The Babesia bovis VESA1 virulence factor subunit 1b is encoded by the 1beta branch of the ves multigene family

Abstract

Babesia bovis, an intraerythrocytic parasite of cattle, establishes persistent infections of extreme duration. This is accomplished, at least in part, through rapid antigenic variation of a heterodimeric virulence factor, the variant erythrocyte surface antigen-1 (VESA1) protein. Previously, the VESA1a subunit was demonstrated to be encoded by a 1alpha member of the ves multigene family. Since its discovery the 1beta branch of this multigene family has been hypothesized to encode the VESA1b polypeptide, but formal evidence for this connection has been lacking. Here, we provide evidence that products of ves1beta genes are rapidly variant in antigenicity and size-polymorphic, matching known VESA1b polypeptides. Importantly, the ves1beta-encoded antigens are co-precipitated with VESA1a during immunoprecipitation with anti-VESA1a monoclonal antibodies, and antisera to ves1beta polypeptide co-precipitate VESA1a. Further, the ves1beta-encoded antigens significantly co-localize with VESA1a on the infected-erythrocyte membrane surface of live cells. These characteristics all match known properties of VESA1b, allowing us to conclude that the ves1beta gene divergently apposing the ves1beta gene within the locus of active ves transcription (LAT) encodes the 1b subunit of the VESA1 cytoadhesion ligand. However, the extent and stoichiometry of VESA1a and 1b co-localization on the surface of individual cells is quite variable, implicating competing effects on transcription, translation, or trafficking of the two subunits. These results provide essential information facilitating further investigation into this parasite virulence factor.

Figure 1. Structure of the LAT ves1β gene and recombinant immunogens

The structure of the LAT-associated ves1β gene is shown in schematic. Exons which are a part of the translated open reading frame are shown as thick white bars and introns as numbered, narrow gray bars. The narrow black line represents 5'- and 3'-untranslated sequences. The orange segments within the exons are patches of sequence conserved among ves genes (adapted from [30]). The segments incorporated into recombinant immunogens are indicated as follows: fragments included in the r-v1β(f1-3) immunogen are indicated by blue arrows labeled “F1”, “F2”, and “F3”, whereas sequences contained in the r-v1β(765) immunogen are indicated by the red arrow labeled “1–765”. The position of the peptide incorporated in the v1β(peptide) immunogen is indicated by a green bar. Note that only exon-encoded sequences taken from a mature mRNA-derived cDNA were included in each immunogen. “CKRD” indicates the position of the cysteine-lysine-rich domain. The black bar near the 3' end indicates the predicted transmembrane domain, and “ATG” and “TAG” are the translational start and stop codons, respectively.

Figure 2. Western blot reactivity of anti-ves1β polypeptide antibodies

Antigen prepared from non-infected erythrocytes, IE materials from B. bovis clonal lines C9.1, CD7, CE11, and MO7, or recombinant E. coli were reacted with the antisera to identify the sizes and variant-specificity of the recognized antigens. (A) Top panel contains antigens reacted with mAb 4D9.1G1 (left half; “4D9”) and R6a-v1β765 (right half; “R6”); bottom panel contains antigens reacted with R5a-v1β765 (left half; “R5”) and pooled preimmune from rabbits R5 and R6 (right half; “Pre”). Lanes are labeled based upon the B. bovis line antigen contained, or “NRBC” for normal, uninfected red blood cell antigens. (B) R6a-v1β765 antiserum was reacted with E. coli expressing various polypeptides, either as soluble antigens or from the BugBuster-insoluble pellet. Samples contained lysate from E. coli expressing recombinant ves1β(1–765) polypeptide (rv1β765); recombinant VESA1a polypeptide (rV1a); glutathione-S-transferase (GST); or non-recombinant E. coli (Ec). The right-most lane shows a lighter exposure of the internal pellet “rv1β765” sample to facilitate visualization of major bands contained within this sample. (C) The top panel shows recognition of VESA1a in various antigenically variant B. bovis clonal lines by mAb 4D9.1G1 (4D9), whereas reactivity of the Chicken a-v1β(f1-3) with the same blot following stripping is shown in the middle panel (Cav1β). The bottom panel is a superimposition of exposures from the two probings. Gel lanes contain antigen from B. bovis IE of the CE11, CD7, and C9.1 lines, or NRBC. Note the size-polymorphism of both VESA1a and the ves1β-encoded proteins. In all panels, black arrowheads indicate VESA1b-size polypeptides, white arrowheads indicate VESA1a polypeptides, and the asterisk indicates an unexpected, size-invariant, IE-associated antigen recognized only by R5a-v1β765. Numbers to the left or right of the figures refer to molecular mass standards (in kDa).

Figure 3. Immunoprecipitation (IP) analysis of metabolically labeled B. bovis IE antigens

(A) Conventional IP analysis of variant B. bovis IE antigens solubilized in NP40 lysis buffer prior to addition of antibody. Above each set of samples is labeled the variant B. bovis line involved (MO7, C9.1, CD7, and CE11), whereas gel lanes are labeled according to the antibody used in the immunoprecipitation. (B) Conventional IP analysis of non-denatured and 8M urea-denatured B. bovis C9.1 line IE antigens. Lanes are labeled to indicate reaction between non-denatured (N) or denatured (D) antigen, and the antibody source. (C) Surface-specific IP analysis of C9.1 and CD7 variant B. bovis clonal line IE. Parasite line is indicated above each sample set. Lanes are labeled according to the antibody used during immunoprecipitation. In all three panels only the region of the gel around the VESA1 bands is shown. The asterisk indicates a presumably unrelated parasite antigen captured from all lines by antibodies present in R6 serum prior to immunization. The white and black arrowheads indicate the VESA1a and 1b subunits, respectively, which were co-precipitated by mAb 4D9.1G1. The antibodies used in this figure include: 4D9, mAb 4D9.1G1; R5, R5a-v1β765, R6, R6a-v1β765; P6, rabbit 6 preimmune serum; MB, anti-Rap1 mAb MBOC79B1; B2442, bovine #2442 infection serum to B. bovis MO7; and P2442, bovine #2442 preimmune serum. “MW std” indicates molecular mass standards. Numbers on the left indicate positions of molecular mass standards (in kDa).

Figure 4. Confirmation of VESA1b identity through re-capture IP of VESA1 subunits

B. bovis C9.1 line VESA1 was immunoprecipitated directly from non-denatured (N) or 8M urea-denatured (D) antigen. Alternatively, antigen was captured from non-denatured antigen with mAb 4D9.1G1, then was released by denaturation with 8M urea, diluted to allow antibody binding, and re-captured with a second antibody of different specificity. The order of reagents used in this experiment is indicated, as well as the nature of the antigens. The designation “U” refers to uncaptured antigens left over after recapture with the indicated antibodies. Only the region of the gel around the VESA1 complex is shown. The lower panel is a lighter exposure of the same region to facilitate visualization of bands in more heavily exposed samples. Antibodies used include: 4D9, mAb 4D9.1G1; R6, R6a-v1β765; P6, rabbit 6 preimmune serum; MBOC, mAb MBOC79B1. Asterisk and numbering on the left have the same meanings as in Figure 3

Figure 5. Live-cell immunofluorescence of B. bovis C9.1 line IE

Mature stage B. bovis C9.1 line IE were reacted with mAb 4D9.1G1 and R6α-v1β765 (panel A; five individual cells of increasing parasite maturity and antigen density are shown) or with mAb 4D9.1G1 and P6 (panel B). Localization of VESA1a by mAb 4D9.1G1 is shown in red and reactivity of the rabbit antisera with VESA1b is shown in green. The top row shows phase contrast images of the individual IE, whereas the second row shows patterns of immunoreactivity superimposed upon the phase contrast images. Note the finely punctate pattern of labeling by rabbit antisera, which is indistinguishable from that of the monoclonal antibody. Co-localization of the two antigens is shown in the bottom row by the superimposition of blue color where red and green signals significantly coincide (ratio ≥0.10 of other signal). Note also the clearly variable ratios of the two antigens, and the occasional signals apparently derived from one antigen only. Single signal foci show up as red or green, whereas co-localized signals appear in varying shades of purple, lavender, and cyan, depending upon the calculated signal ratios. The white bar represents 5 µm; magnification is identical in each image.