The Babesia bovis merozoite surface antigen 2 locus contains four tandemly arranged and expressed genes encoding immunologically distinct proteins

Abstract

Members of the variable merozoite surface antigen (vmsa) gene family of Babesia bovis encode membrane proteins involved in erythrocyte invasion. In this study, we have identified and sequenced the complete 8.3-kb genomic locus containing msa-2, a member of the vmsa family, in the biologically cloned Mexico Mo7 strain. Four tandemly arranged copies of msa-2-related genes were found in the locus. The four genes, designated msa-2a(1) (which corresponds to the originally described msa-2 gene), msa-2a(2), msa-2b, and msa-2c, were shown to be transcribed and expressed and encode proteins with open reading frames ranging in size from 266 (MSA-2c) to 317 (MSA-2a(1)) amino acids. MSA-2a(1) and -2a(2) are the most closely related of the four proteins (90% identity), differing by (i) the number of 24-amino-acid repeats that comprise a surface-exposed B-cell epitope and (ii) the presence of a 32-amino-acid area of recombination between MSA-2a(2) and -2b. In contrast, msa-2c is most closely related to the previously described babr 0.8 gene in Australia strains of B. bovis. Comparison of MSA-2 proteins in the Argentina R1A strain of B. bovis with the Mexico Mo7 clone revealed a relatively high degree of conservation (83.6, 69.4, 79.1, and 88.7% amino acid identity for MSA-2a(1), -2a(2), -2b, and -2c, respectively), in contrast to the extensive MSA-1 sequence variation (52% identity) between the same two strains. Postinfection bovine immune serum contains antibodies that bound to each of the recombinant MSA-2 proteins. Blocking assays demonstrated the presence of unique B-cell epitopes in MSA-2a(1), -2b, and -2c. The results support the evolution of the msa-2 locus through at least two gene duplications, with selection for multiple related but antigenically distinct merozoite surface proteins.

FIG. 1.

The B. bovis Mo7 msa-2 locus has four tandemly arranged msa-2 genes. (A) Southern blot of BamHI-treated genomic DNA from B. bovis Mo7 (lane 1) and DNA from a λ/EMBL-phage clone (λ/EMBL locus) containing an 8.3-kb genomic fragment (lane 2) with the msa-2-specific digoxigenin-labeled oligonucleotide msa-2-F1 (Table 1). Lane 3, DNA ladder. Hybridization was detected by chemiluminescence. (B) Map of the Mo7 msa-2 locus based on sequencing of the 8.3-kb genomic fragment. ORFs are indicated by patterned boxes. Arrows indicate the orientation of the coding DNA strand. orfA shows no significant homology to other known genes, and orfB has strong homology to several thiolases and acetyl-CoA acetyltransferases. Restriction sites for BamHI, HindIII, BglII, and EcoRI are indicated. The subfragments msa2-sub1, msa2-sub2, and msa2-sub3 shown at the bottom were generated by PCR from Mo7 DNA for sequencing.

FIG. 2.

Confirmation of gene arrangement within the msa-2 locus. B. bovis Mo7 DNA was treated with BamHI plus BglII (lane 1) or BamHI (lane 2) and analyzed by Southern blotting with digoxigenin-labeled oligonucleotide probes corresponding in lane 1 to the initial 18 5′ nt of msa-2a₁, -2a₂, and -2b (oligonucleotide msa-2-F1 in Table 1) or in lane 2 to the initial 18 5′ nt of msa-2c (oligonucleotide msa-2c-F in Table 1). Hybridization was detected by chemiluminescence. Size markers in kilobases are shown on the left.

FIG. 3.

Sequence comparisons of B. bovis Mo7 msa-2 genes and encoded proteins. (A) Phylogram of the vmsa family. B. bovis rap1 is used as an outlier, and the most parsimonious tree is shown. Bootstrap values are indicated at branch points. (B) Alignment of MSA-2a₁, -2a₂, and -2b. Areas of amino acid identity among all three proteins are enclosed in black boxes; amino acids conserved between two of the proteins have a gray background, and variant amino acids have a white background. Deletions are indicated by dashed lines. The sequence conserved among MSA-1, -2a₁, and -2a₂ is indicated by a bold line above the sequence, and the predicted site for cleavage of the amino-terminal signal peptide is indicated by an arrow. (C) Fractionated alignment and sequence comparison between Mo7 MSA-2c and Australia K strain BabR 0.8. The amino acid sequences were aligned and divided into three segments according to the degree of conservation. The ORF of BabR 0.8 starts 71 aa downstream from the start codon of MSA-2c. Therefore, the region between aa 1 and 70 of MSA-2c was not included in this alignment. Areas of amino acid identity are enclosed in black boxes, conservative substitutions have a gray background, and variant amino acid substitutions have a white background.

FIG. 4.

Detailed sequence comparison of MSA-2a₁, -2a₂, and -2b. (A) MSA-2a₁ aa 207 to 264 and MSA-2a₂ aa 207 to 240 are aligned to demonstrate changes in the repeat region. Amino acid repeats are enclosed in dark gray boxes. The location of the previously defined B-cell epitope (12) is shown. (B) MSA-2a₁, -2a₂, and -2b aa 118 to 186 are aligned to demonstrate the area of recombination (asterisk and bracket). Conserved regions among all three peptides that flank the recombination site and the conserved YYKK sequence are enclosed in dark gray boxes. (C) Secondary structure prediction of MSA-2a_1. The predicted membrane orientation calculated with TMPred (http://www.ch.embnet.org/software/TMPRED_form.html) is plotted on the y axis against the amino acid position on the x axis. The solid line, i→o, is the calculation performed from N terminal to C terminal. The dashed line, o→i, is the calculation performed C terminal to N terminal. The most hydrophilic region of the molecule, which coincides with the area of recombination, is marked with an asterisk.

FIG. 5.

All four msa-2 genes are transcribed in erythrocytic stages of B. bovis Mo7. Ethidium bromide-stained agarose gel of amplicons of each msa-2 gene or transcript generated by using gene-specific primers (as detailed in Materials and Methods and Table 1) in PCR. Lanes 1, amplification of Mo7 cDNA after reverse transcription of mRNA; lanes 2, same as lane 1, without addition of RT; lanes 3, amplification of Mo7 DNA. Size markers are shown to the right.

FIG. 6.

Reactivity of postinfection and monospecific serum antibodies against msa-2 gene products. (A) Immunoblots showing the reactivity of bovine preimmune and anti-Mo7 postinfection immune serum (1:500) against recombinant MSA-2 antigens. (B) Anti-Mo7 immune serum was blocked with each purified recombinant MSA-2 antigen as indicated, and the reactivity was tested against the different rMSA-2 antigens as in panel A. (C) Immunoblot showing the reactivity of MSA-2a₁ monospecific, polyclonal serum antibodies and MAb 23/70.174 with native B. bovis merozoite antigens. Lane 1, control mouse serum; lane 2, polyclonal mouse serum against rMSA-2a₁; lane 3, MAb 23/70.174; lane 4, polyclonal mouse serum against rMSA-2b run as a size marker; lane 5, control MAb Tryp1E1. The position of the 45-kDa molecular size marker is indicated on the right.

FIG. 7.

Amino acid conservation between MSA-2 peptides from the B. bovis Mo7 and R1A strains. DNA from the Argentina R1A strain was amplified by PCR with primers msa-2-F1 and B42/44R for the msa-2a₁, -2a₂, and -2b genes and msa-2c-F and B42/44R for msa-2c. Amplification products were cloned, and several clones were sequenced to identify R1A orthologues of msa-2a₁, -2a₂, -2b, and -2c based on the degree of sequence identity. The figure shows alignments for each gene product. Areas of amino acid identity are enclosed in black boxes, conservative amino acid substitutions have a gray background, and variant amino acids have a white background. Deletions are indicated with dashed lines. The repeat region in MSA-2a₁ and -2a₂ (bold line above sequence), conserved YYKK sequence (bracket and ∗), and recombination sites between MSA-2a₂ and -2b (∗∗) are indicated.