomes in the three species are very similar in size, content and gene organization. The chromosomes of B. hermsii and B. turicatae are nearly identical to each other and are slightly larger than the B. burgdorferi chromosome. The chromosome of B. burgdorferi encodes 32 to 35 more ORFs than we identified in the chromosome of the two relapsing fever spirochetes. However, most of these ORFs restricted to B. burgdorferi encode hypothetical proteins of 30 amino acids or less, and match nothing in the database.[unreadable] [unreadable] B. hermsii and B. turicatae lack genes in pathways for the synthesis of amino acids, fatty acids, enzyme cofactors and nucleotides. There are 800-plus orthologous genes found on the chromosomes of all three species. There are 29 genes present on the chromosome of the relapsing fever spirochetes that are absent from the genome of B. burgdorferi. A few topics we have pursued with the genomic studies are discussed below.[unreadable] [unreadable] B. hermsii and B. turicatae contain chromosomal ORFs designated bhpA and btpA respectively, which are absent from the genome of B. burgdorferi. These ORFs encode a protein of 62 kDa that contain a predicted signal peptide comprised of the first 17 amino acids. The BhpA amino acid sequence contains a conserved trypsin domain (AA 188 to 333) with a putative His204-Asp244-Ser315 catalytic triad, characteristic of trypsin-like serine proteases. Zymograms with casein showed a single clear band with an apparent molecular mass of 60 kDa with B. hermsii but not B. burgdorferi, suggesting that only the B. hermsii lysate had caseinolytic activity. We speculated that this activity in B. hermsii might be related to the proteolytic activity of BhpA, since casein is usually a generic substrate for such serine proteases. To verify this idea, the synthesis of BhpA by B. hermsii was examined by immunoblot analysis with anti-recombinant BhpA polyclonal antibodies. The immune serum reacted with a 60 kDa protein, suggesting that B. hermsii produced BhpA in vitro. The band recognized by immune serum had the same apparent size of the protein with the unique caseinolytic activity, suggesting they were associated. BhpA antiserum failed to detect the protein in B. burgdorferi, which supports the genome sequence data and demonstrates that the Lyme disease spirochetes do not encode a BhpA ortholog. [unreadable] [unreadable] Immunoblot analysis with polyclonal anti-BhpA antibodies recognized a 60 kDa protein in B. hermsii grown at various temperatures. BhpA antiserum also reacted with a 60 kDa protein in spirochetes collected from the blood of infected mice. Caseinolytic activity was detected with spirochetes grown at various temperatures, and from borreliae collected from mice. These findings suggest that B. hermsii BhpA was produced and active in B. hermsii grown in vitro and during mammalian infection.[unreadable] [unreadable] All attempts to genetically inactivate the B. hermsii bhpA failed. Therefore, to study the possible function of bhpA, we used B. burgdorferi and the shuttle vector pBSV2 to heterologously express the B. hermsii bhpA. Transcripts of bhpA were detected in B. burgdorferi pBSV2+bhpA, but not in B. burgdorferi with only the shuttle vector. Immunoblot analysis demonstrated the synthesis of BhpA by B. burgdorferi transformed with pBSV2+bhpA but not by the spirochetes that contained only the shuttle vector. We also compared B. burgdorferi pBSV2+bhpA and B. burgdorferi pBSV2 for their resistance to oxidative stress. Spirochetes producing BhpA were significantly more resistant to the oxidizing agents diamide and t-butyl peroxide than were spirochetes containing only the shuttle vector. The increased resistance of B. burgdorferi pBSV2+bhpA to oxidative stress suggested that BhpA might protect B. hermsii from the host's innate immune system. To test this hypothesis, we performed in vitro killing assays with human PMNs and compared the survival of B. burgdorferi pBSV2+ bhpA and B. burgdorferi pBSV2 only. There was significantly more survival of B. burgdorferi expressing BhpA compared with B. burgdorferi containing only the shuttle vector. Therefore, heterologous expression of bhpA protected B. burgdorferi against killing by PMNs, possibly through the increased recycling of altered and denatured periplasmic proteins. Therefore, we propose that bhpA encodes a unique and functional serine protease in relapsing fever spirochetes. This periplasmic enzyme may prevent the accumulation of proteins damaged by the innate immune response and contribute to the ability of the relapsing fever spirochetes to achieve high cell densities in blood.[unreadable] [unreadable] Other investigators recently typed spirochetes based on the intergenic spacer (IGS) region of noncoding DNA located between the 16S rRNA and ileT tRNA genes. We examined the IGS locus in 37 isolates of B. hermsii and compared the results to those we obtained by multilocus sequence typing, The IGS sequences separated B. hermsii into two genomic groups (GGI and GGII). The B. hermsii isolates that originated from five patients infected in two nearby cabins on Wild Horse Island, Flathead Lake, Montana in 2002 and 2004 were especially interesting. Both IGS and multilocus sequence analysis typed LAK-4 in GGI and LAK-1, -2, -3 and -5 in GGII. The three patients infected in 2004 slept in the same bed: one child slept with an adult the first night; another child slept with the same adult the next two nights. From these patients we isolated LAK-4 from the adult and LAK-3 and LAK-5 from the two children. Therefore, spirochetes in both genomic groups were isolated from different individuals that slept in the same bed. Among the four GGII isolates, LAK-1 and LAK-2 originated from one cabin and had identical 16S rRNA, flaB, gyrB, glpQ, and IGS sequences. LAK-3 and LAK-5 came from the other cabin and these two isolates also had identical sequences for the five loci examined. However, LAK-1 and LAK-2 contained flaB, glpQ and IGS sequences that each varied by one base when compared to LAK-3 and LAK-5 sequences. The presence of three flaB, glpQ and IGS sequences among the five isolates suggests multiple past introductions of B. hermsii to the island, and that birds might play a role in dispersing these spirochetes in nature. For this to occur, birds must be suitable hosts for O. hermsi. Work is in progress to evaluate the competence of birds as hosts.