The Babesia bovis VESA1 virulence factor subunit 1b is encoded by the 1β 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 1α member of the ves multigene family. Since its discovery the 1β 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 ves1β genes are rapidly variant in antigenicity and size-polymorphic, matching known VESA1b polypeptides. Importantly, the ves1β-encoded antigens are co-precipitated with VESA1a during immunoprecipitation with antiVESA1a monoclonal antibodies, and antisera to ves1β polypeptide co-precipitate VESA1a. Further, the ves1β-encoded antigens significantly co-localize with VESA1a on the infectederythrocyte membrane surface of live cells. These characteristics all match known properties of VESA1b, allowing us to conclude that the ves1β gene divergently apposing the ves1α 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.

Characterization of the Unusual Bidirectional ves Promoters Driving VESA1 Expression and Associated with Antigenic Variation in Babesia bovis

“…Such a situation would seem more likely to result in transcription of either the ves1␣ or ves1␤ gene but not simultaneous transcription of both. This scenario is consistent with the recently reported independent and apparently temporally separated appearance of VESA1a and -1b polypeptides on the IRBC surface during parasite development (58). For the parasite to express VESA1 holoprotein in this case would require a switch in the transcriptional activity from one side of the IGr to the other, perhaps subject to local chromatin remodeling.…”

“…Regardless, these results imply that whatever mechanism underlies silencing of the ves gene family does not depend upon a strict, ves-specific promoter-counting mechanism, such as the exclusionary "transcription body" mechanism proposed to mediate monoallelic transcription of VSG genes in Trypanosoma brucei (36). Indeed, this effect is consistent with the sequential expression of VESA1a and -1b subunits on the IRBC surface (58). Active transcription of ves1␤ may suppress transcription from ves1␣ and vice versa.…”

“…The two subunits of VESA1 are encoded by members of the ves multigene family, with the 1a subunit encoded by ves1␣ genes (5) and the 1b subunit encoded by ves1␤ genes (58). There are approximately 150 members of the ves multigene family in the B. bovis genome (9).…”

“…The divergent, clustered organization of the actively transcribed genes could present the parasite with a mechanism by which to coordinately regulate the expression of the two genes and thus their encoded subunits. Given the adhesive function of this protein, the ability to arrive at a consistent subunit stoichiometry would seem to be beneficial, although this may not always be achieved (58). However, it also creates certain logistical problems and raises fundamental questions about how transcription may be organized to proceed bidirectionally, whether it involves simultaneous transcription of overlapping sequences or whether such overlap occurs within individual parasites.…”

Unusual chromatin structure associated with monoparalogous transcription of the Babesia bovis ves multigene family

“…These functions involve the variant erythrocyte surface antigen-1 (VESA1), a heterodimeric protein comprised of size-polymorphic subunits 1a and 1b (Allred et al, 1994; O’Connor and Allred, 2000). VESA1a and 1b are encoded by ves 1α and ves 1β genes, respectively, from closely related but distinct branches of the ves multigene family, of which there are well over 100 members in total (Allred et al, 2000; Al-Khedery and Allred, 2006; Brayton et al, 2007; Xiao et al, 2010). …”