The antigenically variant major surface protein 2 (MSP2) of is expressed from a 3. infected mammalian host, the infected tick cell line, or within infected ticks. The differential expression of outer membrane proteins from within an operon is usually a novel obtaining in tick-transmitted bacteria, and the regulation of expression may be broadly applicable to understanding how the pathogen adapts ABT-869 novel inhibtior to the mammalian host-tick vector transition. Pathogens in the genera and establish persistent contamination in the mammalian reservoir host that provides a constant source of organisms for tick transmission. Persistent infection within the bovine reservoir host is characterized by rickettsemic cycles, ranging from 102 to 107 bacteria/ml of blood (14). Each rickettsemic cycle is associated with the emergence of new antigenic variants of the immunodominant major surface protein 2 (MSP2) (13). Variation is achieved by gene conversion of the single, full-length expressed copy of utilizing oligonucleotide blocks of at least nine truncated pseudogenes (6, 7). This combinatorial mechanism provides the requisite number of expressed MSP2 variants sufficient for lifelong persistence (7). Whereas pseudogenes are widely dispersed over the bacterial chromosome, the single, full-length copy of is located within an operon made up of three additional open reading frames upstream of (4, 6, 24)). Analysis with the PSORT algorithm predicts that these open reading frames encode outer membrane proteins (4), as predicted and experimentally verified for MSP2 (24). The operon-associated gene 1 (does not resemble any sequences in the databases, whereas predicted proteins encoded by the other two open reading frames, and and (4, 17, 23). The operon linked expression of the three putative outer membrane proteins with MSP2 would be consistent with the functional and structural relatedness of proteins expressed from bacterial operons. The operon is the only source for transcription of full-length and, correspondingly, and the Reynolds Creek strain of expression site in all biologically important stages of However, it remains unknown whether the other open reading frames of the polycistronic operon messageoperon-associated genes are expressed on the bacterial surface, the degree of conservation among strains emerges as an important question. Immunization with purified outer membranes induces protection from disease upon challenge with (9, 32). Notably, outer membrane proteins conserved among strains are targets for CD4+ T lymphocytes derived from protected vaccinates (10). As a result, identification of highly conserved outer membrane proteins is a priority for vaccine development. In the research presented here, expression of the operon-associated genes was examined in mammalian and tick stages of operon-associated genes was analyzed by using genotypically and phenotypically distinct strains. MATERIALS AND METHODS Expression of operon-associated genes. (i) Expression of recombinant proteins. To obtain antigen to be used as positive controls in Western blot analysis and for generation of specific antibody in mice, OpAGs were expressed as His-tagged fusion proteins in by using the pET19b expression system (Novagen). Inserts containing the full-length genes of OpAG1, OpAG2, and OpAG3 of the South Idaho strain of were generated by subcloning from full-length clones obtained in pCR4Blunt-TOPO (Invitrogen), as previously reported (4). operon of the Florida strain (4). Numbering of the primers with the designation Ao is based on the nucleotide sequence of the operon of (GenBank accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”AY132309″,”term_id”:”23168707″,”term_text”:”AY132309″AY132309). cAs published by French et al. (14); numbering is based on pCKR11.2 (24). dUnderlined sequences indicate the OpAG1, -2, and -3 sera. Specific antisera against the predicted proteins encoded by the operon-associated genes were generated in BALB/c mice by immunizations with synthetic peptides or recombinantly expressed OpAG1 (see above). The designations and sequences were as follows: OpAG1-1 (CPSLLRVGGEASGQQ), OpAG1-2 (CSGGGFHAAGGASATR), OpAG2 (CREFAVRENRLTAPSK), OpAG3-1 (CPTRDGFGAHYLPKYENS), and OpAG3-2 (CGTDTEEHAGGGPTLLSTTSSGVPSVDAD). An amino-terminal cysteine was included for conjugation, and 2 ABT-869 novel inhibtior mg of each peptide Rabbit polyclonal to IkB-alpha.NFKB1 (MIM 164011) or NFKB2 (MIM 164012) is bound to REL (MIM 164910), RELA (MIM 164014), or RELB (MIM 604758) to form the NFKB complex.The NFKB complex is inhibited by I-kappa-B proteins (NFKBIA or NFKBIB, MIM 604495), which inactivate NF-kappa-B by trapping it in the cytoplasm. was ABT-869 novel inhibtior cross-linked to maleimide-activated keyhole limpet hemocyanin by using the Imject maleimide activated immunogen conjugation kit (Pierce) according to the manufacturer’s recommendations. Each conjugated peptide or the recombinant OpAG1 was used to immunize three mice subcutaneously with 100 g of antigen emulsified with complete Freunds adjuvant. The mice were boosted by using four immunizations of 100 g of antigen in incomplete Freunds adjuvant. (iii) Western blots. The expression of OpAG1, OpAG2, and OpAG3 in biologically important stages of was examined by Western blot analyses. Expression during mammalian infection was determined with blood obtained from cattle during the acute peak rickettsemia after inoculation with the Florida, South Idaho, Washington-Clarkston, Washington-Okanogan, or Virginia strains of The origin and characterization of the strains have been previously reported (1, 18, 26). To examine expression.