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Microbial Immunity and Vaccines

Identification of Anaplasma marginale Outer Membrane Protein Antigens Conserved between A. marginale Sensu Stricto Strains and the Live A. marginale subsp. centrale Vaccine

Joseph T. Agnes, Kelly A. Brayton, Megan LaFollett, Junzo Norimine, Wendy C. Brown, Guy H. Palmer
R. P. Morrison, Editor
Joseph T. Agnes
Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, and The Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington 99164
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Kelly A. Brayton
Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, and The Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington 99164
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Megan LaFollett
Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, and The Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington 99164
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Junzo Norimine
Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, and The Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington 99164
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Wendy C. Brown
Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, and The Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington 99164
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Guy H. Palmer
Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, and The Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington 99164
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  • For correspondence: gpalmer@vetmed.wsu.edu
R. P. Morrison
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DOI: 10.1128/IAI.01174-10
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  • FIG. 1.
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    FIG. 1.

    Identification of A. marginale strain St. Maries proteins recognized by IgG2 induced by immunization with the A. marginale subsp. centrale vaccine strain. Gels were either stained with Sypro ruby for visualization of protein (top) or used for immunoblotting to detect bound IgG2 present in serum from A. marginale subsp. centrale vaccinate (sera from vaccinate C6175) (middle). As a homologous strain positive control, immunoblotting was performed using serum from an animal infected with the St. Maries strain, vaccinate C6170 (bottom). The pH gradient for all three images is given at the top of the figure. The molecular mass in kilodaltons (kDa) is indicated on the left-hand side of the images. Circles and numbers correspond to protein spots that were identified by LC/MS and are listed in Table 1.

  • FIG. 2.
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    FIG. 2.

    Locus positions of the immunoreactive A. marginale strain St. Maries (St. M) outer membrane proteins compared with orthologues present in the sensu stricto Florida strain (FL) and Israel vaccine strain (IS) of A. marginale subsp. centrale. The complete circular chromosome is shown as linear for illustration purposes, starting with the origin of replication. AM779, AMF575, and ACIS557 are orthologues; AM854, AMF640, and ACIS486 are orthologues. msp2 ES and msp3 ES represent the positions of the msp2 and msp3 expression sites, respectively.

  • FIG. 3.
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    FIG. 3.

    Mapping of IgG2 binding to conserved membrane regions versus variant surface domains of Msp2. Ten picomoles of recombinant full-length Msp2, 9H1 hypervariable region, N-terminal conserved region, and C-terminal conserved region were immunoblotted and probed for serum IgG2 binding from each of the A. marginale subsp. centrale vaccinates: C6171 (A), C6175 (B), C6187 (C), and C6188 (D). Serum from a St. Maries strain-infected animal, C6170 (E), was used as a positive control; preinfection serum from the same animal was used as a negative control (F). Probing with anti-His antibody was used as a positive control for the presence of each recombinant protein domain (G). The position and size of molecular mass standards are indicated to the left of the images.

Tables

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  • TABLE 1.

    Anaplasma marginale St. Maries strain proteins bound by serum IgG2 induced by live vaccination

    Protein typeSpot no.aProtein annotationMascot score(s) (standard)bNo. of peptides with P values of <0.05% sequence coverageMol wt (kDa)Theoretical pI
    Membrane727AM1220 Omp7843941.98.9
    727cAM1221 Omp8421242.99.2
    122, 727cAM1222 Omp979, 422, 16, 243.18.2
    424, 425, 426AM1255 Omp11534, 267, 36510, 5, 935, 23, 3334.86.7
    421, 715AM1258 Omp13139, 653, 16, 1238.07.6
    727AM075 Omp14301243.17.6
    710, 411, 712AM779 surface-expressed protein128, 38, 653, 1, 18, 2, 357.47.2
    724AM854 peptidoglycan-associated lipoprotein13831425.95.3
    123-130AM1144 Msp2132-6402-167-1642.16.0
    30, 112, 113AM1063 Msp3593, 84, 4814, 2, 114, 2, 2104.56.0
    Housekeeping122AM231 succinyl-CoA synthetase beta chain (SucC)9211141.85.5
    121, 122AM254 elongation factor Tu (TufB)647, 42214, 836, 3043.05.4
    702AM1131 isocitrate dehydrogenase (Icd)4011153.55.8
    116, 117AM944 Hsp chaperonin (GroEL)480, 2189, 525, 1858.45.2
    701, 703, 411AM1174 F1-ATP synthase alpha subunit (AtpA)290, 188, 297, 3, 121, 7, 154.66.0
    • ↵a Spot no. refers to the identification illustrated in Fig. 1.

    • ↵b Standard MASCOT scores are the sum of probability-based MASCOT ion scores for each individual peptide.

    • ↵c OMP8 and OMP9 hits for spot no. 727 are from the same peptide.

  • TABLE 2.

    Comparison of Anaplasma marginale subsp. centrale vaccine strain antigens with A. marginale sensu stricto orthologs

    Israel vaccine strain protein annotationSt. Maries strain protein annotation% identitya
    St. Maries strainFlorida strainMississippi strainPuerto Rico strainVirginia strain
    Membrane proteins
        ACIS_00178 Omp7AM1220 Omp770, 10068, 8669, 9467, 9468, 99
        ACIS_00178 Omp7AM1221 Omp866, 10064, 9066, 9076, 10064, 88
        ACIS_00178 Omp7AM1222 Omp964, 10064, 99--64, 98
        ACIS_00140 Omp11AM1255 Omp1160, 10060, 9754, 8761, 97-
        ACIS_00138 Omp13AM1258 Omp1363, 10057, 9860, 9459, 97-
        ACIS_00068 Omp14AM075 Omp1471, 10071, 9773, 99-71, 97
        ACIS_00557 undefined productAM779 surface-expressed protein84, 10084, 10084, 9984, 10084, 100
        ACIS_00486 peptidoglycan-associated lipoproteinAM854 peptidoglycan-associated lipoprotein79, 10079, 10079, 10079, 10079, 100
        ACIS_00229 Msp2 N-terminal conserved regionAM1144 Msp2 N-terminal conserved region84, 10084, 9983, 9984, 10082, 98
        ACIS_00229 Msp2 C-terminal conserved regionAM1144 Msp2 C-terminal conserved region90, 10091, 10086, 9793, 9793, 97
        ACIS_00229 Msp2 hypervariable regionsAM1144 Msp2 hypervariable regions33-74b, 10036-67, 35-100b---
        ACIS_00617 Msp3 N-terminal conserved regionAM1063 Msp3 N-terminal conserved region52, 10051, 7455, 7749, 7753,77
        ACIS_00617 Msp3 C-terminal conserved regionAM1063 Msp3 C-terminal conserved region84, 10085, 9888, 9978, 9671, 88
        ACIS_00617 Msp3 hypervariable regionsAM1063 Msp3 hypervariable regions18-48b, 10013-44, 14-100b---
    Housekeeping proteins
        ACIS_01060 succinyl-CoA synthetase (SucC)AM231 succinyl-CoA synthetase (SucC)98, 10099, 10097, 9998, 99-
        ACIS_01040 elongation factor Tu (TufB)AM254 elongation factor Tu (TufB)97, 10097, 10097, 10098, 10097, 100
        ACIS_00241 isocitrate dehydrogenase (Icd)AM1131 isocitrate dehydrogenase (Icd)99, 100100, 9999, 99100, 99100, 99
        ACIS_00394 chaperonin GroELAM944 60-kD chaperonin (GroEL)99, 10099, 10099, 10099, 10099, 100
        ACIS_00219 Atp synthase subunit alpha (AtpA)AM1174 F1-ATP synthase alpha subunit (AtpA)93, 10093, 10093, 10093, 9993, 100
    • ↵a The St. Maries, Florida, Mississippi, Puerto Rico, and Virginia strains were compared to the Israel and St. Maries strains. The first number indicates sequence identity to the Israel strain, and the second number indicates sequence identity to the St. Maries strain. The Mississippi, Puerto Rico, and Virginia strain comparisons were performed using pyrosequencing data that comprise approximately 96% of the full sequence. Only partial sequences of ortholog gene products were available (12). A dash (-) indicates a partial sequence that does not allow comparison, not the absence of the gene.

    • ↵b For Msp2 and Msp3 hypervariable regions, a range of sequence identities is given that represents the lowest and highest sequence identities observed when comparing all donor alleles in the genome.

  • TABLE 3.

    Amino acid sequence identities between Anaplasma marginale subsp. centrale (Israel vaccine strain) and A. marginale strain St. Maries Msp2 hypervariable region variants

    Israel vaccine Msp2 HVR variant% identity with St. Maries Msp2 HVR variant:
    Ψ1Ψ2a9H1P1bE6/F7
    G1c3648584863
    C3854495354
    F3748515042
    B16648335335
    A224340383842
    AF6154534367
    A17437435246
    • ↵a St. Maries strain donor allele Ψ2 is duplicated, with the duplicate, 3H1, having an identical sequence (7).

    • ↵b St. Maries donor allele Ψ P1 is duplicated, with the duplicate, G11, having an identical sequence (7).

    • ↵c A. marginale subsp. centrale (Israel) donor allele Ψ G1 is duplicated, with the duplicate, G2, having an identical sequence (21).

  • TABLE 4.

    Amino acid sequence identities between Anaplasma marginale subsp. centrale (Israel vaccine strain) and A. marginale strain St. Maries Msp3 hypervariable region variants

    Israel vaccine Msp3 HVR variant% identity with St. Maries Msp3 HVR variant:
    1234567
    130364243331929
    222345341422440
    326383145393441
    430311935343134
    521344829322334
    624202723323323
    727263147443133
    822253718301830
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Identification of Anaplasma marginale Outer Membrane Protein Antigens Conserved between A. marginale Sensu Stricto Strains and the Live A. marginale subsp. centrale Vaccine
Joseph T. Agnes, Kelly A. Brayton, Megan LaFollett, Junzo Norimine, Wendy C. Brown, Guy H. Palmer
Infection and Immunity Feb 2011, 79 (3) 1311-1318; DOI: 10.1128/IAI.01174-10

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Identification of Anaplasma marginale Outer Membrane Protein Antigens Conserved between A. marginale Sensu Stricto Strains and the Live A. marginale subsp. centrale Vaccine
Joseph T. Agnes, Kelly A. Brayton, Megan LaFollett, Junzo Norimine, Wendy C. Brown, Guy H. Palmer
Infection and Immunity Feb 2011, 79 (3) 1311-1318; DOI: 10.1128/IAI.01174-10
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