Anaplasma phagocytophilum is the causative agent of human granulocytic anaplasmosis (formerly named HGE) and granulocytic anaplasmosis in animals. Human, horse, and canine infections are acute and self-limiting 123, whereas ruminant and rodent infections may persist 45. As for Anaplasma marginale, A. phagocytophilum strains from different geographic regions are antigenically variable 6, and the emergence of antigenic variants in vitro and in vivo suggests a role in immune evasion and persistent infection 78910. Antigenic variability results from differential expression of immunodominant surface proteins (Msp2, p44, 44 kDA), encoded by a multigene family 101112. Msp2 expression is polymorphic in vitro, in mice, and in ticks 1314. Based in part on these investigations where changes were characterized under immune selective pressure or in poorly defined polyclonal populations in vitro, the prevailing opinion is that antigenic diversity is generated as a mechanism for immune evasion. However, among 18 unique msp2 paralogs detected in uncloned low passage A. phagocytophilum Webster strain, only 1 of 7 transcripts was dominant in number 15. With the discovery that Msp2 is an A. phagocytophilum adhesin for granulocytes, we hypothesized that Msp2s are also selected by non-immunological processes based on fitness for specific niches, such as growth in mammalian granulocytes. Before such hypotheses can be addressed, it is important to characterize and monitor endogenous changes in msp2 transcription in the absence of selective pressures under defined limits of in vitro propagation. Thus, msp2 transcripts of A. phagocytophilum Webster strain were investigated after 20 in vitro passages, compared to transcripts characterized after only 7 in vitro passages.

Msp2 is the immunodominant protein expressed in both A. marginale and A. phagocytophilum. The encoding msp2s are believed to allow the generation of antigenic variation and persistence in reservoir hosts 89101112151718. In addition, A. phagocytophilum Msp2s are heterogeneous among geographically distinct isolates 6 and this diversity may be important for infection in reservoirs such as rodents, ruminants, and cervids 4519. The mechanisms of persistence of Anaplasma marginale and many other microorganisms involve gene recombination or conversion and expression of new antigens that allow immune evasion 2021222324. However, another hypothesis is whether a non-immunologic functional role for individual msp2 paralogs influences selection when expressed in specific niches such as the mammalian or tick host 1314. Experiments conducted under immune selective pressure, such as in vivo mammalian infection, or with cultures of undefined bacterial clonality are unable to provide supportive data for such alternate roles. Previous characterization showed that very low passage strains of A. phagocytophilum possess a single abundant msp2 transcript 15, which coupled with new data confirming a single expression site for A. phagocytophilum 25, indicates a relative uniform clonal population. If immune selection is the driving force for Msp2 diversity, propagation in vitro in the absence of immune pressure should allow the generation of multiple Msp2 variants. Thus, starting with a low passage, nearly clonal strain of A. phagocytophilum, changes in endogenous msp2 transcription were analyzed when immune selection was absent.

The identification of 4 additional unique msp2 paralog transcripts in A. phagocytophilum Webster strain after prolonged in vitro passage without immune selection suggests a role for either combinatorial recombination or gene conversion, as described for A. marginale 21. However, all 4 new msp2 genes were identified in the genome of clonal low passage A. phagocytophilum excluding the possibility that each was generated by a new recombination event. Although proof of a role for A. phagocytophilum msp2s in immune evasion is lacking, changing Msp2 expression does alter antigenicity, and could contribute to evasion 1314. However, only minimal antigenic changes were observed, and these could equally reflect structural changes anticipated to occur with selection of alternate adhesins.

It is strongly suspected that transcription of msp2 occurs from perhaps a single site in the chromosome, and that msp2 transcriptional changes imply at minimum, emergence of new populations after gene conversion 7825. The incomplete A. phagocytophilum strain HZ genome revealed more than 50 msp2 paralogs and/or pseudogenes of over 100 amino acids. This suggests that a vast capacity for Msp2 diversity is already "hard-wired" and that only simple gene conversion into a transcriptional site is required. Regardless, these findings illustrate that even without immune pressure, spontaneous genetic changes can accrue, but do so in a very limited fashion. It may be that human infection selects for certain Msp2s, explaining the abundance of a single or small number of msp2 transcripts in human HL-60 cell cultures, whereas other Msp2s may more efficiently bind tick cells or neutrophils in reservoir animals. In partial support of this concept, Carlyon et al. recently described an alternative murine neutrophil ligand that differs in scaffold protein from platelet selectin glycoprotein-1 (PSGL-1) on human neutrophils 26.

What evidence is there that Msp2 has a role other than in immune evasion? Although msp2 transcript diversity increases with mammalian cell passage, the effects are not neutral. Given the implied requirement for frequent emergence of variation to resist mammalian immunity 21, one might predict that a larger diversity and a more uniform frequency of msp2 transcripts would appear. Instead, prolonged passage is associated with the appearance of only 2 major msp2 transcripts over 20 or more passages – an interval usually greater than 4 months of continuous in vitro propagation. Moreover, despite the relative lack of selective pressures, the absence of any transcripts that would indicate significant gene recombination suggests that msp2s of individual strains are moderately stable, at least in vitro. Taken together, these data further suggest that some Msp2s have specific functions that are selected for by environmental conditions and niches. Indeed, both Zhi et al. and IJdo et al. have also demonstrated a restricted profile of msp2 transcripts under several conditions including tick infections 1314.

An important function of Msp2 may be to provide bacterial antigenic diversity wherein immune recognition of the abundant bacterial population is targeted. Antigenically diverse bacterial clones temporarily survive and contribute to reservoir persistence. However, the high level of expression, the conserved flanking regions with relative conservation of structure, and highly conserved residues within the hypervariable domain suggest the potential for other roles, such as cell adhesion 272829. Moreover, the possibility that changes in Msp2 expression are associated with changes in A. phagocytophilum pathogenicity has not been investigated. Low passage A. phagocytophilum Webster strain causes disease in horses similar to natural human infection, but prolonged in vitro propagation, which leads to changes in msp2 transcription and Msp2 expression, attenuates clinical signs in the horse model 30. The change in virulence may ultimately be unrelated to changes in Msp2, but as host immune response is the predominant correlate of histopathologic lesions and disease 3132, this hypothesis and the functions of specific Msp2s in various niches warrant further investigation.

Msp2 expression and msp2 transcription in A. phagocytophilum display limited diversity under nonimmune-selective conditions despite long intervals of passage, suggesting that Msp2 has other functions beyond generation of antigenic variation for immune evasion. With the recognition of its role in adhesion to host cells, Msp2 diversity could also be an important adaptation to alternate niches such as small mammal or cervid reservoir neutrophils or tick cells.

DGS performed molecular biological techniques and sequence analysis, alignment, interpretation, and manuscript preparation. KC performed some amplification and cloning reactions and assisted in initial design. HO performed bioinformatics manipulations to assess the genomic content of the A. phagocytophilum HZ strain. JHP assisted in design of the study and assisted with molecular techniques. JSD was responsible for overall design of the study, analysis and interpretation of data, and assisted with manuscript preparation.