Universal primers suitable to assess population dynamics reveal apparent mutually exclusive transcription of the Babesia bovis ves1α gene

Department of Infectious Diseases and Pathology, University of Florida, Gainesville, FL 32611-0880, USA.
Molecular and Biochemical Parasitology (Impact Factor: 1.79). 08/2009; 166(1):47-53. DOI: 10.1016/j.molbiopara.2009.02.008
Source: PubMed


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.

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    • "Expression of the surface antigen is commonly restricted to a specific locus, while most or all other gene copies are silenced. Available evidence suggests this is the case for ves1 genes in B. bovis (25) and perhaps ves genes among Babesia spp. in general (this study). Periodic switching of a new variant antigen gene, in whole or in part, into the expression site from silent loci, or epigenetic transcriptional switching among members of the multigene family, then results in antigenic variation on the parasite (or IRBC) surface. "
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    ABSTRACT: Babesia spp. are tick-borne, intraerythrocytic hemoparasites that use antigenic variation to resist host immunity, through sequential modification of the parasite-derived variant erythrocyte surface antigen (VESA) expressed on the infected red blood cell surface. We identified the genomic processes driving antigenic diversity in genes encoding VESA (ves1) through comparative analysis within and between three Babesia species, (B. bigemina, B. divergens and B. bovis). Ves1 structure diverges rapidly after speciation, notably through the evolution of shortened forms (ves2) from 5′ ends of canonical ves1 genes. Phylogenetic analyses show that ves1 genes are transposed between loci routinely, whereas ves2 genes are not. Similarly, analysis of sequence mosaicism shows that recombination drives variation in ves1 sequences, but less so for ves2, indicating the adoption of different mechanisms for variation of the two families. Proteomic analysis of the B. bigemina PR isolate shows that two dominant VESA1 proteins are expressed in the population, whereas numerous VESA2 proteins are co-expressed, consistent with differential transcriptional regulation of each family. Hence, VESA2 proteins are abundant and previously unrecognized elements of Babesia biology, with evolutionary dynamics consistently different to those of VESA1, suggesting that their functions are distinct.
    Nucleic Acids Research 05/2014; 42(11). DOI:10.1093/nar/gku322 · 9.11 Impact Factor
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    • "Also interestingly is that only a single ves divergently oriented αβ pair was detected to be significantly upregulated in L17_V as opposed to three upregulated divergently oriented ves αβ pairs in T2Bo_V. As mutually exclusive transcription of this gene family occurs [28], the detection of three ves αβ pairs may suggest that the parasites which eventually express these “functional” VESA1 heterodimers represent the dominant members within the heterologous T2Bo virulent population while the detection of a single ves αβ pair in L17_V sample may imply that only one dominant parasite which expresses functional VESA1 αβ is present within the L17 virulent population. If divergently oriented ves αβ pair’s transcription and subsequent expression are associated with virulence severity, then T2B0_V may be more virulent than L17_V. "
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    ABSTRACT: Loss of virulence is a phenotypic adaptation commonly seen in prokaryotic and eukaryotic pathogens. This mechanism is not well studied, especially in organisms with multiple host and life cycle stages such as Babesia, a tick-transmitted hemoparasite of humans and animals. B. bovis, which infects cattle, has naturally occurring virulent strains that can be reliably attenuated in vivo. Previous studies suggest the virulence loss mechanism may involve post-genomic modification. We investigated the transcriptome profiles of two geographically distinct B. bovis virulent and attenuated strain pairs to better understand virulence loss and to gain insight into pathogen adaptation strategies. Expression microarray and RNA-sequencing approaches were employed to compare transcriptome profiles of two B. bovis strain pairs, with each pair consisting of a virulent parental and its attenuated derivative strain. Differentially regulated transcripts were identified within each strain pair. These included genes encoding for VESA1, SmORFs, undefined membrane and hypothetical proteins. The majority of individual specific gene transcripts differentially regulated within a strain were not shared between the two strains. There was a disproportionately greater number of ves genes upregulated in the virulent parental strains. When compared with their attenuated derivatives, divergently oriented ves genes were included among the upregulated ves genes in the virulent strains, while none of the upregulated ves genes in the attenuated derivatives were oriented head to head. One gene family whose specific members were consistently and significantly upregulated in expression in both attenuated strains was spherical body protein (SBP) 2 encoding gene where SBP2 truncated copies 7, 9 and 11 transcripts were all upregulated. We conclude that ves heterodimer pair upregulation and overall higher frequency of ves gene expressions in the virulent strains is consistent with the involvement of this gene family in virulence. This is logical given the role of VESA1 proteins in cytoadherence of infected cells to other infected erythrocytes or to endothelial cells. However, upregulation of some ves genes in the attenuated derivatives suggests that the consequence of upregulation is gene-specific. Furthermore, upregulation of the spherical body protein 2 gene family may play a role in the attenuated phenotype. Exactly how these two gene families may contribute to the loss or gain of virulence is discussed.
    BMC Genomics 11/2013; 14(1):763. DOI:10.1186/1471-2164-14-763 · 3.99 Impact Factor
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    • "It would have been less likely in this study to identify transcripts from smorf genes potentially present in the gap using gene-specific primers, and it is possible that the entire repertoire of smorf transcription is larger than found if potential genes in the gap are also transcriptionally active. Transcription occurred from both paired and unpaired smorf genes, unlike the mutually exclusive transcription that has been identified in the ves1alpha gene family of B. bovis (Zupanska et al., 2009). Since the T2Bo strain is an oligoclonal population (Perez-Llaneza et al., 2010), we analysed transcription from a clonal line of B. bovis grown in vitro. "
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    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 · 3.87 Impact Factor
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