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Infect Immun, August 1998, p. 3711-3718, Vol. 66, No. 8
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Major Antigenic Proteins of the Agent of Human Granulocytic Ehrlichiosis Are Encoded by Members of a Multigene Family

Cheryl I. Murphy,1 * James R. Storey,1 Joanne Recchia,1 Linda A. Doros-Richert,1 Cindy Gingrich-Baker,1 Kenneth Munroe,1 Johan S. Bakken,2 Richard T. Coughlin,1 and Gerald A. Beltz1

Aquila Biopharmaceuticals, Inc., Worcester, Massachusetts 01605,1 and Section of Infectious Diseases, The Duluth Clinic, Duluth, Minnesota 558052

Received 23 October 1997/Returned for modification 3 December 1997/Accepted 28 May 1998

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.

* Corresponding author. Mailing address: Aquila Biopharmaceuticals, 365 Plantation St., Worcester, MA 01605. Phone: (508) 797-5777, ext. 188. Fax: (508) 797-4014. E-mail: molbio{at}splusnet.com.

Infect Immun, August 1998, p. 3711-3718, Vol. 66, No. 8
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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