MAJOR ANTIGENIC PROTEINS OF THE AGENT OF HUMAN GRANULOCYTIC EHRLICHIOSIS ARE ENCODED BY MEMBERS OF A MULTIGENE FAMILY

Western blot analysis of proteins from a cell culture isolate (USG3) o f the human granulocytic ehrlichiosis (HGE) agent has identified a num ber of immunoreactive proteins, including major antigenic proteins of 43 and 45 kDa, Peptides derived from the 43- and 45-kDa proteins were sequenced, and degenerate PCR primers based on these sequences were us ed to amplify DNA from USG3, Sequencing of a 550-bp PCR product reveal ed that it encodes a protein homologous to the MSP-2 proteins of Anapl asma marginale, Concurrently, an expression library made from USG3 gen omic DNA was screened with granulocytic Ehrlichia (GE)-positive immune sera. Analysis of two clones showed that they contain one partial and three full-length highly related genes, suggesting that they are part of a multigene family. Amino acid alignment showed conserved amino- a nd carboxyterminal regions which flank a variable region. The conserve d regions of these proteins are also homologous to the MSP-2 proteins of A. marginale; thus, they were designated GE MSP-2A (45 kDa), MSP-2B (34 kDa), and MSP-2C (38 kDa). The PCR fragment obtained as a result of peptide sequencing was completely contained within the msp-2A clone , and all of the sequenced peptides were found in the GE MSP-2 protein s. Recombinant MSP-2B protein and an MSP-2A fusion protein were expres sed in Escherichia coli and reacted with human sera positive for the H GE agent by immunofluorescence assay. These data suggest that the 43- and 45-kDa proteins of the HGE agent are encoded by members of the GE MSP-2 multigene family.