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

Abstract

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 inEscherichia 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.

Emergence of Anaplasma marginale Antigenic Variants during Persistent Rickettsemia

“…The semivariable, N-terminal, and C-terminal variable regions defined by analysis of genes encoding MSP2 homologues in the genogroup I ehrlichiae (E. chaffeensis, E. canis, and C. ruminantium) were not detected in any of the A. marginale MSP2 variants. In contrast, these regions were all highly conserved among the expressed A. marginale MSP2 variants, as well as among the proteins encoded by the previously reported msp2 genes that are polymorphic within and between HGE strains (8,13,28). Our analysis excluding the central region that is hypervariable within each genogroup II species revealed a much higher similarity between A. marginale and HGE MSP2 than previously reported.…”

“…The MSP2 homologues have been defined within genogroup I and II ehrlichiae based on overall nucleotide and amino acid similarity. The amino acid similarity between the MSP2 homologues of A. marginale and HGE has been reported as approximately 60% (8,13,28). However, this similarity is skewed by the presence of the hypervariable regions.…”

Invasion and survival strategies ofAnaplasma phagocytophilum

“…Studies by Park et al . (2003) suggest that a paralogue of the P44 (MSP2) family of outer surface proteins (Ijdo et al ., 1998; Murphy et al ., 1998; Zhi et al ., 1998) may facilitate A. phagocytophilum binding to human PSGL‐1. A. phagocytophilum usage of PSGL‐1 and α1,3‐fucosylated, α2,3‐sialylated glycans as a receptor‐mediated pathway for cellular adhesion and entry explains not only its tropism for neutrophils but also one means by which it evades destruction by the normal neutrophil phagocytic pathway.…”

Outer Membrane Protein-Specific Monoclonal Antibodies Protect SCID Mice from Fatal Infection by the Obligate Intracellular Bacterial Pathogen Ehrlichia chaffeensis

“…Thus, the OMPs are major targets of the protective immune response in the mouse, and are also likely to be important in human immunity (26). The OMPs in E. chaffeensis and related ehrlichiae are encoded by multigene families (10,21,22,(27)(28)(29)(30), so it is likely that variation in the expression of different OMPs contribute to immune evasion in the natural host for E. chaffeensis, the white tailed deer (31). Indeed, cyclic rickettsemia has been reported to occur in cattle infected with the related erythrocyte tropic ehrlichial pathogen Anaplasma marginale (32,33).…”