To this end, we infected five SCID mice with 50 cells each on day 0
To this end, we infected five SCID mice with 50 cells each on day 0. orthologous to BHA007 were found on large plasmids of other RF species, including the coding sequences for the CihC proteins of and species. Recombinant BHA007 bound both human and bovine fibronectin with (dissociation constant) values of 22 and 33 nM, respectively, and bound to C4-binding protein with less affinity. The distant homology of BHA007 and its orthologs to BBK32 proteins of Lyme disease species, as well as to previously described BBK32-like proteins in relapsing fever species, indicates that BHA007 is usually a member of a large family of multifunctional proteins in species that bind to fibronectin as well as other host proteins. INTRODUCTION Relapsing fever (RF) is an arthropod-borne disease that is caused by several species of the spirochete genus (1). It is found in every geographic region except Antarctica and Oceania. At present, most cases of human RF in the world occur in sub-Saharan Africa, where it is underdiagnosed and often misdiagnosed as malaria (2, 3). It is a neglected tropical disease and affects predominantly rural poor and disadvantaged urban populations in Africa (3,C5). The etiologic brokers in Africa are the tick-borne species and and the sole species transmitted by lice, and (8). Comparatively little is known about the surface proteins expressed by RF spirochetes during a mammalian contamination. Besides the variable membrane proteins Vsp and Vlp, CGP 57380 which are the basis of antigen variation during relapsing fever (9), as well as various subsurface structural proteins and enzymes common to many bacteria (10), proteins identified as being expressed during experimental or clinical relapsing fever include glycerophosphodiester phosphodiesterase (GlpQ) (11), factor H-binding protein A (FhbA) (12), and immunogenic protein (BipA) (13). Alp is usually another protein that has been identified as being expressed in mice, although it is usually expressed mainly in ticks (14). Our objective was to identify additional proteins expressed by RF spirochetes in the blood of a mammalian host using a mouse model of contamination. For a set of CGP 57380 candidates of a manageable number and that might be enriched for outer membrane-associated proteins, we used CGP 57380 an approach that was previously used to identify circulating antigens of the bacterial pathogens and in the serum of infected mice (15), namely, by immunizing immunocompetent mice with a cell-free filtrate of blood from immunodeficient mice bacteremic with spp. We further characterized EIF4EBP1 this protein with regard to surface exposure, immunogenicity, the elicitation of protective immunity, and binding activities for selected host proteins. (This material was presented in part at the American Association for the Advancement of Science annual meeting, San Diego, CA, CGP 57380 18 to 22 February 2010. ) MATERIALS AND METHODS Organisms and cultivation. strains HS1 from Washington, CC1 from California, and LPO from Idaho (14); strain HR1 from California; strain 91E135 from Texas (16); and strain B31 (ATCC 35210) were used. HS1 serotype 7 expresses Vlp7, and serotype 33 expresses Vtp (formerly Vsp33). A spontaneous Vtp? mutant of strain HS was previously described (14). Spirochetes were cultivated at 34C in Barbour-Stoenner-Kelly II (BSK II) medium (17). Bacterial stocks in BSK II medium or infected mouse plasma were kept frozen at ?80C in 10% dimethyl sulfoxide. Cells were counted in a Petroff-Hausser chamber by phase-contrast microscopy. Spirochetes were harvested by centrifugation at 9,500 for 20 min and washed twice with phosphate-buffered saline (PBS)C5 mM MgCl2 at pH 7.4 (PBS-Mg). strains TOP10, BL21(DE3), and BL21(DE3)/pLysS were cultivated with Luria-Bertani (LB) broth or solid medium made up of 50 g/ml kanamycin or ampicillin at 37C. Generation of the Vtp? knockout mutant. The construction of the inactivation plasmid pOKvtpKO (GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”EF488747″,”term_id”:”154223484″,”term_text”:”EF488747″EF488747), the preparation of electrocompetent HS1 serotype 33 cells, and the transformation of these cells with pOKvtpKO were carried out according to a procedure described previously by Battisti et al. (18). The insertion of the gene was confirmed by PCR with DNA polymerase (New England BioLabs, Inc., Ipswich, MA) and primer sets A, B, and C. Conditions for PCR were 5 min of activation at 95C followed by 30 cycles of 30 s at 95C, 60 s at 60C, and 2.5 min at 72C and then a final extension step at 72C for 7 min, with the exceptions that annealing temperatures were 56C and 63C for primer sets B and C, respectively. Set A primers (5-ATGAAGAAGAATACATTAAGTGCGA-3 and 5-TTAACAGGGGTCGCTGG-3) amplified a 623-bp product if was intact or a 1,237-bp product if was insertionally disrupted. Set.