Because the anti-VirB7 antibody did not consistently bind to a native protein of the predicted molecular weight, we focused on using antibodies to VirB9-1, VirB9-2, and VirB10 to identify interacting native protein partners using immunoprecipitation of solubilized OM (Fig. restriction fragment length polymorphism types 8/23, 3/16, and 16/27 lacked T-cell responses to VirB9-1, VirB9-1 and VirB9-2, or VirB9-2 and VirB10, respectively. For SB-423562 these animals, VirB9-1-, VirB9-2-, and VirB10-specific IgG production may be associated with T-cell help provided by responses to an interacting protein partner(s). Interacting protein partners indicated by far-Western blotting were confirmed by immunoprecipitation assays and revealed, for the first time, specific interactions of VirB9-1 with VirB9-2 and VirB10. The immunogenicity and interactions of VirB9-1, VirB9-2, and VirB10 justify their testing as a linked protein vaccine against is an obligate intraerythrocytic Gram-negative bacterium causing bovine anaplasmosis. Immunization of cattle with outer membranes (OM) and cross-linked OM proteins can induce complete protection against disease and contamination (10, 43, 57). However, the well-characterized immunodominant major surface proteins 1 to 5 (MSP1 to MSP5), which include antigenically variant MSP2 and MSP3, do not induce equivalent protection against contamination (1, 46, 47, 49, 50). Because immunodominant MSPs are not protective, we have focused on identifying subdominant and conserved surface proteins, including those that associate within the membrane that may be important components of the protective OM vaccine. Among the subdominant antigens identified in the OM are type IV secretion system (T4SS) proteins. Several T4SS proteins induced CD4 T cell responses, including gamma interferon (IFN-) production, and were recognized by IgG2 in cattle immunized with the OM vaccine (32C34, 56). SB-423562 These type 1 immune responses are associated with protection against contamination in cattle immunized with OM (9, 10, 50). HMOX1 The T4SS is usually a 1.1-MDa protein complex that spans the outer and inner bacterial membranes and has been most widely studied in (13, 17). Typically, the T4SS is made up of 12 interacting VirB/D membrane proteins, several of which are likely to be surface exposed (14) and are thus targets for neutralizing and protective immune responses. The T4SS core complex is made up of 14 copies of interacting VirB7, VirB9, and VirB10, which form a complex that was crystallized and studied with VirB homologues TraN (VirB7), TraO (VirB9), and TraF (VirB10) (13, 17). The VirB proteins also assemble into a pilus made up of VirB2, VirB5, and VirB7 (28, 29, 53, 54). There are three nucleotide triphosphate (NTP)-utilizing T4SS proteinsVirB4, VirB11, and VirD4that supply energy for substrate translocation, and VirD4 specifically acts as the substrate-coupling protein (3, 11). VirB6 and VirB8 assemble the T4SS apparatus SB-423562 at the cell pole and also make up the core complex (24, 25, 27). The T4SS proteins harbored by and other members of the family are unique because they are encoded by two copies of and have not been identified (7, 19, 20). The function of the T4SS in has not been determined, but in a pathogen that has undergone reductive evolution, retention of these genes indicates their requirement for invasion and survival within erythrocytes and/or tick cells (19, 20). Because lack lipopolysaccharides (7, 20), several T4SS proteins may be surface uncovered where they could be targeted by neutralizing antibody. Furthermore, T4SS proteins interact within the bacterial membrane so associated proteins could provide linked recognition for T-cellCB-cell interactions. Linked recognition was described in the 1970s by Mitchison, who revealed that both T cells and B cells must recognize antigenic determinants on the same molecule for B-cell activation to occur (39C41). T cells help B cells to undergo somatic hypermutation and isotype switching through cognate conversation and cytokine secretion. However, linked recognition can also occur with two.