Immunodominant Major Outer Membrane Proteins of Ehrlichia chaffeensis Are Encoded by a Polymorphic Multigene Family

Several immunodominant major proteins ranging from 23 to 30 kDa were identified in the outer membrane fractions of Ehrlichia chaffeensis and Ehrlichia canis. The N-terminal amino acid sequence of a 28-kDa protein of E. chaffeensis (one of the major proteins) was determined. The gene (p28), almost full length, encoding the 28-kDa protein was cloned by PCR with primers designed based on the N-terminal sequence of the E. chaffeensis 28-kDa protein and the consensus sequence between the C termini of the Cowdria ruminantium MAP-1 and Anaplasma marginale MSP-4 proteins. The p28 gene was overexpressed, and antibody to the recombinant protein was raised in a rabbit. The antibody and serum from a patient infected with E. chaffeensis reacted with the recombinant protein, three proteins (29, 28, and 25 kDa) of E. chaffeensis, and a 30-kDa protein of E. canis. Immunoelectron microscopy with the rabbit antibody revealed that the antigenic epitope of the 28-kDa protein was exposed on the surface of E. chaffeensis. Southern blot analysis with a ³²P-labeled p28 gene probe revealed multiple copies of genes homologous to p28 in the E. chaffeensis genome. Six copies of the p28 gene were cloned and sequenced from the genomic DNA by using the same probe. The open reading frames of these gene copies were tandemly arranged with intergenic spaces. They were nonidentical genes and contained a semivariable region and three hypervariable regions in the predicted protein molecules. One of the gene copies encoded a protein with an internal amino acid sequence identical to the chemically determined N-terminal amino acid sequence of a 23-kDa protein of E. chaffeensis. Immunization with the recombinant P28 protein protected mice from infection with E. chaffeensis. These findings suggest that the 30-kDa-range proteins of E. chaffeensis represent a family of antigenically related homologous proteins encoded by a single gene family.

Ehrlichia chaffeensis, which causes human monocytic ehrlichiosis, is an obligate intracellular bacterium of monocytes and macrophages and belongs to the family Rickettsiaceae. Human ehrlichiosis is a tick-borne illness and was first reported in 1987 in the United States (21). Most patients have fever, chills, headache, arthralgia, myalgia, and hematologic abnormalities, including thrombocytopenia and leukopenia. Elevation of liver enzymes occurs in most patients. Since 1987, over 400 cases of human ehrlichiosis, detected primarily by serological means, have been reported in 30 states (3, 14, 16).Recently, several protein antigens of E. chaffeensis were identified by Western blot analysis with naturally infected human sera, experimentally inoculated dog sera, or monoclonal antibodies (7–10, 13, 30, 35, 40–42). Two of these antigens, namely, a heat shock protein (HSP) 60 homolog (35) and a 120-kDa protein (41, 42), have been cloned, sequenced, and expressed. Two E. chaffeensis proteins ranging from 28 to 30 kDa were shown to be dominant antigens and were cross-reactive between two Ehrlichia spp.: E. chaffeensis and E. canis (7, 30). Studies with monoclonal antibodies (MAbs) against E. chaffeensis showed that two or three proteins of from 22 to 30 kDa react with three MAbs by Western blotting and that these antigens are exposed on the surface of the organism as determined by immunogold labeling of negatively staining ehrlichiae (8–10, 40). However, why multiple proteins of different molecular sizes react with the MAbs has not been answered. These E. chaffeensis antigens in the 30-kDa range have not been examined at the molecular level.In this study, we demonstrated that a potentially immunoprotective 28-kDa protein (designated P28) located on the E. chaffeensis surface and antigenically cross-reactive proteins in the 30-kDa range are encoded by a multigene family.