Variable and Variant Protein Multigene Families in Babesia bovis Persistence

Abstract

Cattle infected with Babesia bovis face a bifurcated fate: Either die of the severe acute infection, or survive and carry for many years a highly persistent but generally asymptomatic infection. In this review, the author describes known and potential contributions of three variable or highly variant multigene-encoded families of proteins to persistence in the bovine host, and the mechanisms by which variability arises among these families. Ramifications arising from this variability are discussed.

Keywords: Babesia; antigenic variation; cytoadhesion; immune evasion; immunodominance; persistent infection.

Figure 1

Comparative structures of RAP-1, VESA1, and SmORF proteins. Comparative structures and significant domains of stereotypic examples of each of the protein families discussed in this review is provided here. (A) RAP-1 proteins have an N-terminal signal sequence (SS) for entrance into the endoplasmic reticulum and may have a site for post-translational covalent modification (PCM) that results in membrane anchoring. These proteins partition with integral membrane proteins but do not possess a transmembrane domain [6]. (B) VESA1 proteins do not possess an N-terminal signal sequence, but do have a C-terminal transmembrane domain (TM). VESA1a contains a cysteine-lysine-rich domain (CKRD), variable domain conserved sequences (VDCS), and a C-terminal cysteine-rich domain (CTC). VESA1a frequently contains coiled-coil sequences (thin blue lines beneath the VDCS domain). VESA1b possesses a CKRD, CTC, and TM domain but no VDCS domain. On the other hand, they contain a centrally located low-complexity variant domain (LCV) [15,31,55]. (C) SmORF proteins possess an SS domain for entry into the endoplasmic reticulum, and hypervariable regions (HVR) in the N-terminal third and near the C-terminus. Type A and B contain a central repeat domain (A/B Rpt), whereas type B possess an additional HVR and a second (or more) repeat region (A Rpt) [16]. Proteins are depicted roughly to scale relative to one another. SmORF proteins additionally possess a PEXEL-like motif not found in the other two families that is proteolytically processed as a part of trafficking and proper staging.

Figure 2

Predicted three-dimensional structure of RAP-1 polypeptide. The three-dimensional structure of RAP-1 was predicted using Robetta [56,57] and displayed with Chimera software [58]. The predicted structure is one of a flattened helix comprised of alternating α-helices in opposing orientations, with an overall curve. The sequences shown in orange are the mapped B-cell epitopes recognized by monoclonal antibodies 23/38.120 and MBOC79, respectively [43], whereas residues colored yellow represent a slightly degenerate form that is repeated eight times, only two copies of which appear to be accessible.

Figure 3

Immunolocalization of VESA1a and SmORF in Babesia bovis-infected erythrocytes. (A) B. bovis-infected bovine erythrocytes are shown by phase contrast, revealing paired merozoites. (B) Immunofluorescence was used to localize VESA1a proteins with monoclonal antibody 4D9.1G1 (red), and a model SmORF-GFP fusion protein with a rabbit anti-GFP antibody (green). Parasite nuclei are revealed by counterstaining with DAPI (blue). (C) Overlay of immunofluorescence and phase-contrast images to enable visualization of label relative to structures. Intensity ranges have been adjusted (uniformly) to facilitate visualization of faint fluorescence signals arising from within the erythrocyte cytoplasm. The separate movement of both proteins through the infected erythrocyte cytoplasm as small “packets”, many associated with vesicular structures (examples identified with arrowheads), is easily seen (described in [37]).

Figure 4

Segmental gene conversion of ves genes. Much of the ves gene family is organized as pairs of genes apposing single, bidirectional promoters (bent arrows). Transcription of ves genes occurs from a single locus at a time, which is referred to as the “Locus of active ves transcription” (LAT), although there are numerous silent ves loci with the potential to become an LAT through in situ switching. The ves1α and ves1β genes within the LAT are both progressively modified over time by SGC, a replacement of sequences by a gene conversion-like process involving short segments copied from silent ves loci (shown only for the ves1α gene). This is indicated by colored patches derived from silent loci that remain unchanged by the process of donation. The donor locus may be on the same or any of the other three nuclear chromosomes.