Expression of Multiple Outer Membrane Protein Sequence Variants from a Single Genomic Locus of Anaplasma phagocytophilum

Abstract

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.

Complete genome sequencing of Anaplasma marginale reveals that the surface is skewed to two superfamilies of outer membrane proteins

“…The orthologs of msp2 in members of the genus Ehrlichia, E. ruminantium, E. canis and E. chaffeensis (41)(42)(43) are arranged as tandemly repeated full-length genes in one (E. ruminantium, E. chaffeensis) or two (E. canis) loci containing 16-25 paralogs. There is synteny between the arrangement of these ehrlichial genes and part of the msp2 superfamily in the region of the msp2 operon in both A. marginale and A. phagocytophilum (30,44). Interestingly, the mechanism for generating antigenic variation in these immunodominant OMPs is very different between these two genera: Erhlichial species use multiple genes from the tandem array of OMPs, whereas A. marginale and A. phagocytophilum use a recombination mechanism (41-43).…”

“…Notably, A. marginale has genes defined as functional pseudogenes: truncated copies of genes that are only expressed as part of a functional full-length protein after recombination into a unique expression site (11,28,29). Similar functional pseudogenes are also present in A. phagocytophilum (30) proteins (OMPs) respectively, missing many of the known A. marginale OMPs. By sequence identity to previously defined OMPs, 13 CDSs were assigned as OMPs.…”

Tick cell lines: tools for tick and tick-borne disease research

“…In A. phagocytophilum, the immunodominant p44 antigen predominated in human cells but not in tick cells and might be involved in regulatory changes that mediate survival of the pathogen by immune modulation after tick transmission [46]. The p44 gene expression site was found to be polymorphic in human and tick cells, with sequence changes in p44 variants being influenced by host cell type and culture conditions [47]. Although all 16 members of the E. ruminantium major antigenic protein 1 (map1) multigene family were transcribed in vitro in mammalian cells, between 4 and 11 paralogs were transcribed in different tick cell lines [48].…”

Reconstructing immune phylogeny: new perspectives

“…In various pathogens, including bacteria and protozoan parasites, several fundamental mechanisms of DNA change are used during invasion of the host. DNA recombination [148][149][150][151] , gene conversion [152][153][154] and gene hypermutation can lead to the evasion of host detection and to the subsequent clonal selection of the pathogenic variant. For example, in trypanosomes, DNA recombination and gene conversion rapidly modify variant surface glycoproteins and allow evasion of host detection 155,156 .…”