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 structures and are encoded by polymorphic multigene families. We show here 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.Ehrlichiae are major causes of tick-borne diseases, including the recently emergent human monocytic and two granulocytic ehrlichioses and the most prevalent tick-borne infection in cattle worldwide, anaplasmosis (6). These pathogens, classified in genogroups I and II of the tribe Ehrlichieae, have a complex life cycle characterized by acute and persistent infection in the mammalian host and several replicative and developmental stages within the tick vector (10, 17). Notably, this complexity is achieved using a small genome, only 0.8 to 1.5 Mb in a single chromosome (1,25). Persistence of Anaplasma marginale in cattle, which is fundamental for continued transmission, reflects sequential expression of antigenically variant outer membrane proteins that are encoded by the msp2 multigene family (21). The outer membrane proteins of different ehrlichial organisms are significantly similar to one another in amino acid sequence, are all encoded by multigene families, and possess one to four variable regions (8, 13, 18-20, 22, 26, 29, 32). In A. marginale-infected cattle, three to six MSP2 variants are expressed in each sequential rickettsemic cycle, which recur every 4 to 8 weeks during persistent infection (8,9,15,16). Thus, over the 7-year period in which A. marginale has been shown to persist, over 500 variants may be expressed. Although the cyclic emergence and immune control of A. marginale is similar to that occurring in African trypanosomiasis, the mechanisms used by the organism to persist in mammalian hosts are unknown. We show here that variation of msp2 in erythrocyte stages of A. marginale proceeds through the formation of different sequence mosaics in a polycistronic expression site. MATERIALS AND METHODSIsolation and sequencing of A. marginale genomic DNA. Florida and South Idaho strains of A. marginale were maintained as liquid nitrogen-cryopreserved stabilates of infected bovine erythrocytes in dimethyl sulfoxide-phosphate-buffered saline that were then used to infect cattle (20). A. marginale genomic DNA ...

SummaryThe 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.

Anaplasma phagocytophilum is the causative agent of an emerging tick-borne zoonosis in the United States and Europe. The organism causes a febrile illness accompanied by other nonspecific symptoms and can be fatal, especially if treatment is delayed. Persistence of A. phagocytophilum within mammalian reservoir hosts is important for ensuring continued disease transmission. In the related organism Anaplasma marginale, persistence is associated with antigenic variation of the immunoprotective outer membrane protein MSP2. Extensive diversity of MSP2 is achieved by combinatorial gene conversion of a genomic expression site by truncated pseudogenes. The major outer membrane protein of A. phagocytophilum, MSP2(P44), is homologous to MSP2 of A. marginale, has a similar organization of conserved and variable regions, and is also encoded by a multigene family containing some truncated gene copies. This suggests that the two organisms could use similar mechanisms to generate diversity in outer membrane proteins from their small genomes. We define here a genomic expression site for MSP2(P44) in A. phagocytophilum. As in A. marginale, the msp2(p44) gene in this expression site is polymorphic in all populations of organisms we have examined, whether organisms are obtained from in vitro culture in human HL-60 cells, from culture in the tick cell line ISE6, or from infected human blood. Changes in culture conditions were found to favor the growth and predominance of certain msp2(p44) variants. Insertions, deletions, and substitutions in the region of the genomic expression site encoding the central hypervariable region matched sequence polymorphisms in msp2(p44) mRNA. These data suggest that, similarly to A. marginale, A. phagocytophilum uses combinatorial mechanisms to generate a large array of outer membrane protein variants. Such gene polymorphism has profound implications for the design of vaccines, diagnostic tests, and therapy.

The rickettsial pathogen Anaplasma marginale expresses a variable immunodominant outer membrane protein, major surface protein 2 (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 humans, domestic animals, and reservoir hosts.

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