Expression of multiple outer membrane protein sequence variants from a single genomic locus of Anaplasma phagocytophilum.

Anaplasma phagocytophilum is the causative agent of an emerging tick-borne zoonosis in the United States and Europe. The organism causes a febrile illness accompanied by other nonspecific symptoms and can be fatal, especially if treatment is delayed. Persistence of A. phagocytophilum within mammalian reservoir hosts is important for ensuring continued disease transmission. In the related organism Anaplasma marginale, persistence is associated with antigenic variation of the immunoprotective outer membrane protein MSP2. Extensive diversity of MSP2 is achieved by combinatorial gene conversion of a genomic expression site by truncated pseudogenes. The major outer membrane protein of A. phagocytophilum, MSP2(P44), is homologous to MSP2 of A. marginale, has a similar organization of conserved and variable regions, and is also encoded by a multigene family containing some truncated gene copies. This suggests that the two organisms could use similar mechanisms to generate diversity in outer membrane proteins from their small genomes. We define here a genomic expression site for MSP2(P44) in A. phagocytophilum. As in A. marginale, the msp2(p44) gene in this expression site is polymorphic in all populations of organisms we have examined, whether organisms are obtained from in vitro culture in human HL-60 cells, from culture in the tick cell line ISE6, or from infected human blood. Changes in culture conditions were found to favor the growth and predominance of certain msp2(p44) variants. Insertions, deletions, and substitutions in the region of the genomic expression site encoding the central hypervariable region matched sequence polymorphisms in msp2(p44) mRNA. These data suggest that, similarly to A. marginale, A. phagocytophilum uses combinatorial mechanisms to generate a large array of outer membrane protein variants. Such gene polymorphism has profound implications for the design of vaccines, diagnostic tests, and therapy.

Recombinant major antigenic protein 2 of Ehrlichia canis: a potential diagnostic tool.

The major antigenic protein 2 (MAP2) of Ehrlichia canis was cloned and expressed. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of canine monocytic ehrlichiosis. The recombinant protein, which contained a C-terminal polyhistidine tag, had a molecular mass of approximately 26 kDa. The antigen was clearly identified by Western immunoblotting using antihistidine antibody and immune serum from an experimentally infected dog. The recombinant MAP2 (rMAP2) was tested in an ELISA format using 141 serum samples from E. canis immunofluorescent antibody (IFA)-positive and IFA-negative dogs. Fifty-five of the serum samples were from dogs experimentally or naturally infected with E. canis and were previously demonstrated to contain antibodies reactive with E. canis by indirect immunofluorescence assays. The remaining 86 samples, 33 of which were from dogs infected with microorganisms other than E. canis, were seronegative. All of the samples from experimentally infected animals and 36 of the 37 samples from naturally infected animals were found to contain antibodies against rMAP2 of E. canis in the ELISA. Only 3 of 53 IFA-negative samples tested positive on the rMAP2 ELISA. There was 100% agreement among IFA-positive samples from experimentally infected animals, 97.3% agreement among IFA-positive samples from naturally infected animals, and 94.3% agreement among IFA-negative samples, resulting in a 97.2% overall agreement between the two assays. These data suggest that rMAP2 of E. canis could be used as a recombinant test antigen for the serodiagnosis of canine monocytic ehrlichiosis.

Expression of a gene encoding the major antigenic protein 2 homolog of ehrlichia chaffeensis and potential application for serodiagnosis.

The major antigenic protein 2 (MAP2) homolog of Ehrlichia chaffeensis was cloned and expressed. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of human monocytic ehrlichiosis. The recombinant protein, which contained a C-terminal polyhistidine tag, had a molecular mass of approximately 26 kDa. The antigen was clearly identified by Western immunoblotting using antihistidine antibody. However, immune sera failed to react with the recombinant on immunoblots when the antigen was denatured by heat or reduced using beta-mercaptoethanol. The recombinant MAP2 (rMAP2) was used in an ELISA format with 60 blinded serum samples. Twenty of the serum samples were previously demonstrated to contain antibodies reactive with E. chaffeensis by indirect immunofluorescence assays (IFAs). The remaining 40 samples were seronegative. All samples negative by IFA were also found to be negative for antibodies against the rMAP2 of E. chaffeensis by using the ELISA. Only 1 of 20 IFA-positive samples tested negative in the rMAP2 ELISA. There was 100% agreement using IFA-negative samples and 95% agreement using IFA-positive samples, resulting in a 97.5% overall agreement between the two assays. These data suggest that the rMAP2 homolog of E. chaffeensis may have potential as a test antigen for the serodiagnosis of human monocytic ehrlichiosis. To our knowledge, this recombinant is unique because it is thus far the only E. chaffeensis recombinant antigen that has been shown to work in an ELISA format.

Sequence and expression analysis of a surface antigen gene family of the rickettsia Anaplasma marginale.

The tick-borne rickettsial organism, Anaplasma marginale, causes a disease in cattle of world-wide economic significance. This disease, anaplasmosis, is characterized by severe hemolytic anemia, high levels of rickettsemia and, often, death in animals over 3years of age. Animals that survive acute infection remain carriers, with continuous sub-microscopic cycles of rickettsemia that can persist for the lifetime of the animal. In the search for potential recombinant immunogens, it was discovered that several surface proteins of A. marginale encode polymorphic multigene families. Despite the small size of the genome (approx. 1250kb), these surface antigen gene families comprise greater than 2% of the genome. We present here a mapping, sequencing and expression analysis of five complete or partial genes encoding MSP1b in a Florida strain of A. marginale. Two genes are complete; they encode mRNA that is translated into polypeptide products. Three genes are incomplete and appear to be derived from the complete genes by a series of segmental intragenic recombinations. In two of the incomplete genes, 5' sequence in the incomplete genes is 3' sequence in the complete genes. Recombination within these gene families may generate diversity in surface antigens through combinatorial rearrangements. This could contribute to persistence in the chronic infections caused by A. marginale and related rickettsiae.

Potential value of major antigenic protein 2 for serological diagnosis of heartwater and related ehrlichial infections.

Cowdria ruminantium is the etiologic agent of heartwater, a disease causing major economic loss in ruminants in sub-Saharan Africa and the Caribbean. Development of a serodiagnostic test is essential for determining the carrier status of animals from regions where heartwater is endemic, but most available tests give false-positive reactions with sera against related Erhlichia species. Current approaches rely on molecular methods to define proteins and epitopes that may allow specific diagnosis. Two major antigenic proteins (MAPs), MAP1 and MAP2, have been examined for their use as antigens in the serodiagnosis of heartwater. The objectives of this study were (i) to determine if MAP2 is conserved among five geographically divergent strains of C. ruminantium and (ii) to determine if MAP2 homologs are present in Ehrlichia canis, the causative agent of canine ehrlichiosis, and Ehrlichia chaffeensis, the organism responsible for human monocytic ehrlichiosis. These two agents are closely related to C. ruminantium. The map2 gene from four strains of C. ruminantium was cloned, sequenced, and compared with the previously reported map2 gene from the Crystal Springs strain. Only 10 nucleic acid differences between the strains were identified, and they translate to only 3 amino acid changes, indicating that MAP2 is highly conserved. Genes encoding MAP2 homologs from E. canis and E. chaffeensis also were cloned and sequenced. Amino acid analysis of MAP2 homologs of E. chaffeensis and E. canis with MAP2 of C. ruminantium revealed 83.4 and 84.4% identities, respectively. Further analysis of MAP2 and its homologs revealed that the whole protein lacks specificity for heartwater diagnosis. The development of epitope-specific assays using this sequence information may produce diagnostic tests suitable for C. ruminantium and also other related rickettsiae.

Molecular cloning of a gene encoding the immunogenic 21 kDa protein of Cowdria ruminantium.

Major immunogenic polypeptides (21, 32, 40, 46, 58, 85 and 160 kDa) of Cowdria ruminantium were identified by immunoprecipitation and immunoblotting. A pUC13 library of C. ruminantium genomic DNA was screened with hyperimmune sheep serum to identify Escherichia coli colonies which expressed genes encoding these immunogenic proteins. A recombinant E. coli colony, F5.2, was identified containing plasmid insert DNA of 2773 bp. The cloned DNA insert contained two long open reading frames (ORFs) of 627 bp (complete) and 831 bp (incomplete), both potentially encoding proteins containing an N-terminal signal peptide. Deletion experiments suggested that the hyperimmune sheep serum recognized a protein that was encoded by the 627 bp ORF. The 627 bp ORF was amplified by polymerase chain reaction (PCR), subcloned and expressed to a high level in E. coli. A sheep antiserum made to the expressed recombinant fusion protein recognized a 21 kDa protein of all strains of C. ruminantium tested, confirming that the 627 bp ORF encodes a native 21 kDa protein in C. ruminantium. Similarly, the recombinant protein was recognized by all sera tested from heartwater-infected animals. The antigenic conservation of the 21 kDa protein and its immunogenic nature are reasons for further testing of this recombinant protein in subunit diagnostic tests.