Progress 01/01/00 to 12/31/04
Outputs
Anaplasma marginale maintains two separate systems to generate antigenic variation, MSP2 and MSP3. Each system is characterized by a single expression site encoding a polycistronic mRNA and functional pseudogenes that can recombine into that expression site. We have compared these systems with related Anaplasma and Ehrlichia organisms. In both Ehrlichia and Anaplasma phagocytophilum there are orthologous genes to MSP2 encoding outer membrane proteins. In Anaplasma phagocytophilum there is also an expression site that encodes polymorphic MSP2-related outer membrane proteins and a larger number of functional pseudogenes than in A. marginale. Hypervariable region segments of A. phagocytophilum also recombine into the expression site. We have identified and established the function of three promoter elements in the loci encoding major outer membrane protein expression sites in both Anaplasma marginale and Anaplasma phagocytophilum. Gene expression from this locus involves
both classical and atypical polycistronic transcripts. The identified promoter elements have a structure similar to that defined in Escherichia coli and are functional in driving protein expression in a prokaryotic cell-free transcription and translation system and in recombinant E. coli. The two strongest promoters identified in vitro and with recombinant E. coli were also shown to be functional in A. marginale infected cells, as determined by quantification of downstream transcripts. The promoters in both A. marginale and A. phagocytophilum have similar structure and activity, supporting the conclusion that the two loci are syntenic with conservation of function. In addition, they share structural elements within the promoters that appear to be likely sites for regulation. Interestingly, the 5' structure of the expression locus is conserved in the omp1 locus of Ehrlichia chaffeensis and the p30 gene locus of E. canis despite marked divergence between genera in the 3' region of the
loci. This supports the hypothesis that the expression sites of these immunogenic proteins are derived from a common precursor with later divergent evolution along genus lines.
Impacts
These studies show evolution of highly variable genome segments within small-genome pathogens capable of resisting host immune responses. They suggest that vaccines cannot be targeted to the most immunogenic proteins of A. marginale and provide sterile immunity.
Publications
- Lohr CV, Brayton KA, Barbet AF, Palmer GH: Characterization of the Anaplasma marginale msp2 locus and its synteny with the omp1/p30 loci of Ehrlichia chaffeensis and E. canis. Gene 325: 115-121, 2004.
- Barbet AF, Agnes JT, Moreland AL, Lundgren AM, Alleman AR, Noh SM, Brayton KA, Munderloh UG, Palmer GH: Identification of functional promoters in the msp2 expression loci of Anaplasma marginale and Anaplasma phagocytophilum. (submitted for publication), 2005.
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Progress 01/01/03 to 12/31/03
Outputs
Anaplasma marginale establishes persistent infections with sequential cycles of parasitemia in which new antigenic variants emerge. We have demonstrated previously that recombination of portions of msp2 pseudogenes into an msp2 expression site provide for a combinatorial number of possible msp2 variants sufficient for lifelong persistence. However, what is the involvement of other surface proteins of the organism and why could an immune response against these not be protective? A partial answer to this question was provided by studies on msp3. The two most immunodominant proteins of A. marginale are msp2 and msp3, in terms of both antibody and T cell immunoreactivity. We demonstrated that msp2 and msp3 are each encoded by a distinct family of related genes. Each family has maintained a similar mechanism to generate structurally and antigenically polymorphic surface antigens. Like msp2, msp3 is expressed from a single locus in which variation of the expressed msp3 gene
is generated by recombination using msp3 pseudogenes. Each of the msp3 pseudogenes encodes a central variable region and flanking conserved regions. Msp3 expression site variation appears to be generated by gene conversion, as with msp2. Thus, A. marginale maintains two large, separate systems to generate antigenic variation. Analogous structural elements in the msp2 and msp3 suggest a common mechanism. Further studies showed that there was simultaneous variation of both msp2 and msp3 followed by clearance and emergence of new variants during A. marginale persistence in cattle.
Impacts
These studies show evolution of highly variable genome segments within small-genome pathogens capable of resisting host immune responses. They suggest that vaccines cannot be targeted to the most immunogenic proteins of A. marginale and provide sterile immunity.
Publications
- Meeus PFM, Brayton KA, Palmer GH, Barbet AF. Conservation of a gene conversion mechanism in two distantly related paralogues of Anaplasma marginale. Molecular Microbiology 47:633-643, 2003.
- Brayton KA, Meeus PFM, Barbet AF, Palmer GH. Simultaneous variation of the immunodominant outer membrane proteins MSP2 and MSP3, during Anaplasma marginale persistence In Vivo. Infection and Immunity 71:6627-6632, 2003.
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Progress 01/01/02 to 12/31/02
Outputs
The rickettsial pathogen Anaplasma marginale establishes lifelong persistent infection in the mammalian reservoir host, during which time immune escape variants continually arise in part because of variation in the expressed copy of the immunodominant outer membrane protein MSP2. A key question is how the small 1.2 Mb A. marginale genome generates sufficient variants to allow long-term persistence in an immunocompetent reservoir host. The recombination of whole pseudogenes into the single msp2 expression site has been previously identified as one method of generating variants, but is inadequate to generate the number of variants required for persistent infection. In the present study, we demonstrate that recombination of a whole pseudogene is followed by a second level of variation in which small segments of pseudogenes recombine into the expression site by gene conversion. Evidence for four short sequential changes in the hypervariable region of msp2 coupled with the
identification of nine pseudogenes from a single strain of A. marginale provides for a combinatorial number of possible expressed MSP2 variants sufficient for lifelong persistence.
Impacts
The above study provides a mechanism by which small-genome organisms, such as A. marginale, can make a sufficient number of variable surface proteins to continually avoid the host immune response.
Publications
- Brayton KA, Palmer GH, Lundgren A, Yi, J, Barbet AF. Antigenic variation of Anaplasma marginale msp2 occurs by combinatorial gene conversion. Molecular Microbiology 43:1151-1159, 2002.
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Progress 01/01/01 to 12/31/01
Outputs
The rickettsial pathogen Anaplasma marginale expresses a variable immunodominant outer membrane protein, MSP2, involved in antigenic variation and long-term persistence of the organism in carrier animals. MSP2 contains a central hypervariable region of about 100 amino acids that encodes immunogenic B cell epitopes that induce variant-specific antibodies during infection. Previously, we have shown that MSP2 is encoded on a polycistronic mRNA transcript in erythrocyte stages of A. marginale, and defined the structure of the genomic expression site for this transcript. In this study, we show that the same expression site is utilized in stages of A. marginale infecting tick salivary glands. We also analyzed the variability of this genomic expression site in Oklahoma strain A. marginale transmitted from in vitro cultures to cattle and between cattle and ticks. The structure of the expression site and flanking regions was conserved except for sequence that encoded the MSP2
hypervariable region. At least three different MSP2 variants were encoded in each A. marginale population. The major sequence variants did not change on passage of A. marginale between culture, acute erythrocyte stage infections and tick salivary glands, but did change during persistent infections of cattle. The variant types found in tick salivary glands most closely resembled those present in bovine blood at the time of acquisition of infection, whether infection was acquired from an acute or from a persistent rickettsemia. These variations in structure of an expression site for a major, immunoprotective outer membrane protein have important implications for vaccine development and for obtaining an improved understanding of the mechanisms of persistence of ehrlichial infections in man, domestic animals and reservoir hosts. In a separate study we also examined conservation of the major surface protein 1 genes during cyclic transmission. Protein sequences of MSP1a, MSP1b1 and MSP1b2
were conserved during the life cycle of the rickettsia. No amino acid changes were observed in MSP1a. Small variations were found in MSP1b1 and MSP1b2, which could be attributed to recombination, selection for subpopulations of A. marginale and/or PCR errors. Several isolate specific sequences were also observed. Based on this information, the MSP1 protein appears to be fairly well conserved and a potential vaccine candidate.
Impacts
Attempts to develop vaccines against anaplasmosis, or to develop disease free herds, have not been successful because of the persistent infections caused by A. marginale. It has been unclear what is the basis for successful evasion of the host immune response by the rickettsia. This study suggests that the molecular basis for persistence lies in extensive sequence change in a central hypervariable region of MSP2. Other surface proteins, such as MSP1, are more conserved and may be better vaccine antigens.
Publications
- Barbet, A.F., Yi, J., Lundgren, A., McEwen, B.R., Blouin, E.F., Kocan, K.M. 2001. Antigenic variation of Anaplasma marginale: major surface protein 2 diversity during cyclic transmission between ticks and cattle. Infection and Immunity 69:3057-3066.
- Bowie, M.V., de la Fuente, J., Kocan, K.M., Blouin, E.F., Barbet, A.F. 2002. Conservation of major surface protein 1 genes of Anaplasma marginale during cyclic transmission between ticks and cattle. Gene 282:95-102.
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Progress 01/01/00 to 12/31/00
Outputs
Anaplasma marginale is a tick-borne pathogen, one of several closely related ehrlichial organisms that cause disease in animals and humans. These Ehrlichia species have complex life cycles that require, in addition to replication and development within the tick vector, evasion of the immune system in order to persist in the mammalian reservoir host. This complexity requires efficient use of the small ehrlichial genome. A. marginale and related ehrlichiae express immunoprotective, variable, outer membrane proteins that have similar structure and are encoded by polymorphic multigene families. We have shown that the major outer membrane protein of A. marginale, MSP2, is encoded on a polycistronic mRNA. The genomic expression site for this mRNA is polymorphic and encodes numerous amino acid sequence variants in bloodstream populations of A. marginale. A potential mechanism for persistence is segmental gene conversion of the expression site to link hypervariable msp2
sequences to the promoter and polycistron.
Impacts
Attempts to develop vaccines against anaplasmosis, or to develop disease free herds, have not been successful because of the persistent infections caused by A. marginale. It has been unclear what is the basis for successful evasion of the host immune response by the rickettsia. This study suggests the molecular basis for persistence.
Publications
- Barbet AF, Lundgren AL, Yi J, Rurangirwa FR, Palmer GH. Antigenic variation of Anaplasma marginale by expression of MSP2 mosaics. Infect. Immun. 68:6133-6138, 2000.
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