Segmental variation in a duplicated msp2 pseudogene generates Anaplasma marginale antigenic variants.

Anaplasma marginale is a prototypical highly antigenically variant bacterial pathogen, dependent on sequential generation of Major Surface Protein-2 (Msp2) outer membrane variants to establish persistent infection. Msp2 is encoded by a single expression site and diversity is achieved by gene conversion of chromosomally encoded msp2 pseudogenes. Analysis of the full complement of msp2 pseudogenes in the St. Maries strain revealed identical sequences in different loci. The Florida strain shared the same locus structure but in the loci where the St. Maries strain had two identical pseudogenes, the Florida strain had one that was identical to the St. Maries sequences, while the sequence of the second pseudogene differed. Consequently, we hypothesized that the msp2 pseudogene repertoire arose via gene duplication, allowing structural variation to occur in one copy while retaining the utility of the other. Using comparative genomics, we first established that duplication of msp2 pseudogenes is common among A. marginale strains: all seven examined strains had at least one duplicated pair, either maintained as identical copies or containing segmental changes. We then demonstrated that a minimal segmental change in a duplicated pseudogene locus is sufficient for immune escape from the broad antibody response generated in a natural host as is a completely divergent pseudogene sequence in an otherwise conserved locus. The results support a model in which a locus first duplicates, resulting in a second identical copy, then progressively incorporates changes to generate an msp2 repertoire capable of generating sufficient antigenic variants to escape immunity and establish persistent infection.