Analysis of the Anaplasma marginale major surface protein 1 complex protein composition by tandem mass spectrometry

The protective major surface protein 1 (MSP1) complex of Anaplasma marginale is a heteromer of MSP1a and MSP1b, encoded by a multigene family. The msp1beta sequences were highly conserved throughout infection. However, liquid chromatography-tandem mass spectrometry analysis identified only a single MSP1b protein, MSP1b1, within the MSP1 complex.     

The hypervariable region of Anaplasma marginale major surface protein 2 (MSP2) contains multiple immunodominant CD4+ T lymphocyte epitopes that elicit variant-specific proliferative and IFN-gamma responses in MSP2 vaccinates

Major surface protein 2 (MSP2) is an immunodominant outer membrane protein of Anaplasma marginale and Anaplasma phagocytophilum pathogens that cause bovine anaplasmosis and human granulocytic ehrlichiosis, respectively. MSP2 has a central hypervariable region (HVR) flanked by highly conserved amino and carboxyl termini. During A. marginale infection, dynamic and extensive amino acid sequence variation in MSP2 occurs through recombination of msp2 pseudogenes into the msp2 expression site, followed by sequential segmental gene conversions to generate additional variants. We hypothesized that MSP2 variation leads to significant changes in Th cell recognition of epitopes in the HVR. T cell epitopes were mapped using T cells from native MSP2-immunized cattle and overlapping peptides spanning the most abundant of five different MSP2 HVRs in the immunogen. Several epitopes elicited potent effector/memory Th cell proliferative and IFN-gamma responses, including those in three discreet blocks of sequence that undergo segmental gene conversion. Th cell clones specific for an epitope in the block 1 region of the predominant MSP2 variant type failed to respond to naturally occurring variants. However, some of these variants were recognized by oligoclonal T cell lines from MSP2 vaccinates, indicating that the variant sequences contain immunogenic CD4(+) T cell epitopes. In competition/antagonism assays, the nonstimulatory variants were not inhibitory for CD4(+) T cells specific for the agonist peptide. Dynamic amino acid sequence variation in MSP2 results in escape from recognition by some effector/memory MSP2-specific Th cells. Antigenic variation in MSP2 Th cell and B cell epitopes may contribute to immune evasion that allows long-term persistence of A. marginale in the mammalian reservoir.     

Conservation of a gene conversion mechanism in two distantly related paralogues of Anaplasma marginale

Anaplasmataceae, the causative agents of anaplasmosis and ehrlichiosis, persist in the bloodstream of their mammalian hosts, allowing acquisition and transmission by tick vectors. Anaplasma marginale establishes persistent infection characterized by sequential cycles of rickettsaemia in which new antigenic variants emerge. The two most immunodominant outer membrane proteins, MSP2 and MSP3, are paralogues, each encoded by a distinct family of related genes. This study demonstrates that, although the two gene families have diverged substantially, each has maintained a similar mechanism to generate structurally and antigenically polymorphic surface antigens. Like MSP2, MSP3 is expressed from a single locus in which variation of the expressed msp3 gene is generated by recombination using msp3 pseudogenes. Each of the msp3 pseudogenes encodes a unique central variable region (CVR) flanked by conserved 5' and 3' regions. Changes in the CVR of the expressed msp3, concomitant with invariance of the pseudogenes, indicate that expression site variation is generated using gene conversion. A. marginale thus maintains two large, separate systems within its small genome to generate antigenic variation of its surface proteins, while analogous structural elements indicate a common mechanism.     

Differential adhesion of major surface proteins 1a and 1b of the ehrlichial cattle pathogen Anaplasma marginale to bovine erythrocytes and tick cells

Anaplasma marginale is a tick-borne ehrlichial pathogen of cattle for which six major surface proteins (MSPs) have been described. The MSP1 complex, a heterodimer composed of MSP1a and MSP1b, was shown to induce a protective immune response in cattle and both proteins have been identified as putative adhesins for bovine erythrocytes. In this study the role of MSP1a and MSP1b as adhesins for bovine erythrocytes and tick cells was defined. msp1alpha and msp1beta1 genes from the Oklahoma isolate of A. marginale were cloned and expressed in Escherichia coli K-12 under the control of endogenous and tac promoters for both low and high level protein expression. Expression of the recombinant polypeptides was confirmed and localised on the surface of transformed E. coli. The adhesion properties of MSP1a and MSP1b were determined by allowing recombinant E. coli expressing these surface polypetides to react with bovine erythrocytes, Dermacentor variabilis gut cells and cultured tick cells derived from embryonic Ixodes scapularis. Adhesion of the recombinant E. coli to the three cell types was determined using recovery adhesion and microtiter haemagglutination assays, and by light and electron microscopy. MSP1a was shown by all methods tested to be an adhesin for bovine erythrocytes and both native and cultured tick cells. In contrast, recombinant E. coli expressing MSP1b adhered only to bovine erythrocytes and not to tick cells. When low expression vectors were used, single E. coli expressing MSP1a was seen adhered to individual tick cells while reaction of tick cells with the E. coli/MSP1a/high expression vector resulted in adhesion of multiple bacteria per cell. With electron microscopy, fusion of E. coli cell membranes expressing MSP1a or MSP1b with erythrocyte membranes was observed, as well as fusion of tick cell membranes with E. coli membranes expressing MSP1a. These studies demonstrated differential adhesion for MSP1a and MSP1b for which MSP1a is an A. marginale adhesin for both bovine erythrocytes and tick cells while MSP1b is an adhesin only for bovine erythrocytes. The role of the MSP1 complex, therefore, appears to vary among vertebrate and invertebrate hosts.     

Cloning, sequencing, expression, and antigenic characterization of rMSP4 from Anaplasma marginale isolated from Paraná State, Brazil

Anaplasmosis is a bovine intraerythrocytic disease caused by the bacterium Anaplasma marginale; it causes significant economic losses in tropical and subtropical regions, worldwide. The msp4 gene of an A. marginale strain isolated in Paran , Brazil, was amplified by PCR and sequenced; its cloning into the pET102/D-TOPO vector produced an msp4-6xHis-V5-HP thioredoxin fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21(DE-3); the expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in the cell lysate. The inclusion bodies were solubilized with urea and the recombinant protein was purified by Ni-NTA column and dialyzed. This method produced a relatively high yield of rMSP4, which was used to immunize rabbits. The deduced amino acid sequence encoded by MSP4 showed 99% homology to A. marginale isolates from Florida, USA, and from Minas Gerais, Brazil. Both rMSP4 and native MSP4 were recognized by post-immunization rabbit serum, showing that rMSP4 has conserved epitopes. As antigenicity was preserved, rMSP4 might be useful for the development of vaccine against anaplasmosis.     

Antigenic variation of Anaplasma marginale msp2 occurs by combinatorial gene conversion

The rickettsial pathogen Anaplasma marginale establishes lifelong persistent infection in the mammalian reservoir host, during which time immune escape variants continually arise in part because of variation in the expressed copy of the immunodominant outer membrane protein MSP2. A key question is how the small 1.2 Mb A. marginale genome generates sufficient variants to allow long-term persistence in an immunocompetent reservoir host. The recombination of whole pseudogenes into the single msp2 expression site has been previously identified as one method of generating variants, but is inadequate to generate the number of variants required for persistent infection. In the present study, we demonstrate that recombination of a whole pseudogene is followed by a second level of variation in which small segments of pseudogenes recombine into the expression site by gene conversion. Evidence for four short sequential changes in the hypervariable region of msp2 coupled with the identification of nine pseudogenes from a single strain of A. marginale provides for a combinatorial number of possible expressed MSP2 variants sufficient for lifelong persistence.     

恶性疟裂殖子表面蛋白1合成基因在毕赤酵母中的表达

恶性疟原虫裂殖子表面蛋白1是当今疟疾疫苗主要的候选抗原。由于天然MSP1基因AT含量异常高(为74%),使得克隆全长天然基因无法实现。本文已全合成了msp1基因(4940bp),解决了该天然基因在异源系统中不稳定的问题。为制备大量msp1重组蛋白进行疫苗有效性试验,本研究建立了msp1基因在毕赤酵母中的表达,将合成的msp1基因克隆到毕赤酵母胞内表达载体pPIC3.5,构建了重组质粒pPIC3.5/msp1,用电击转化毕赤酵母得到重组转化子,经PCR证实msp1基因已整合于毕赤酵母染色体中。含有重组表达质粒的毕赤酵母菌经甲醇诱导后表达出全长msp1重组蛋白。表达产物能与识别MSP1分子二硫键依赖构象表位的特异性单抗发生很强的反应,表明msp1重组蛋白至少在该表位构象上与天然蛋白一致。从毕赤酵母中分离得到大量msp1为开展该蛋白的结构与功能,特别是测定其疟疾保护性免疫提供可能。     

Structural basis for segmental gene conversion in generation of Anaplasma marginale outer membrane protein variants

Bacterial pathogens in the genus Anaplasma generate surface coat variants by gene conversion of chromosomal pseudogenes into single-expression sites. These pseudogenes encode unique surface-exposed hypervariable regions flanked by conserved domains, which are identical to the expression site flanking domains. In addition, Anaplasma marginale generates variants by recombination of oligonucleotide segments derived from the pseudogenes into the existing expression site copy, resulting in a combinatorial increase in variant diversity. Using the A. marginale genome sequence to track the origin of sequences recombined into the msp2 expression site, we demonstrated that the complexity of the expressed msp2 increases during infection, reflecting a shift from recombination of the complete hypervariable region of a given pseudogene to complex mosaics with segments derived from hypervariable regions of different pseudogenes. Examination of the complete set of 1183 variants with segmental changes revealed that 99% could be explained by one of the recombination sites occurring in the conserved flanking domains and the other within the hypervariable region. Consequently, we propose an 'anchoring' model for segmental gene conversion whereby the conserved flanking sequences tightly align and anchor the expression site sequence to the pseudogene. Associated with the recombination sites were deletions, insertions and substitutions; however, these are a relatively minor contribution to variant generation as these occurred in less than 2% of the variants. Importantly, the anchoring model, which can account for more variants than a strict segmental sequence identity mechanism, is consistent with the number of msp2 variants predicted and empirically identified during persistent infection.     

重组裂殖子表面蛋白质1特异性抗体有效抑制恶性疟原虫体外生长

42kD恶性疟原虫裂殖子表面蛋白质 1C末端片段 (MSP1 42 )是当今重要的疟疾疫苗候选抗原。为获得大量构象正确的MSP1 42重组蛋白进行疫苗有效性试验 ,在毕氏酵母系统中分泌表达了MSP1 42重组蛋白。通过与一组特异性识别构象表位的单抗反应 ,该重组蛋白在重要构象表位上与天然蛋白质一致。由该蛋白质诱生的抗体能有效地抑制恶性疟原虫的体外生长 ,这些结果为进一步开展MSP1 42重组蛋白疫苗有效性试验提供了基础     

Linkage arrangement of Na,K-ATPase genes in the tetraploid-derived genome of the rainbow trout (Oncorhynchus mykiss)

As part of our efforts to characterize Na,K-ATPase isoforms in salmonid fish, we investigated the linkage arrangement of genes coding for the alpha and beta-subunits of the enzyme complex in the tetraploid-derived genome of the rainbow trout (Oncorhynchus mykiss). Genetic markers were developed from four of five previously characterized alpha-subunit isoforms (alpha1b, alpha1c, alpha2 and alpha3) and four expressed sequence tags derived from yet undescribed beta-subunit isoforms (beta1a, beta1b, beta3a and beta3b). Sex-specific linkage analysis of polymorphic loci in a reference meiotic panel revealed that Na,K-ATPase genes are generally dispersed throughout the rainbow trout genome. A notable exception was the colocalization of two alpha-subunit genes and one beta-subunit gene on linkage group RT-12, which may thus share a conserved orthologous segment with linkage group 1 in zebrafish (Danio rerio). Consistent with previously reported homeologous relationships among the chromosomes of the rainbow trout, primers designed from the alpha3-isoform detected a pair of duplicated genes on linkage groups RT-27 and RT-31. Similarly, the evolutionary conservation of homeologous regions on linkage groups RT-12 and RT-16 was further supported by the map localization of gene duplicates for the beta1b isoform. The detection of homeologs within each gene family also raises the possibility that novel isoforms may be discovered as functional duplicates.     

Evolution of antigen variation in the tick-borne pathogen Anaplasma phagocytophilum

Anaplasma phagocytophilum is an obligately intracellular tick-transmitted bacterial pathogen of humans and other animals. During the course of infection, A. phagocytophilum utilizes gene conversion to shuffle ～100 functional pseudogenes into a single expression cassette of the msp2(p44) gene, which codes for the major surface antigen and major surface protein 2 (MSP2). The role and extent of msp2(p44) recombination, particularly in hosts that only experience acute infections, is not clear. In the present study, we explored patterns of recombination and expression of the msp2(p44) gene of A. phagocytophilum in a serially infected mouse model. Even though the bacterium was passed rapidly among mice, minimizing the opportunities for the host to develop adaptive immunity, we detected the emergence of 34 unique msp2(p44) expression cassette variants. The expression of msp2(p44) pseudogenes did not follow a consistent pattern among different groups of mice, although some pseudogenes were expressed more frequently than others. In addition, among 263 expressed pseudogenes, 3 mosaic sequences each consisting of 2 different pseudogenes were identified. Population genetic analysis showed that genetic diversity and subpopulation differentiation tended to increase over time until stationarity was reached but that the variance that was observed in allele (expressed pseudogene) frequency could occur by drift alone only if a high variance in bacterial reproduction could be assumed. These findings suggest that evolutionary forces influencing antigen variation in A. phagocytophilum may comprise random genetic drift as well as some innate but apparently nonpurifying selection prior to the strong frequency-dependent selection that occurs cyclically after hosts develop strong adaptive immunity.     

Genetic characterization of Anaplasma ovis strains from bighorn sheep in Montana

Wildlife reservoir species and genetic diversity of Anaplasma ovis (Rickettsiales: Anaplasmataceae) have been poorly characterized. Bighorn sheep (Ovis canadensis), captured in Montana from December 2004 to January 2005, were tested for antibodies to Anaplasma spp.; the presence of A. ovis was determined by the characterization of major surface protein msp4 sequences. Anaplasma antibodies were detected in 25/180 (14%) sampled bighorn sheep and A. ovis msp4 sequences were amplified by polymerase chain reaction (PCR) and sequenced from 9/23 (39%) of seropositive animals. All animals were negative by PCR for the related pathogens, Anaplasma phagocytophilum and Anaplasma marginale. All msp4 sequences identified in the bighorn sheep were identical and corresponded to a single A. ovis genotype that was identical to a sheep isolate reported previously from Idaho. The finding of a single genotype of A. ovis in this wild herd of bighorn sheep was in contrast to the genetic diversity reported for A. marginale in cattle herds in the western United States and worldwide. These results demonstrated that bighorn sheep may be a wildlife reservoir of A. ovis in Montana.     

Functional and Immunological Relevance of Anaplasma marginale Major Surface Protein 1a Sequence and Structural Analysis

Bovine anaplasmosis is caused by cattle infection with the tick-borne bacterium, Anaplasma marginale. The major surface protein 1a (MSP1a) has been used as a genetic marker for identifying A. marginale strains based on N-terminal tandem repeats and a 5′-UTR microsatellite located in the msp1a gene. The MSP1a tandem repeats contain immune relevant elements and functional domains that bind to bovine erythrocytes and tick cells, thus providing information about the evolution of host-pathogen and vector-pathogen interactions. Here we propose one nomenclature for A. marginale strain classification based on MSP1a. All tandem repeats among A. marginale strains were classified and the amino acid variability/frequency in each position was determined. The sequence variation at immunodominant B cell epitopes was determined and the secondary (2D) structure of the tandem repeats was modeled. A total of 224 different strains of A. marginale were classified, showing 11 genotypes based on the 5′-UTR microsatellite and 193 different tandem repeats with high amino acid variability per position. Our results showed phylogenetic correlation between MSP1a sequence, secondary structure, B-cell epitope composition and tick transmissibility of A. marginale strains. The analysis of MSP1a sequences provides relevant information about the biology of A. marginale to design vaccines with a cross-protective capacity based on MSP1a B-cell epitopes.     

水稻msp1-4突变体的鉴定及其UDT1和GAMYB基因的表达分析

通过对粳稻‘9522’辐射诱变,得到一隐性雄性不育突变体msp1-4（MULTIPLE SPOROCYTE）,用遗传定位方法将该基因座位定位在分子标记WY-4和WY-8之间,相距0．8cM,物理距离247kb。测序分析证明这247kb区间中的MSP1基因的编码区在第758bp到767bp之间发生了10个碱基的缺失。形态学观察结果表明该突变体和已经报告过的msp1突变体的表型基本一致。为分析水稻其它与花药发育相关的基因在msp1-4中的表达变化,用半定量RT-PCR技术检测到影响绒毡层和花粉发育的重要基因UDT1和GAMYB的表达在突变体中比在野生型水稻中低,说明这2个基因可能位于MSP1基因的下游。     

Rapid and long-term disappearance of CD4+ T lymphocyte responses specific for Anaplasma marginale major surface protein-2 (MSP2) in MSP2 vaccinates following challenge with live A. marginale

In humans and ruminants infected with Anaplasma, the major surface protein 2 (MSP2) is immunodominant. Numerous CD4(+) T cell epitopes in the hypervariable and conserved regions of MSP2 contribute to this immunodominance. Antigenic variation in MSP2 occurs throughout acute and persistent infection, and sequentially emerging variants are thought to be controlled by variant-specific Ab. This study tested the hypothesis that challenge of cattle with Anaplasma marginale expressing MSP2 variants to which the animals had been immunized, would stimulate variant epitope-specific recall CD4(+) T cell and IgG responses and organism clearance. MSP2-specific T lymphocyte responses, determined by IFN-gamma ELISPOT and proliferation assays, were strong before and for 3 wk postchallenge. Surprisingly, these responses became undetectable by the peak of rickettsemia, composed predominantly of organisms expressing the same MSP2 variants used for immunization. Immune responsiveness remained insignificant during subsequent persistent A. marginale infection up to 1 year. The suppressed response was specific for A. marginale, as responses to Clostridium vaccine Ag were consistently observed. CD4(+)CD25(+) T cells and cytokines IL-10 and TGF-beta1 did not increase after challenge. Furthermore, a suppressive effect of nonresponding cells was not observed. Lymphocyte proliferation and viability were lost in vitro in the presence of physiologically relevant numbers of A. marginale organisms. These results suggest that loss of memory T cell responses following A. marginale infection is due to a mechanism other than induction of T regulatory cells, such as peripheral deletion of MSP2-specific T cells.     

Cell surface expression of hybrid murine/human MHC class II beta alpha dimers. Key influence of residues in the amino-terminal portion of the beta 1 domain

To aid in the identification of key residues responsible for the control of class II MHC beta-alpha dimer assembly and expression, a series of cotransfections of human plus mouse beta- and alpha-genes was performed. The resulting expression data were correlated with the sequences of the relevant proteins to identify residues that played critical roles in these processes. For the I-E/DR homologues good expression was seen for both E beta DR alpha and DR beta E alpha combinations involving several allelically variable beta-chains of each species. These results are consistent with the sequence conservation seen for I-E and DR gene products, and indicate that the species-specific differences that do exist play little role in controlling dimer formation or transport. For A beta chains, a more complex picture was seen. A beta d, but not A beta k or A beta b, was found to coexpress with human alpha-chains. Not only did A beta d show expression with the homologous DQ alpha-chain, but it also was expressed with DR alpha and DP alpha. These data indicate that species-specific residues do not control dimer expression under these conditions and confirm that allelically polymorphic residues have a crucial role in this process. Mapping studies using recombinant A beta genes established the importance of the residues in the amino-terminal half of the beta 1 domain in the differences observed among the A beta alleles. Sequence comparison of DR beta, DP beta, DQ beta, E beta, and A beta chains in this region revealed a single residue (position 12) conserved in most chains and differing in a nonconservative fashion between A beta d vs A beta b or k. A beta d has the conserved lysine at this position, whereas A beta b has methionine and A beta k has glutamine. To test whether this residue actually was important physiologically, a lysine codon was created in a recombinant A beta gene possessing the amino-terminal sequence of the kappa haplotype, and the ability of this mutant chain to be expressed with various mouse A alpha-chains was examined. This mutant chain was shown to gain the ability to be efficiently expressed with A alpha d without losing its ability to be expressed with A alpha k. These data reemphasize the special role played by allelically polymorphic residues in Ia expression and identify one such polymorphic site as position 12.(ABSTRACT TRUNCATED AT 400 WORDS)