Monica J. Pedroni

Structure and evolution of a proviral locus of Glyptapanteles indiensis bracovirus

BackgroundBracoviruses (BVs), a group of double-stranded DNA viruses with segmented genomes, are mutualistic endosymbionts of parasitoid wasps. Virus particles are replication deficient and are produced only by female wasps from proviral sequences integrated into the wasp genome. Virus particles are injected along with eggs into caterpillar hosts, where viral gene expression facilitates parasitoid survival and therefore perpetuation of proviral DNA. Here we describe a 223 kbp region of Glyptapanteles indiensis genomic DNA which contains a part of the G. indiensis bracovirus (GiBV) proviral genome.ResultsEighteen of ~24 GiBV viral segment sequences are encoded by 7 non-overlapping sets of BAC clones, revealing that some proviral segment sequences are separated by long stretches of intervening DNA. Two overlapping BACs, which contain a locus of 8 tandemly arrayed proviral segments flanked on either side by ~35 kbp of non-packaged DNA, were sequenced and annotated. Structural and compositional analyses of this cluster revealed it exhibits a G+C and nucleotide composition distinct from the flanking DNA. By analyzing sequence polymorphisms in the 8 GiBV viral segment sequences, we found evidence for widespread selection acting on both protein-coding and non-coding DNA. Comparative analysis of viral and proviral segment sequences revealed a sequence motif involved in the excision of proviral genome segments which is highly conserved in two other bracoviruses.ConclusionContrary to current concepts of bracovirus proviral genome organization our results demonstrate that some but not all GiBV proviral segment sequences exist in a tandem array. Unexpectedly, non-coding DNA in the 8 proviral genome segments which typically occupies ~70% of BV viral genomes is under selection pressure suggesting it serves some function(s). We hypothesize that selection acting on GiBV proviral sequences maintains the genetic island-like nature of the cluster of proviral genome segments described herein. In contrast to large differences in the predicted gene composition of BV genomes, sequences that appear to mediate processes of viral segment formation, such as proviral segment excision and circularization, appear to be highly conserved, supporting the hypothesis of a single origin for BVs.

Attenuation of virulence in an apicomplexan hemoparasite results in reduced genome diversity at the population level

BackgroundVirulence acquisition and loss is a dynamic adaptation of pathogens to thrive in changing milieus. We investigated the mechanisms of virulence loss at the whole genome level using Babesia bovis as a model apicomplexan in which genetically related attenuated parasites can be reliably derived from virulent parental strains in the natural host. We expected virulence loss to be accompanied by consistent changes at the gene level, and that such changes would be shared among attenuated parasites of diverse geographic and genetic background.ResultsSurprisingly, while single nucleotide polymorphisms in 14 genes distinguished all attenuated parasites from their virulent parental strains, all non-synonymous changes resulted in no deleterious amino acid modification that could consistently be associated with attenuation (or virulence) in this hemoparasite. Interestingly, however, attenuation significantly reduced the overall populations genome diversity with 81% of base pairs shared among attenuated strains, compared to only 60% of base pairs common among virulent parental parasites. There were significantly fewer genes that were unique to their geographical origins among the attenuated parasites, resulting in a simplified population structure among the attenuated strains.ConclusionsThis simplified structure includes reduced diversity of the variant erythrocyte surface 1 (ves) multigene family repertoire among attenuated parasites when compared to virulent parental strains, possibly suggesting that overall variance in large protein families such as Variant Erythrocyte Surface Antigens has a critical role in expression of the virulence phenotype. In addition, the results suggest that virulence (or attenuation) mechanisms may not be shared among all populations of parasites at the gene level, but instead may reflect expansion or contraction of the population structure in response to shifting milieus.

Comparative transcriptome analysis of geographically distinct virulent and attenuated Babesia bovis strains reveals similar gene expression changes through attenuation

BackgroundLoss 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.ResultsExpression 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.ConclusionsWe 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 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.

Genotypic diversity of merozoite surface antigen 1 of Babesia bovis within an endemic population

Graphical abstract Investigation of the population dynamics of pathogens contributes to a better understanding of disease transmission. Herein, the extent of Babesia bovis genotypic strain diversity within a defined cohort is reported.

Genome-wide analysis of peptidase content and expression in a virulent and attenuated Babesia bovis strain pair

Graphical abstract Bovipain-2 (in red), a Babesia bovis cysteine protease, is released into the host erythrocyte. Bovipain-2 is expressed in both virulent and attenuated B. bovis. DAPI stains the nuclei of two B. bovis. Highlights ► Genome sequence of the virulent T2Bo strain reveals 66 peptidases which constitute 2% of the annotated coding sequences. ► Each of the peptidases identified in the virulent parent was represented in the attenuated daughter strain. ► Microarrays analyses of transcript levels for all 66 peptidase-encoding genes were investigated. ► Our study discusses our findings in regards to peptidase involvement in in vivo attenuation of B. bovis.

Bumped kinase inhibitor prohibits egression in Babesia bovis

Babesiosis is a global zoonotic disease acquired by the bite of a Babesia-infected Ixodes tick or through blood transfusion with clinical relevance affecting humans and animals. In this study, we evaluated a series of small molecule compounds that have previously been shown to target specific apicomplexan enzymes in Plasmodium, Toxoplasma and Cryptosporidium. The compounds, bumped kinase inhibitors (BKIs), have strong therapeutic potential targeting apicomplexa-specific calcium dependent protein kinases (CDPKs). We investigated if BKIs also show inhibitory activities against piroplasms such as Babesia. Using a subset of BKIs that have promising inhibitory activities to Plasmodium and Toxoplasma, we determined that their actions ranged from 100% and no inhibition against Babesia bovis blood stages. One specific BKI, RM-1-152, showed complete inhibition against B. bovis within 48h and was the only BKI that showed noticeable phenotypic changes to the parasites. Focusing our study on this BKI, we further demonstrated that RM-1-152 has Babesia-static activity and involves the prohibition of merozoite egress while replication and re-invasion of host cells are unaffected. The distinct, abnormal phenotype induced by RM-1-152 suggests that this BKI can be used to investigate less studied cellular processes such as egression in piroplasm.

Babesia bovis: a bipartite signal directs the glutamyl-tRNA synthetase to the apicoplast.

Graphical abstract Highlights ► Protein traffic into the apicoplastic lumen requires a bipartite signal. ► Glutamyl tRNA synthetase (GltX) is a putative apicoplast targeted protein. ► GltX contains a bipartite signal sequence, a pre-requisite for apicoplast traffic. ► GltX bipartite sequence successfully directed GFP into the apicoplast. ► Native GltX may utilize its bipartite sequence for apicoplast entry.

Target specific-trisubstituted pyrrole inhibits Babesia bovis erythrocytic growth

Babesiosis, a significant veterinary disease and an emerging zoonotic human infection, is caused by certain species of the protozoan parasite, Babesia. Here we report that a trisubstituted pyrrole is a potent inhibitor of Babesia bovis, a bovine parasite. Furthermore, B. bovis expresses the known target of the compound, the cGMP dependent protein kinase. Target conservation and the in vitro efficacy support further investigation of this compound and validation of Babesia cGMP dependent protein kinase as its in vivo target.