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Infection and Immunity, March 2007, p. 1502-1506, Vol. 75, No. 3
0019-9567/07/$08.00+0     doi:10.1128/IAI.01801-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Selection for Simple Major Surface Protein 2 Variants during Anaplasma marginale Transmission to Immunologically Naïve Animals

Guy H. Palmer,^1* James E. Futse,¹ Christina K. Leverich,¹ Donald P. Knowles Jr.,² Fred R. Rurangirwa,¹ and Kelly A. Brayton¹

Program in Vector-borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University,¹ Animal Diseases Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Pullman, Washington²

Received 10 November 2006/ Returned for modification 1 December 2006/ Accepted 7 December 2006

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Anaplasma marginale, a rickettsial pathogen, evades clearance in the animal host by antigenic variation. Under immune selection, A. marginale expresses complex major surface protein 2 mosaics, derived from multiple donor sequences. However, these mosaics have a selective advantage only in the presence of adaptive immunity and are rapidly replaced by simple variants following transmission.

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Persistent infection is fundamental to the epidemiology of vector-borne pathogens, providing a reservoir for transmission during periods of peak arthropod activity. African trypanosomes, which use a large repertoire of variable surface glycoprotein (vsg) sequences to generate diversity in their bloodstream form surface coats, classically illustrate how antigenic variation allows evasion of the host immune response and persistence within an immunocompetent mammalian reservoir host (4, 18). The importance of this process in vector-borne pathogen persistence, and thus transmission, is evident by the use of a very similar mechanism in evolutionarily distinct pathogens, most notably the tick-borne bacteria in the genera Anaplasma and Borrelia (3). Anaplasma marginale, the most globally prevalent tick-borne pathogen of livestock, closely mimics the trypanosome in that it uses duplication of chromosomal donor sequences into the expression site (7, 8). A. marginale, with a 1.2-Mb genome, less than 1/20 the size of the Trypanosoma brucei genome (5, 6), represents a simplified model of this convergent mechanism of antigenic variation, since all major surface protein-2 (MSP2) surface coat variants are generated by recombination from 5 to 7 chromosomal donor sequences, termed functional pseudogenes, into a single expression site (Fig. 1A). The evolutionary selection for gene duplication, resulting in the repertoire of the donor msp2 and vsg sequences in A. marginale and T. brucei, respectively, is evident: this repository must be sufficiently diverse to generate numerous antigenic variants and allow persistent infection in the face of a rapidly responding immune system.

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FIG. 1. (A) Genomic structure of the Anaplasma marginale msp2 pseudogene and expression site loci. The 1.2-Mb genome is a single circular chromosome and encodes a total of seven msp2 pseudogenes, two of which are exact duplicates (3H1 and 2; P1 and G11), and a single expression site (ES). The five unique pseudogenes are each indicated by a different color. The expression site msp2 variant, in this example, corresponds to pseudogene 9H1. (B) Gene conversion generates unique msp2 expression site variants. Sequential rounds of recombination result in replacement of the existing expression site copy by either a whole pseudogene donor sequence (as shown in the first gene conversion event) or progressive modification of the existing expression site copy by segmental gene conversion. Over time, this process of segmental gene conversion generates complex expression site mosaics derived from multiple distinct pseudogene donors. The light blue regions flanking the expression site represent the conserved 5' and 3' domains; identical, truncated domains flank each pseudogene and direct recombination (14).

In addition to this selective pressure for surface coat diversity, there is a balancing selection for growth fitness. At a structural level, this is evident by conservation of the transmembrane domains among MSP2 variants in A. marginale and among VSG variants in T. brucei (7, 10, 23); however, the extracellular domains are also very likely under growth fitness selection. While the large genome size, the complexity of the vsg families, and the multiple expression sites have, to date, precluded comprehensive testing in T. brucei, A. marginale variants expressing the complete extracellular domain derived from each of the unique pseudogenes of the St. Maries strain can be identified early in infection (14). This expression indicates that each of the chromosomally encoded extracellular MSP2 domains confers in vivo fitness. A. marginale also generates expression site mosaics following recombination of oligonucleotide segments derived from two or more donor sequence loci (Fig. 1B) (1, 8, 14). These mosaics, quantified in terms of complexity based on the number of expression site segments derived from different donor pseudogene sequences, emerge over time during infection and dominate the variant population following months of persistence (14). Similar mosaics are generated by T. brucei and the related Trypanosoma equiperdum, in which mosaics emerge over time during infection (16, 20, 23). The ability to generate mosaics tremendously expands the repertoire of possible antigenic variants, allowing continual immune evasion and persistence. However, unlike the stable chromosomal donor sequence loci, A. marginale expression site mosaics themselves are not subject to long-term selection, since they are not permanently represented in the genome and are lost from the genome when they are replaced in the expression site by a subsequent recombination event (1, 8, 12, 13). Thus, we hypothesize that while complex mosaics emerge under strong immune selective pressure, they do not represent in vivo growth fitness selection, and in the absence of immunity, simple type variants will predominate.

To test this hypothesis, we established persistent A. marginale infection in a natural animal reservoir host, confirmed that the msp2 expression site encoded complex mosaic variants, and transmitted these complex MSP2 variants to immunologically naïve animals. Briefly, infection of calf 983 was initiated by transmission feeding of adult male Dermacentor andersoni ticks infected with the St. Maries strain of A. marginale (22). This strain was selected because it has been completely sequenced (6), and thus each expression site sequence can be mapped to one or more specific msp2 donor pseudogenes. In this analysis, msp2 expression site variants are amplified, sequenced, and either mapped to a whole-donor msp2 pseudogene or, in the case of mosaics, each oligonucleotide segment in the expression site is mapped to its specific-donor pseudogene (see Fig. 1B) (14). Acute infection of calf 983 with high-level bacteremia (10⁹ A. marginale parasites per ml) was confirmed by microscopic detection of infected erythrocytes in Giemsa-stained blood smears. Expression of the unique extracellular domains derived from recombination of each intact whole pseudogene (Fig. 1A) was detected within the first 60 days of infection (Fig. 2), consistent with their preferential use early in infection. Persistent infection was tracked using quantitative real-time msp5 PCR, as previously described in detail (15), and was maintained at levels of 10⁷ organisms per ml of blood from 3 months post-tick transmission through the end of the study. During persistent infection, the population structure of the expressed MSP2 was confirmed to be mosaic (Fig. 2) (14). Analysis of at least 30 sequences provides a >90% probability that all sequences representing at least 5% of the variant population will be detected (14). A complexity score of zero reflects the presence of a single whole pseudogene sequence in the expression site, a score of 1 indicates the recombination of a segment derived from a distinct donor pseudogene, and scores of 2 and 3 apply when, respectively, two and three segments from distinct pseudogenes are recombined into the expression site (Fig. 1B). The complexity score of calf 983 progressively increased during infection to a mean score of >2.0 (Fig. 2), indicating that the expressed MSP2 was derived from a mosaic of multiple distinct pseudogenes, a result consistent with the prior analysis of multiple calves during long-term persistent infection (14).

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FIG. 2. Development of complex MSP2 mosaics during persistent infection of animal 983. Complexity, measured by the number of expression site segments derived from different donor pseudogenes (14), is plotted on the y axis, and duration of infection (in months) is shown on the x axis. At 24 months of infection (solid bar), this calf was used both for acquisition feeding of D. andersoni for tick transmission and as the source for direct transmission by intravenous inoculation.

Dermacentor andersoni adult males were then allowed to feed on calf 983 for 7 days to acquire infection. The mean complexity of MSP2 variants in the blood during acquisition tick feeding was 2.2 ± 0.69. Following 48 h of incubation at 26°C and 98% relative humidity, ticks were transmission fed on each of four age-matched calves (calves 1104, 1113, 1118, and 1121), which had been found seronegative by competitive indirect enzyme-linked immunosorbent assay (VMRD, Pullman, WA), for an additional 7 days. From the earliest detectable time point following transmission through the first 3 weeks of acute infection, the complexity score for all calves was <1.0, reflecting a significantly less complex variant structure (Fig. 3). Both the overall complexity scores for the emergent organisms in the four calves combined (0.45 ± 0.29) and those for the emergent organisms in each calf (Fig. 3) were significantly lower (P < 0.05) than those present in the blood of persistently infected calf 983 during tick acquisition feeding. For each of the transmission calves and at each time point, 100% of the 121 total variants examined were simple variants (complexity score, 1.0) derived from recombination of a whole pseudogene hypervariable region or single segment into the expression site, in contrast to the overwhelming predominance (>90%) of complex variants (complexity score, 2.0) in the blood of persistently infected calf 983 at the time of acquisition tick feeding.

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FIG. 3. Preferential expression of simple MSP2 variants following tick transmission to immunologically naïve animals. Solid bar, complexity score of A. marginale msp2 variants in persistently infected calf 983 during the acquisition feeding of ticks. Open bars, complexity of the variants arising during acute infection following tick transmission to each of four immunologically naïve calves (calves 1104, 1113, 1118, and 1121).

The emergence of A. marginale simple variants during early infection in immunologically naïve animals is consistent with the progressive development of complex mosaic variants during persistent infection only under the selective pressure of immunity: in the absence of immune memory, these complex variants have no intrinsic advantage. To the contrary, the data indicates that these variants are at a disadvantage, since neutral selection would predict that the population early in infection of the naïve animals would reflect that of calf 983 at the time of acquisition feeding. To address where this selection for simple variants occurs, we examined two nonmutually exclusive possibilities: (i) during invasion, replication, and transmission by the tick vector and (ii) during invasion and replication in the naïve calf. For the first, A. marginale msp2 expression site variants were amplified and sequenced from the tick salivary glands (21) obtained on the last day of transmission feeding on calves 1104, 1113, 1118, and 1121, since these organisms should best represent the cumulative selective events during replication and development prior to and at the time of transmission. The msp2 expression site variants from individual ticks fed on each animal were amplified and sequenced. The mean complexity score from all expressed variant sequences (n = 200) was 0.49, and that of ticks fed on each individual calf ranged from a mean of 0.26 to 0.74. These salivary gland variants were of the simple type, reflected in the significantly lower complexity score (P < 0.05), compared to the MSP2 variants present during the tick acquisition feeding (Fig. 4). For the second possibility, 10 ml of blood, collected from calf 983 during tick acquisition feeding and containing MSP2 variants with a mean complexity score of 2.2, was inoculated intravenously directly into calf 1125, and the msp2 expression site variants early in infection were determined as described above for the calves following tick transmission. Examination of 192 variants collected in the first month of acute infection revealed a mean complexity score of 0.42 ± 0.47, with a score of 0.29 ± 0.47 at the earliest time point examined (7 days postinoculation). All time points were again significantly different (P < 0.05) from the inoculated A. marginale both in complexity score (Fig. 5) and in the usage of recombined whole pseudogenes.

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FIG. 4. Preferential expression of simple MSP2 variants in the tick salivary gland. Solid bar, complexity score of A. marginale MSP2 variants in persistently infected calf 983 during the acquisition feeding of ticks. Open bars, complexity of the variants in the salivary glands of ticks transmission fed on each of four calves (calves 1104, 1113, 1118, and 1121).

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FIG. 5. Preferential expression of simple MSP2 variants following direct inoculation of complex variants into an immunologically naïve animal. Solid bar, complexity score of A. marginale variants in persistently infected calf 983. These were inoculated intravenously into immunologically naïve calf 1125. Open bars, complexity of the MSP2 variants emergent during the 3 weeks of acute infection.

We accept the hypothesis that in the absence of the selective pressure of the mammalian adaptive immune response, MSP2 simple variants have a competitive fitness advantage and predominate. The rapid emergence of very simple variants within the first week of infection following direct inoculation demonstrates the rigor of selection for optimal in vivo growth. Unlike the mosaics, which are not maintained in the genome and thus are not under specific evolutionary selection, the individual whole-pseudogene sequences, originally generated by gene duplication, can be under selection both for ability to generate antigenic variants and for growth fitness following recombination and expression. Consistent with this dual selection, one or more identical msp2 pseudogenes are conserved among otherwise genetically different A. marginale strains (6-8, 19).

Interestingly, the selection for growth of simple variants occurred both within the tick and in the mammalian host following transmission. Previously, we proposed that there is selection for specific MSP2 variants within the tick (21). While our new data cannot exclude a role for specific variants in development within the tick, the most parsimonious explanation for the results is that A. marginale simple variants have a fitness advantage that is manifest in the absence of adaptive immunity, be it in the tick or in the immunologically naïve animal host. This explanation is consistent with prior studies, although previously interpreted differently, using two additional strains of A. marginale. Variants that can now be identified as simple type MSP2 variants in the South Idaho strain (following sequencing of the pseudogene repertoire in this strain) were detected in the tick midgut within 3 days following acquisition feeding (17). In a separate study, these South Idaho strain simple variants were shown to predominate in the tick salivary gland and, following transmission, in naïve calves (21). Independent studies using the Oklahoma strain of A. marginale revealed that for splenectomized calves, in which immune pressure is abrogated, the same MSP2 types were identified in the splenectomized calf used for tick acquisition feeding, the salivary glands of ticks fed on this calf, and, again following transmission, in the naïve calf (2). Although not examined, our results would predict that these represent simple variants with a fitness advantage in the absence of immune selection.

Whether the fitness disadvantage of organisms expressing complex mosaic variant coats contributes to the lower bacteremia levels observed during persistent A. marginale infection is unknown. Unlike the peak bacteremia levels of 10⁹ per ml during acute infection, A. marginale variants emergent during persistent infection of immunocompetent animals are controlled at levels below 10⁷ per ml (11-13). This has been proposed to reflect more rapid development of variant-specific immune responses due to linked conserved epitopes (9); however, a significantly lower replication rate would also provide more time for the immune system to respond to and control emergent variants. We emphasize that it remains unknown if A. marginale parasites expressing simple MSP2 variants have a significantly higher replication rate that those expressing complex mosaics, since in vivo fitness reflects both growth and survival. African trypanosomiasis follows a similar course, with initial peak parasitemia levels followed by a gradual reduction in the peak of subsequent cycles in which novel VSG variants sequentially arise and are controlled. Whether duration of persistence and amplitude of parasitemic peaks are associated with the emergence of complex VSG mosaics in trypanosomiasis awaits investigation. However, the development of mosaics as a mechanism to generate sufficient antigenic variants to evade immune clearance, even when this is associated with a significant fitness disadvantage, illustrates the overriding importance of persistent infection for efficient onward transmission of vector-borne pathogens.

 
This research was supported by NIH R01 AI44005, USDA ARS-CRIS 5348-32000-016-00D, and USDA-ARS Cooperative Agreement 58-5348-3-212.

The technical assistance of Ralph Horn, Beverly Hunter, and James Allison is gratefully acknowledged.

 
* Corresponding author. Mailing address: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040. Phone: (509) 335-6033. Fax: (509) 335-8529. E-mail: gpalmer{at}vetmed.wsu.edu.

Published ahead of print on 18 December 2006.

Editor: R. P. Morrison

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Infection and Immunity, March 2007, p. 1502-1506, Vol. 75, No. 3
0019-9567/07/$08.00+0     doi:10.1128/IAI.01801-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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• Sarkar, M., Troese, M. J., Kearns, S. A., Yang, T., Reneer, D. V., Carlyon, J. A. (2008). Anaplasma phagocytophilum MSP2(P44)-18 Predominates and Is Modified into Multiple Isoforms in Human Myeloid Cells. Infect. Immun. 76: 2090-2098 [Abstract] [Full Text]  
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