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) of the human granulocytic ehrlichiosis (HGE) agent has identified a number 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 used to amplify DNA from USG3. Sequencing of a 550-bp PCR product revealed that it encodes a protein homologous to the MSP-2 proteins of Anaplasma marginale. Concurrently, an expression library made from USG3 genomic 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- and carboxy-terminal regions which flank a variable region. The conserved 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 proteins. Recombinant MSP-2B protein and an MSP-2A fusion protein were expressed in Escherichia coli and reacted with human sera positive for the HGE 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.
Murphy
C I
CI
Aquila Biopharmaceuticals, Inc., Worcester, Massachusetts 01605, USA.
Storey
J R
JR
Recchia
J
J
Doros-Richert
L A
LA
Gingrich-Baker
C
C
Munroe
K
K
Bakken
J S
JS
Coughlin
R T
RT
Beltz
G A
GA
D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D004269:DNA, Bacterial; D016208:Databases, Factual; D018469:Ehrlichia chaffeensis / Q000276:immunology*; D016873:Ehrlichiosis / Q000097:blood / Q000382:microbiology*; D006041:Goats; D006098:Granulocytes / Q000382:microbiology*; D006801:Humans; D008969:Molecular Sequence Data; D005810:Multigene Family; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; C103348:msp2 protein, Anaplasma marginale