Audrey O.T. Lau

Genome Sequence of Babesia bovis and Comparative Analysis of Apicomplexan Hemoprotozoa

Babesia bovis is an apicomplexan tick-transmitted pathogen of cattle imposing a global risk and severe constraints to livestock health and economic development. The complete genome sequence was undertaken to facilitate vaccine antigen discovery, and to allow for comparative analysis with the related apicomplexan hemoprotozoa Theileria parva and Plasmodium falciparum. At 8.2 Mbp, the B. bovis genome is similar in size to that of Theileria spp. Structural features of the B. bovis and T. parva genomes are remarkably similar, and extensive synteny is present despite several chromosomal rearrangements. In contrast, B. bovis and P. falciparum, which have similar clinical and pathological features, have major differences in genome size, chromosome number, and gene complement. Chromosomal synteny with P. falciparum is limited to microregions. The B. bovis genome sequence has allowed wide scale analyses of the polymorphic variant erythrocyte surface antigen protein (ves1 gene) family that, similar to the P. falciparum var genes, is postulated to play a role in cytoadhesion, sequestration, and immune evasion. The ∼150 ves1 genes are found in clusters that are distributed throughout each chromosome, with an increased concentration adjacent to a physical gap on chromosome 1 that contains multiple ves1-like sequences. ves1 clusters are frequently linked to a novel family of variant genes termed smorfs that may themselves contribute to immune evasion, may play a role in variant erythrocyte surface antigen protein biology, or both. Initial expression analysis of ves1 and smorf genes indicates coincident transcription of multiple variants. B. bovis displays a limited metabolic potential, with numerous missing pathways, including two pathways previously described for the P. falciparum apicoplast. This reduced metabolic potential is reflected in the B. bovis apicoplast, which appears to have fewer nuclear genes targeted to it than other apicoplast containing organisms. Finally, comparative analyses have identified several novel vaccine candidates including a positional homolog of p67 and SPAG-1, Theileria sporozoite antigens targeted for vaccine development. The genome sequence provides a greater understanding of B. bovis metabolism and potential avenues for drug therapies and vaccine development.

Comparative genomic analysis and phylogenetic position of Theileria equi

BackgroundTransmission of arthropod-borne apicomplexan parasites that cause disease and result in death or persistent infection represents a major challenge to global human and animal health. First described in 1901 as Piroplasma equi, this re-emergent apicomplexan parasite was renamed Babesia equi and subsequently Theileria equi, reflecting an uncertain taxonomy. Understanding mechanisms by which apicomplexan parasites evade immune or chemotherapeutic elimination is required for development of effective vaccines or chemotherapeutics. The continued risk of transmission of T. equi from clinically silent, persistently infected equids impedes the goal of returning the U. S. to non-endemic status. Therefore comparative genomic analysis of T. equi was undertaken to: 1) identify genes contributing to immune evasion and persistence in equid hosts, 2) identify genes involved in PBMC infection biology and 3) define the phylogenetic position of T. equi relative to sequenced apicomplexan parasites.ResultsThe known immunodominant proteins, EMA1, 2 and 3 were discovered to belong to a ten member gene family with a mean amino acid identity, in pairwise comparisons, of 39%. Importantly, the amino acid diversity of EMAs is distributed throughout the length of the proteins. Eight of the EMA genes were simultaneously transcribed. As the agents that cause bovine theileriosis infect and transform host cell PBMCs, we confirmed that T. equi infects equine PBMCs, however, there is no evidence of host cell transformation. Indeed, a number of genes identified as potential manipulators of the host cell phenotype are absent from the T. equi genome. Comparative genomic analysis of T. equi revealed the phylogenetic positioning relative to seven apicomplexan parasites using deduced amino acid sequences from 150 genes placed it as a sister taxon to Theileria spp.ConclusionsThe EMA family does not fit the paradigm for classical antigenic variation, and we propose a novel model describing the role of the EMA family in persistence. T. equi has lost the putative genes for host cell transformation, or the genes were acquired by T. parva and T. annulata after divergence from T. equi. Our analysis identified 50 genes that will be useful for definitive phylogenetic classification of T. equi and closely related organisms.

An overview of the Babesia, Plasmodium and Theileria genomes: A comparative perspective

Babesia, Plasmodium and Theileria form a triad of apicomplexan hemoparasites and are accountable for significant mortality and morbidity to humans and animals globally. Understanding the pathobiology of these three genera is crucial as multiple drug resistant strains continue to arise in endemic areas along with pesticide and acaricide resistant vector hosts. Vastly improved sequencing technology has produced whole genome sequences of several apicomplexan species and subsequent comparative analyses of these genomes have identified unique as well as common features among the different species, information that will help in the pursuit of alternative therapies, management and perhaps elimination of the disease. This review, therefore, summarizes comparisons of genome structure, protein families, metabolic pathways and organelle biology in these three apicomplexans and how such knowledge has and will continue to enhance the field.

ApicoAP: The First Computational Model for Identifying Apicoplast-Targeted Proteins in Multiple Species of Apicomplexa

Background Most of the parasites of the phylum Apicomplexa contain a relict prokaryotic-derived plastid called the apicoplast. This organelle is important not only for the survival of the parasite, but its unique properties make it an ideal drug target. The majority of apicoplast-associated proteins are nuclear encoded and targeted post-translationally to the organellar lumen via a bipartite signaling mechanism that requires an N-terminal signal and transit peptide (TP). Attempts to define a consensus motif that universally identifies apicoplast TPs have failed. Methodology/Principal Findings In this study, we propose a generalized rule-based classification model to identify apicoplast-targeted proteins (ApicoTPs) that use a bipartite signaling mechanism. Given a training set specific to an organism, this model, called ApicoAP, incorporates a procedure based on a genetic algorithm to tailor a discriminating rule that exploits the known characteristics of ApicoTPs. Performance of ApicoAP is evaluated for four labeled datasets of Plasmodium falciparum, Plasmodium yoelii, Babesia bovis, and Toxoplasma gondii proteins. ApicoAP improves the classification accuracy of the published dataset for P. falciparum to 94%, originally 90% using PlasmoAP. Conclusions/Significance We present a parametric model for ApicoTPs and a procedure to optimize the model parameters for a given training set. A major asset of this model is that it is customizable to different parasite genomes. The ApicoAP prediction software is available at http://code.google.com/p/apicoap/ and http://bcb.eecs.wsu.edu.

The gatekeeper residue and beyond: homologous calcium-dependent protein kinases as drug development targets for veterinarian Apicomplexa parasites.

Specific roles of individual CDPKs vary, but in general they mediate essential biological functions necessary for parasite survival. A comparative analysis of the structure-activity relationships (SAR) of Neospora caninum, Eimeria tenella and Babesia bovis calcium-dependent protein kinases (CDPKs) together with those of Plasmodium falciparum, Cryptosporidium parvum and Toxoplasma gondii was performed by screening against 333 bumped kinase inhibitors (BKIs). Structural modelling and experimental data revealed that residues other than the gatekeeper influence compound-protein interactions resulting in distinct sensitivity profiles. We subsequently defined potential amino-acid structural influences within the ATP-binding cavity for each orthologue necessary for consideration in the development of broad-spectrum apicomplexan CDPK inhibitors. Although the BKI library was developed for specific inhibition of glycine gatekeeper CDPKs combined with low inhibition of threonine gatekeeper human SRC kinase, some library compounds exhibit activity against serine- or threonine-containing CDPKs. Divergent BKI sensitivity of CDPK homologues could be explained on the basis of differences in the size and orientation of the hydrophobic pocket and specific variation at other amino-acid positions within the ATP-binding cavity. In particular, BbCDPK4 and PfCDPK1 are sensitive to a larger fraction of compounds than EtCDPK1 despite the presence of a threonine gatekeeper in all three CDPKs.

Babesia bovis: a comprehensive phylogenetic analysis of plastid-encoded genes supports green algal origin of apicoplasts.

Apicomplexan parasites commonly contain a unique, non-photosynthetic plastid-like organelle termed the apicoplast. Previous analyses of other plastid-containing organisms suggest that apicoplasts were derived from a red algal ancestor. In this report, we present an extensive phylogenetic study of apicoplast origins using multiple previously reported apicoplast sequences as well as several sequences recently reported. Phylogenetic analysis of amino acid sequences was used to determine the evolutionary origin of the organelle. A total of nine plastid genes from 37 species were incorporated in our study. The data strongly support a green algal origin for apicoplasts and Euglenozoan plastids. Further, the nearest green algae lineage to the Apicomplexans is the parasite Helicosporidium, suggesting that apicoplasts may have originated by lateral transfer from green algal parasite lineages. The results also substantiate earlier findings that plastids found in Heterokonts such as Odontella and Thalassiosira were derived from a separate secondary endosymbiotic event likely originating from a red algal lineage.

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.

Characterization of acyl carrier protein and LytB in Babesia bovis apicoplast

Graphical abstract Investigation of type II fatty acid and isoprenoid biosyntheses in Babesia resulted in the identification of two major components within the apicoplastic lumen. Highlights ► This study illustrates a four membrane babesid apicoplast. ► Babesia bovis apicoplast resides adjacent to the nucleus. ► Acyl carrier protein and LytB are transcribed and translated in Babesia bovis. ► Isoprenoid biosynthesis likely exists in Babesia bovis. ► Type II fatty acid biosynthesis may not be present in Babesia bovis.