Article

Universal primers suitable to assess population dynamics reveal apparent mutually exclusive transcription of the Babesia bovis ves1alpha gene.

Department of Infectious Diseases and Pathology, University of Florida, Gainesville, FL 32611-0880, USA.
Molecular and Biochemical Parasitology (Impact Factor: 2.24). 08/2009; 166(1):47-53. DOI: 10.1016/j.molbiopara.2009.02.008
Source: PubMed

ABSTRACT Babesia bovis is an intraerythrocytic hemoparasite of widespread distribution, which adversely affects livestock production in many regions of the world. This parasite establishes persistent infections of long duration, at least in part through rapid antigenic variation of the VESA1 protein on the infected-erythrocyte surface. To understand the dynamics of in vivo antigenic variation among the parasite population it is necessary to have sensitive and broadly applicable tools enabling monitoring of variation events in parasite antigen genes. To address this need for B. bovis, "universal" primers for the polymerase chain reaction have been designed for the ves1alpha gene, spanning from exon 2 to near the 3' end of cysteine-lysine-rich domain (CKRD) sequences in exon 3. These primers robustly amplified this segment, with minimal bias, from essentially the entire repertoire of full-length ves1alpha sequences in the B. bovis Mexico isolate genome, and are equivalently present in other isolates. On purified genomic DNA, this primer set can achieve a sensitivity of 10 genome equivalents or less. When applied to the amplification of cDNA derived from the B. bovis C9.1 clonal line evidence consistent with mutually exclusive transcription of the ves1alpha gene was obtained, concomitant with detection of numerous mutational events among members of the parasite population. These characteristics of the primers will facilitate the application of polymerase chain reaction-based methodologies to the study of B. bovis population and antigenic switching dynamics.

Download full-text

Full-text

Available from: David R Allred, Jul 06, 2015
0 Followers
 · 
152 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Small open reading frame (smorf) genes comprise the second largest Babesia bovis multigene family. All known 44 variant smorf genes are located in close chromosomal proximity to ves1 genes, which encode proteins that mediate cytoadhesion and contribute to immune evasion. In this study, we characterised the general topology of smorf genes and investigated the gene repertoire, transcriptional profile and SMORF expression in two distinct strains, T2Bo and Mo7. Sequence analysis using degenerate primers identified additional smorf genes in each strain and demonstrated that the smorf gene repertoire varies between strains, with conserved and unique genes in both. Smorf genes have multiple semi-conserved and variable blocks, and a large hypervariable insertion in 20 of the 44 genes defines two major branches of the family, termed smorf A and smorf B. A total of 32 smorf genes are simultaneously transcribed in T2Bo strain B. bovis merozoites obtained from deep brain tissue of an acutely infected animal. SMORF peptide-specific antiserum bound in immunoblots to multiple proteins with a range of sizes predicted by smorf genes, confirming translation of smorf gene products from these transcripts. These results indicate that the smorf multigene family is larger than previously described and demonstrate that smorf genes are expressed and are undergoing variation, both within strains and in a lineage-specific pattern independent of strain specificity. The function of these novel proteins is unknown.
    International journal for parasitology 11/2011; 42(2):131-8. DOI:10.1016/j.ijpara.2011.10.004 · 3.40 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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.
    Molecular and Biochemical Parasitology 03/2010; 171(2):81-8. DOI:10.1016/j.molbiopara.2010.03.001 · 2.24 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Over the past decade or so, our understanding of the biology of apicomplexan parasites has increased dramatically, particularly in the case of malaria. Notable achievements are the availability of complete genome sequences, transcriptome and proteome profiles and the establishment of in vitro transfection techniques for asexual-stage malaria parasites. Interestingly, despite their major economic importance and striking similarities with malaria, Babesia parasites have been relatively ignored, but change is on the horizon. Here, we bring together recent work on Babesia bovis parasites which are beginning to unravel the molecular mechanisms that underlie the pathogenesis of babesiosis and highlight some opportunities and challenges that lie ahead.
    Trends in Parasitology 12/2010; 26(12):591-9. DOI:10.1016/j.pt.2010.06.012 · 6.22 Impact Factor