Strain diversity in major surface protein 2 expression during tick transmission of Anaplasma marginale

Specific major surface protein 2 (MSP2) variants are expressed by Anaplasma marginale within the tick salivary gland and, following transmission, are expressed during acute rickettsemia. In previous work, we have shown that a restricted pattern of MSP2 variants is expressed in the salivary glands of Dermacentor andersoni ticks infected with the South Idaho strain of A. marginale. Now we demonstrate that the identical restriction does not apply to two other strains of A. marginale, and that different variants are also expressed when the same strain is transmitted by different Dermacentor spp. This indicates that antigenic diversity among strains is maintained in tick transmission and may be a significant constraint to MSP2 vaccine development.     

Specific Expression of Anaplasma marginale Major Surface Protein 2 Salivary Gland Variants Occurs in the Midgut and Is an Early Event during Tick Transmission

Infectivity of Anaplasma spp. develops when infected ticks feed on a mammalian host (transmission feed). Specific Anaplasma marginale major surface protein 2 (MSP2) variants are selected for within the tick and are expressed within the salivary glands. The aims of this study were to determine when and where MSP2 variant selection occurs in the tick, how MSP2 expression is regulated in salivary glands of transmission-feeding ticks, and whether the number of A. marginale organisms per salivary gland is significantly increased during transmission feeding. The South Idaho strain of A. marginale was used, as MSP2 expression is restricted to two variants, SGV1 and SGV2, in Dermacentor andersoni. Using Western blot, real-time PCR, and DNA sequencing analyses it was shown that restriction and expression of MSP2 occurs early in the midgut within the first 48 h of the blood meal, when ticks acquire infection. A. marginale is present in the tick salivary glands before transmission feeding is initiated, but the msp2 mRNA and MSP2 protein levels per A. marginale organism increase only minimally and transiently in salivary glands of transmission-feeding ticks compared to that of unfed ticks. A. marginale numbers per tick increase gradually in salivary glands of both transmission-fed and unfed ticks. It is concluded that MSP2 variant selection is an early event in the tick and that MSP2 variants SGV1 and SGV2 are expressed both in the midgut and salivary glands. While MSP2 may be required for infectivity, there is no strict temporal correlation between MSP2 expression and the development of infectivity.     

Expression of Anaplasma marginale Major Surface Protein 2 Variants in Persistently Infected Ticks

Anaplasma marginale, an intraerythrocytic ehrlichial pathogen of cattle, establishes persistent infections in both vertebrate (cattle) and invertebrate (tick) hosts. The ability of A. marginale to persist in cattle has been shown to be due, in part, to major surface protein 2 (MSP2) variants which are hypothesized to emerge in response to the bovine immune response. MSP2 antigenic variation has not been studied in persistently infected ticks. In this study we analyzed MSP2 in A. marginale populations from the salivary glands of male Dermacentor variabilis persistently infected with A. marginale after feeding successively on one susceptible bovine and three sheep. New MSP2 variants appeared in each A. marginale population, and sequence alignment of the MSP2 variants revealed multiple amino acid substitutions, insertions, and deletions. These results suggest that selection pressure on MSP2 occurred in tick salivary glands independent of the bovine immune response.     

Tick-borne transmission of two genetically distinct Anaplasma marginale strains following superinfection of the mammalian reservoir host

Strain superinfection affects the dynamics of epidemiological spread of pathogens through a host population. Superinfection has recently been shown to occur for two genetically distinct strains of the tick-borne pathogen Anaplasma marginale that encode distinctly different surface protein variants. Superinfected animals could serve as a reservoir for onward transmission of both strains if the tick vector is capable of acquiring and transmitting both strains. Whether competition among strains during development within the tick vector, which requires sequential invasion and replication events, limits colonization and subsequent transmission to a single strain is unknown. We tested this possibility by acquisition feeding Dermacentor andersoni ticks on a reservoir host superinfected with the genetically distinct St. Maries and EMPhi strains. Although the St. Maries strain consistently maintained higher bacteremia levels in the mammalian host and the EMPhi strain had an early advantage in colonization of the tick salivary glands, individual ticks were coinfected, and there was successful transmission of both strains. These results indicate that a genetically distinct A. marginale strain capable of superinfecting the mammalian host can subsequently be cotransmitted and become established within the host population despite the presence of an existing established strain.     

Presence of common antigens, including major surface protein epitopes, between the cattle (intraerythrocytic) and tick stages of Anaplasma marginale.

Epitopes of major surface proteins of the intraerythrocytic cattle stage of Anaplasma marginale were demonstrated in the midgut stage of the organism within the infective tick host Dermacentor andersoni. These proteins were common to all A. marginale isolates tested and at all stages of parasitemia. Sera from cattle immunized with the tick midgut stage of A. marginale immunoprecipitated multiple-erythrocyte-stage proteins, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The major proteins recognized (primarily greater than 14 and less than 200 kilodaltons [kDa]) included two major-erythrocyte-stage surface proteins of 36 and 105 kDa molecular size. To confirm the presence of common tick and erythrocyte A. marginale antigens with the immunized cattle sera, we purified the 36-kDa erythrocyte-stage protein by monoclonal immunoaffinity chromatography and developed an enzyme-linked immunosorbent assay based on the purified protein. All sera from cattle immunized with tick-stage A. marginale and cattle infected with various isolates of A. marginale developed antibodies to the 36-kDa protein. The potential immunoprophylactic, diagnostic, and epidemiologic value of the major epitopes common to both the invertebrate and mammalian stages of A. marginale, especially the 36-kDa protein, is discussed.     

Anaplasma marginale major surface protein 3 is encoded by a polymorphic, multigene family.

The immunodominant surface protein, MSP3, is structurally and antigenically polymorphic among strains of Anaplasma marginale. In this study we show that a polymorphic multigene family is at least partially responsible for the variation seen in MSP3. The A. marginale msp3 gene msp3-12 was cloned and expressed in Escherichia coli. With msp3-12 as a probe, multiple, partially homologous gene copies were identified in the genomes of three A. marginale strains. These copies were widely distributed throughout the chromosome. Sequence analysis of three unique msp3 genes, msp3-12, msp3-11, and msp3-19, revealed both conserved and variant regions within the open reading frames. Importantly, msp3 contains amino acid blocks related to another polymorphic multigene family product, MSP2. These data, in conjunction with data presented in previous studies, suggest that multigene families are used to vary important antigenic surface proteins of A. marginale. These findings may provide a basis for studying antigenic variation of the organism in persistently infected carrier cattle.     

Evaluation of Anaplasma marginale major surface protein 3 (MSP3) as a diagnostic test antigen.

An immunodominant surface protein, major surface protein 3 (MSP3), has been proposed as an antigen suitable for use in the diagnosis of bovine anaplasmosis. We further characterized MSP3 to examine its potential as a test antigen for the serological diagnosis of carrier cattle. The specificity of this antigen in detecting infected cattle as well as the conservation of MSP3 between strains of Anaplasma marginale was evaluated by using immunoblots of A. marginale proteins separated by one- and two-dimensional polyacrylamide gel electrophoreses. Immune sera from animals infected with Anaplasma ovis, Ehrlichia risticii, and Ehrlichia ewingii reacted with the MSP3 antigen of A. marginale. One-dimensional gel electrophoresis of A. marginale proteins demonstrated size polymorphism of MSP3 between different geographic isolates. Two-dimensional gel electrophoresis revealed at least three different antigens migrating at the 86-kDa molecular size, and sera from animals infected with different strains of A. marginale reacted with different 86-kDa antigens. These results indicate that MSP3 may share cross-reactive epitopes with antigens found in A. ovis and some Ehrlichia spp. In addition, MSP3 is not conserved between different isolates of A. marginale, and at least in the isolate from Florida, what was previously identified as MSP3 is actually a group of three or more 86-kDa antigens with different isoelectric points. The cross-reactivity of MSP3 with some Ehrlichia spp., the variability of MSP3 between isolates, and the multiple 86-kDa antigens recognized by various sera suggest that MSP3 is not a suitable candidate for use as a recombinant test antigen.     

Glycosylation of Anaplasma marginale major surface protein 1a and its putative role in adhesion to tick cells

Anaplasma marginale, the causative agent of bovine anaplasmosis, is a tick-borne rickettsial pathogen of cattle that multiplies in erythrocytes and tick cells. Major surface protein 1a (MSP1a) and MSP1b form the MSP1 complex of A. marginale, which is involved in adhesion of the pathogen to host cells. In this study we tested the hypothesis that MSP1a and MSP1b were glycosylated, because the observed molecular weights of both proteins were greater than the deduced molecular masses. We further hypothesized that the glycosylation of MSP1a plays a role in adhesion of A. marginale to tick cells. Native and Escherichia coli-derived recombinant MSP1a and MSP1b proteins were shown by gas chromatography to be glycosylated and to contain neutral sugars. Glycosylation of MSP1a appeared to be mainly O-linked to Ser/Thr residues in the N-terminal repeated peptides. Glycosylation may play a role in adhesion of A. marginale to tick cells because chemical deglycosylation of MSP1a significantly reduced its adhesive properties. Although the MSP1a polypeptide backbone alone was adherent to tick cell extract, the glycans in the N-terminal repeats appeared to enhance binding and may cooperatively interact with one or more surface molecules on host cells. These results demonstrated that MSP1a and MSP1b are glycosylated and suggest that the glycosylation of MSP1a plays a role in the adhesion of A. marginale to tick cells.     

Expression of multiple outer membrane protein sequence variants from a single genomic locus of Anaplasma phagocytophilum

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.     

Conservation of transmission phenotype of Anaplasma marginale (Rickettsiales: Anaplasmataceae) strains among Dermacentor and Rhipicephalus ticks (Acari: Ixodidae)

Before the eradication of Boophilus ticks from the United States, Rhipicephalus (Boophilus) microplus (Canestrini) and Rhipicephalus (Boophilus) annulatus (Say) were important biological vectors of the cattle pathogen Anaplasma marginale Theiler. In the absence of Boophilus ticks, A. marginale continues to be transmitted by Dermacentor ticks. However, a few U.S. strains are not transmissible by Dermacentor andersoni Stiles, Dermacentor variabilis (Say), or both, raising the question of how these strains evolved and how they are maintained. We hypothesize that the U.S. non-Dermacentor-transmissible strains of A. marginale were formerly Boophilus-transmitted strains that have been maintained by a combination of persistent infection and mechanical transmission since the eradication of their biological vector from the United States. To test this hypothesis, we attempted to transmit a well-documented non-Dermacentor-transmissible A. marginale strain (Florida), by using D. andersoni and the two Boophilus species that formerly occurred in the United States. For comparison, we examined tick-borne transmission of a strain of A. marginale (Puerto Rico), which has previously been shown to be transmissible by both D. andersoni and B. microplus. All three species of tick transmitted the Puerto Rico strain, and immunohistochemical (IHC) analysis confirmed the presence ofA. marginale colonies in their salivary glands. All three tick species failed to transmit the Florida strain. Although both D. andersoni and B. microplus acquired transient midgut and salivary gland infections after acquisition feeding, we were unable to detect colonies of the Florida strain in the salivary glands with IHC. This demonstrates that the transmission phenotype ofA. marginale strains is conserved among tick species, and it suggests that the failure of the Florida strain to be transmitted by ticks is related to a general inability to efficiently invade or replicate in tick cells, rather than to a failure to invade or replicate in cells of a specific tick species.     

Identification of novel antigenic proteins in a complex Anaplasma marginale outer membrane immunogen by mass spectrometry and genomic mapping

Immunization with purified Anaplasma marginale outer membranes induces complete protection against infection that is associated with CD4+ T-lymphocyte-mediated gamma interferon secretion and immunoglobulin G2 (IgG2) antibody titers. However, knowledge of the composition of the outer membrane immunogen is limited. Recent sequencing and annotation of the A. marginale genome predicts at least 62 outer membrane proteins (OMP), enabling a proteomic and genomic approach for identification of novel OMP by use of IgG serum antibody from outer membrane vaccinates. Outer membrane proteins were separated by two-dimensional electrophoresis, and proteins recognized by total IgG and IgG2 in immune sera of outer membrane-vaccinated cattle were detected by immunoblotting. Immunoreactive protein spots were excised and subjected to liquid chromatography-tandem mass spectrometry. A database search of the A. marginale genome identified 24 antigenic proteins that were predicted to be outer membrane, inner membrane, or membrane-associated proteins. These included the previously characterized surface-exposed outer membrane proteins MSP2, operon associated gene 2 (OpAG2), MSP3, and MSP5 as well as recently identified appendage-associated proteins. Among the 21 newly described antigenic proteins, 14 are annotated in the A. marginale genome and include type IV secretion system proteins, elongation factor Tu, and members of the MSP2 superfamily. The identification of these novel antigenic proteins markedly expands current understanding of the composition of the protective immunogen and provides new candidates for vaccine development.     

Identification of midgut and salivary glands as specific and distinct barriers to efficient tick-borne transmission of Anaplasma marginale

Understanding the determinants of efficient tick-borne microbial transmission is needed to better predict the emergence of highly transmissible pathogen strains and disease outbreaks. Although the basic developmental cycle of Anaplasma and Ehrlichia spp. within the tick has been delineated, there are marked differences in the ability of specific strains to be efficiently tick transmitted. Using the highly transmissible St. Maries strain of Anaplasma marginale in Dermacentor andersoni as a positive control and two unrelated nontransmissible strains, we identified distinct barriers to efficient transmission within the tick. The Mississippi strain was unable to establish infection at the level of the midgut epithelium despite successful ingestion of infected blood following acquisition feeding on a bacteremic animal host. This inability to colonize the midgut epithelium prevented subsequent development within the salivary glands and transmission. In contrast, A. marginale subsp. centrale colonized the midgut and then the salivary glands, replicating to a titer indistinguishable from that of the highly transmissible St. Maries strain and at least 100 times greater than that previously associated with successful transmission. Nonetheless, A. marginale subsp. centrale was not transmitted, even when a large number of infected ticks was used for transmission feeding. These results establish that there are at least two specific barriers to efficient tick-borne transmission, the midgut and salivary glands, and highlight the complexity of the pathogen-tick interaction.     

Simultaneous variation of the immunodominant outer membrane proteins, MSP2 and MSP3, during anaplasma marginale persistence in vivo

Vector-borne bacterial pathogens persist in the mammalian host by varying surface antigens to evade the existing immune response. To test whether the model of surface coat switching and immune evasion can be extended to a vector-borne bacterial pathogen with multiple immunodominant surface proteins, we examined Anaplasma marginale, a rickettsia with two highly immunogenic outer membrane proteins, major surface protein 2 (MSP2) and MSP3. The simultaneous clearance of variants of the two most immunodominant surface proteins of A. marginale followed by emergence of unique variants indicates that the switch rates and immune selection for MSP2 and MSP3 are sufficiently similar to explain the cyclic bacteremia observed during infection in the immunocompetent host.     

Expression of Anaplasma marginale ankyrin repeat-containing proteins during infection of the mammalian host and tick vector

Transmission of tick-borne pathogens requires transition between distinct host environments with infection and replication in host-specific cell types. Anaplasma marginale illustrates this transition: in the mammalian host, the bacterium infects and replicates in mature (nonnucleated) erythrocytes, while in the tick vector, replication occurs in nucleated epithelial cells. We hypothesized that proteins containing ankyrin motifs would be expressed by A. marginale only in tick cells and would traffic to the infected host cell nucleus. A. marginale encodes three proteins containing ankyrin motifs, an AnkA orthologue (the AM705 protein), AnkB (the AM926 protein), and AnkC (the AM638 protein). All three A. marginale Anks were confirmed to be expressed during intracellular infection: AnkA is expressed at significantly higher levels in erythrocytes, AnkB is expressed equally by both infected erythrocytes and tick cells, and AnkC is expressed exclusively in tick cells. There was no evidence of any of the Ank proteins trafficking to the nucleus. Thus, the hypothesis that ankyrin-containing motifs were predictive of cell type expression and nuclear localization was rejected. In contrast, AnkA orthologues in the closely related A. phagocytophilum and Ehrlichia chaffeensis have been shown to localize to the host cell nucleus. This difference, together with the lack of a nuclear localization signal in any of the AnkA orthologues, suggests that trafficking may be mediated by a separate transporter rather than by endogenous signals. Selection for divergence in Ank function among Anaplasma and Ehrlichia spp. is supported by both locus and allelic analyses of genes encoding orthologous proteins and their ankyrin motif compositions.     

Composition of the surface proteome of Anaplasma marginale and its role in protective immunity induced by outer membrane immunization

Surface proteins of tick-borne, intracellular bacterial pathogens mediate functions essential for invasion and colonization. Consequently, the surface proteome of these organisms is specifically relevant from two biological perspectives, induction of protective immunity in the mammalian host and understanding the transition from the mammalian host to the tick vector. In this study, the surface proteome of Anaplasma marginale, a tick-transmitted bacterial pathogen, was targeted by using surface-specific cross-linking to form intermolecular bonds between adjacent proteins. Liquid chromatography and tandem mass spectroscopy were then employed to characterize the specific protein composition of the resulting complexes. The surface complexes of A. marginale isolated from erythrocytes of the mammalian host were composed of multiple membrane proteins, most of which belong to a protein family, pfam01617, which is conserved among bacteria in the genus Anaplasma and the closely related genus Ehrlichia. In contrast, the surface proteome of A. marginale isolated from tick cells was much less complex and contained a novel protein, AM778, not identified within the surface proteome of organisms from the mammalian host. Immunization using the cross-linked surface complex induced protection against high-level bacteremia and anemia upon A. marginale challenge of cattle and effectively recapitulated the protection induced by immunization with whole outer membranes. These results indicate that a surface protein subset of the outer membrane is capable of inducing protective immunity and serves to direct vaccine development. Furthermore, the data support that remodeling of the surface proteome accompanies the transition between mammalian and arthropod hosts and identify novel targets for blocking transmission.     

Antigenic variation of Anaplasma marginale by expression of MSP2 mosaics

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.     

Strain composition of the ehrlichia Anaplasma marginale within persistently infected cattle, a mammalian reservoir for tick transmission

Tick-borne ehrlichial pathogens of animals and humans require a mammalian reservoir of infection from which ticks acquire the organism for subsequent transmission. In the present study, we examined the strain structure of Anaplasma marginale, a genogroup II ehrlichial pathogen, in both an acute outbreak and in persistently infected cattle that serve as a reservoir for tick transmission. Using the msp1alpha genotype as a stable strain marker, only a single genotype was detected in a disease outbreak in a previously uninfected herd. In contrast, a diverse set of genotypes was detected in a persistently infected reservoir herd within a region where A. marginale is endemic. Genotypic diversity did not appear to be rapidly generated within an individual animal, because only a single genotype, identical to that of the inoculating strain, was detected at time points up to 2 years after experimental infection, and only a single identical genotype was found in repeat sampling of individual naturally infected cattle. Similarly, only a single genotype, identical to that of the experimentally inoculated St. Maries or South Idaho strain, was identified in the bloodmeal taken by Dermacentor andersoni ticks, in the midgut and salivary glands of the infected ticks, and in the blood of acutely infected cattle following tick transmission. The results show that mammalian reservoirs harbor genetically heterogeneous A. marginale and suggest that different genotypes are maintained by transmission within the reservoir population.