Up-regulated expression of spherical body protein 2 truncated copy 11 in Babesia bovis is associated with reduced cytoadhesion to vascular endothelial cells.

The factors involved in gain or loss of virulence in Babesia bovis are unknown. Spherical body protein 2 truncated copy 11 (sbp2t11) transcripts in B. bovis were recently reported to be a marker of attenuation for B. bovis strains. Increased cytoadhesion of B. bovis-infected red blood cells (iRBC) to vascular endothelial cells is associated with severe disease outcomes and an indicator of parasite virulence. Here, we created a stable B. bovis transfected line over-expressing sbp2t11 to determine whether up-regulation of sbp2t11 is associated with changes in cytoadhesion. This line was designated sbp2t11up and five B. bovis clonal lines were derived from the sbp2t11up line by limiting dilution for characterisation. We compared the ability of iRBCs from the sbp2t11up line and its five derivative clonal lines to adhere to bovine brain endothelial cells, using an in vitro cytoadhesion assay. The same lines were selected for in vitro cytoadhesion and the levels of sbp2t11 transcripts in each selected line were quantified. Our results demonstrate that up-regulation of sbp2t11 is accompanied by a statistically significant reduction in cytoadhesion. Confirmed up-regulation of sbp2t11 in B. bovis concomitant with the reduction of iRBC in vitro cytoadhesion to bovine brain endothelial cell is consistent with our previous finding that up-regulation of sbp2t11 is an attenuation marker in B. bovis and suggests the involvement of sbp2t11 transcription in B. bovis virulence.

Extended-spectrum antiprotozoal bumped kinase inhibitors: A review.

Many life-cycle processes in parasites are regulated by protein phosphorylation. Hence, disruption of essential protein kinase function has been explored for therapy of parasitic diseases. However, the difficulty of inhibiting parasite protein kinases to the exclusion of host orthologues poses a practical challenge. A possible path around this difficulty is the use of bumped kinase inhibitors for targeting calcium-dependent protein kinases that contain atypically small gatekeeper residues and are crucial for pathogenic apicomplexan parasites' survival and proliferation. In this article, we review efficacy against the kinase target, parasite growth in vitro, and in animal infection models, as well as the relevant pharmacokinetic and safety parameters of bumped kinase inhibitors.

Expression of 6-Cys Gene Superfamily Defines Babesia bovis Sexual Stage Development within Rhipicephalus microplus.

Babesia bovis, an intra-erythrocytic tick-borne apicomplexan protozoan, is one of the causative agents of bovine babesiosis. Its life cycle includes sexual reproduction within cattle fever ticks, Rhipicephalus spp. Six B. bovis 6-Cys gene superfamily members were previously identified (A, B, C, D, E, F) where their orthologues in Plasmodium parasite have been shown to encode for proteins required for the development of sexual stages. The current study identified four additional 6-Cys genes (G, H, I, J) in the B. bovis genome. These four genes are described in the context of the complete ten 6-Cys gene superfamily. The proteins expressed by this gene family are predicted to be secreted or surface membrane directed. Genetic analysis comparing the 6-Cys superfamily among five distinct B. bovis strains shows limited sequence variation. Additionally, A, B, E, H, I and J genes were transcribed in B. bovis infected tick midgut while genes A, B and E were also transcribed in the subsequent B. bovis kinete stage. Transcription of gene C was found exclusively in the kinete. In contrast, transcription of genes D, F and G in either B. bovis infected midguts or kinetes was not detected. None of the 6-Cys transcripts were detected in B. bovis blood stages. Subsequent protein analysis of 6-Cys A and B is concordant with their transcript profile. The collective data indicate as in Plasmodium parasite, certain B. bovis 6-Cys family members are uniquely expressed during sexual stages and therefore, they are likely required for parasite reproduction. Within B. bovis specifically, proteins encoded by 6-Cys genes A and B are markers for sexual stages and candidate antigens for developing novel vaccines able to interfere with the development of B. bovis within the tick vector.

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.

Tick passage results in enhanced attenuation of Babesia bovis.

Serial blood passage of virulent Babesia bovis in splenectomized cattle results in attenuated derivatives that do not cause neurologic disease. Tick transmissibility can be lost with attenuation, but when retained, attenuated B. bovis can revert to virulence following tick passage. This study provides data showing that tick passage of the partially attenuated B. bovis T2Bo derivative strain further decreased virulence compared with intravenous inoculation of the same strain in infected animals. Ticks that acquired virulent or attenuated parasites by feeding on infected cattle were transmission fed on naive, splenectomized animals. While there was no significant difference between groups in the number of parasites in the midgut, hemolymph, or eggs of replete female ticks after acquisition feeding, animals infected with the attenuated parasites after tick transmission showed no clinical signs of babesiosis, unlike those receiving intravenous challenge with the same attenuated strain prior to tick passage. Additionally, there were significantly fewer parasites in blood and tissues of animals infected with tick-passaged attenuated parasites. Sequencing analysis of select B. bovis genes before and after tick passage showed significant differences in parasite genotypes in both peripheral blood and cerebral samples. These results provide evidence that not only is tick transmissibility retained by the attenuated T2Bo strain, but also it results in enhanced attenuation and is accompanied by expansion of parasite subpopulations during tick passage that may be associated with the change in disease phenotype.

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.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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 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.

ApicoAMP: the first computational model for identifying apicoplast-targeted transmembrane proteins in Apicomplexa.

BACKGROUND: Computational identification of apicoplast-targeted proteins is important in drug target determination for diseases such as malaria. While there are established methods for identifying proteins with a bipartite signal in multiple species of Apicomplexa, not all apicoplast-targeted proteins possess this bipartite signature. The publication of recent experimental findings of apicoplast membrane proteins, called transmembrane proteins, that do not possess a bipartite signal has made it feasible to devise a machine learning approach for identifying this new class of apicoplast-targeted proteins computationally. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we develop a method for predicting apicoplast-targeted transmembrane proteins for multiple species of Apicomplexa, whereby several classifiers trained on different feature sets and based on different algorithms are evaluated and combined in an ensemble classification model to obtain the best expected performance. The feature sets considered are the hydrophobicity and composition characteristics of amino acids over transmembrane domains, the existence of short sequence motifs over cytosolically disposed regions, and Gene Ontology (GO) terms associated with given proteins. Our model, ApicoAMP, is an ensemble classification model that combines decisions of classifiers following the majority vote principle. ApicoAMP is trained on a set of proteins from 11 apicomplexan species and achieves 91% overall expected accuracy. CONCLUSIONS/SIGNIFICANCE: ApicoAMP is the first computational model capable of identifying apicoplast-targeted transmembrane proteins in Apicomplexa. The ApicoAMP prediction software is available at http://code.google.com/p/apicoamp/ and http://bcb.eecs.wsu.edu.

Loss of neurovirulence is associated with reduction of cerebral capillary sequestration during acute Babesia bovis infection.

BACKGROUND: Severe neurological signs that develop during acute infection by virulent strains of Babesia bovis are associated with sequestration of infected erythrocytes in cerebral capillaries. Serial passage of virulent strains in cattle results in attenuated derivatives that do not cause neurologic disease. We evaluated whether serial passage also results in a loss of cerebral capillary sequestration by examining brain biopsies during acute disease and at necropsy. FINDINGS: Cerebral biopsies of spleen intact calves inoculated intravenously with a virulent or attenuated strain pair of B. bovis were evaluated for capillary sequestration at the onset of babesiosis and during severe disease. In calves infected with the virulent strain, there was a significant increase in sequestration between the first and second biopsy timepoint. The attenuated strain was still capable of sequestration, but at a reduced level, and did not change significantly between the first and second biopsy. Necropsy examination confirmed the second biopsy results and demonstrated that sequestration identified at necropsy reflects pathologic changes occurring in live animals. CONCLUSIONS: Loss of neurovirulence after serial in vivo passage of the highly virulent T2Bo strain of B. bovis in splenectomized animals is associated with a significant reduction of cerebral capillary sequestration. Previous genomic analysis of this and two other strain pairs suggests that this observation could be related to genomic complexity, particularly of the ves gene family, rather than consistent gene specific differences. Additional experiments will examine whether differential gene expression of ves genes is also associated with reduced cerebral sequestration and neurovirulence in attenuated strains.

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.

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.

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

Babesia bovis contains a prokaryotic derived organelle known as the apicoplast. Many participants of the metabolic pathways within the apicoplast are encoded in the nuclear genome and post-translationally imported with the help of a bipartite signal. Recently, an all encompassing algorithm was derived to predict apicoplast targeted proteins for many non-Plasmodium apicomplexans in which it reported the presence of 260 apicoplast targeted proteins in Babesia. One of these proteins is glutamyl tRNA synthetase (GltX). This study investigates if the putative bipartite signal of GltX alone is sufficient to direct proteins into the apicoplast. Using a transient transfection system consisting of a green fluorescent protein as the reporter, we tested the signal and transit portions of the bipartite signal in apicoplastic transport. We first identified the transcript of gltX to be expressed during the asexual blood stages and subsequently confirmed that the complete bipartite signal is responsible for directing the reporter protein into a compartment distinct from the nucleus and the mitochondrion. As GltX bipartite signal successfully guided the reporter protein into the apicoplast, our finding implies that it also directs native GltX into the same organelle.

Characterization of acyl carrier protein and LytB in Babesia bovis apicoplast.

The apicoplast is a highly specialized organelle that mediates required functions in the growth and replication of apicomplexan parasites. Despite structural conservation of the apicoplast among different parasite genera and species, there are also critical differences in the metabolic requirements of different parasites and at different stages of the life cycle. To specifically compare apicoplast pathways between parasites that have both common and unique stages, we characterized the apicoplast in Babesia bovis, which has only intraerythrocytic asexual stages in the mammalian host, and compared it to that of Plasmodium falciparum, which has both asexual intraerythrocytic and hepatic stages. Specifically focusing on the type II fatty acid (FASII) and isoprenoid (MEP) biosynthesis pathways, we searched for pathway components and retention of active sites within the genome, localized key components [acyl carrier protein (ACP) and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB)] to the apicoplast, and demonstrated that the N-terminal bipartite signals of both proteins are required and sufficient for trafficking to the apicoplast lumen. Using specific pharmacologic inhibition, we demonstrated that MEP biosynthesis may be disrupted and its presence is required for intraerythrocytic growth of B. bovis asexual stages, consistent with the genomic pathway analysis and with its requirement in the asexual erythrocytic stages of P. falciparum. In contrast, FASII biosynthesis may or may not be present and specific drug targets did not have any inhibitory effect to B. bovis intraerythrocytic growth, which is consistent with the lack of requirement for P. falciparum intraerythrocytic growth. However, genomic analysis revealed the loss of FASII pathway components in B. bovis whereas the pathway is intact for P. falciparum but regulated to be expressed when needed (hepatic stages) and silent when not (intraerythrocytic stages). The results indicate specialized molding of apicoplast biosynthetic pathways to meet the requirements of individual apicomplexan parasites and their unique intracellular niches.

Expression and strain variation of the novel "small open reading frame" (smorf) multigene family in Babesia bovis.

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.

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

Identifying virulence determinants in Apicomplexan parasites remains a major gap in knowledge for members within this phylum. We hypothesized that peptidases would segregate with virulence between a virulent parent Babesia bovis strain and an attenuated daughter strain derived by rapid in vivo passage. Using the complete genome sequence of the virulent T2Bo strain, 66 peptidases were identified and active sites confirmed. The presence, sequence identity and expression levels were tested for each of the 66 peptidases in the virulent parent and attenuated daughter T2Bo strains using whole genome, targeted sequencing approaches and microarrays analyses. Quantitative PCR revealed that there was no significant difference in peptidase expression between the virulent and attenuated strains. We conclude that while peptidases may well play a required role in B. bovis pathogenesis, neither loss of peptidase gene content nor reduced gene expression underlies the loss of virulence associated with in vivo passage and attenuation.

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

Multiple genetically distinct strains of a pathogen circulate and compete for dominance within populations of animal reservoir hosts. Understanding the basis for genotypic strain structure is critical for predicting how pathogens respond to selective pressures and how shifts in pathogen population structure can lead to disease outbreaks. Evidence from related Apicomplexans such as Plasmodium, Toxoplasma, Cryptosporidium and Theileria suggests that various patterns of population dynamics exist, including but not limited to clonal, oligoclonal, panmictic and epidemic genotypic strain structures. In Babesia bovis, genetic diversity of variable merozoite surface antigen (VMSA) genes has been associated with disease outbreaks, including in previously vaccinated animals. However, the extent of VMSA diversity within a defined population in an endemic area has not been examined. We analyzed genotypic diversity and temporal change of MSA-1, a member of the VMSA family, in individual infected animals within a reservoir host population. Twenty-eight distinct MSA-1 genotypes were identified within the herd. All genotypically distinct MSA-1 sequences clustered into three groups based on sequence similarity. Two thirds of the animals tested changed their dominant MSA-1 genotypes during a 6-month period. Five animals within the population contained multiple genotypes. Interestingly, the predominant genotypes within those five animals also changed over the 6-month sampling period, suggesting ongoing transmission or emergence of variant MSA-1 genotypes within the herd. This study demonstrated an unexpected level of diversity for a single copy gene in a haploid genome, and illustrates the dynamic genotype structure of B. bovis within an individual animal in an endemic region. Co-infection with multiple diverse MSA-1 genotypes provides a basis for more extensive genotypic shifts that characterizes outbreak strains.

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.

Babesia bovis: transcriptional analysis of rRNA gene unit expression.

The complex life cycle of Babesia bovis includes erythrocytic stages in the bovine host and other stages occurring inside its common tick vector Rhipicephalus microplus. In related apicomplexa, changing environmental conditions affect the expression of ribosomal RNA, but it remained unknown whether the polymorphic A, B, and C rRNA coding units of B. bovis are differentially expressed. Northern blot analysis confirmed that polymorphic regions in the B. bovis 18s and ITS-2 rRNA coding units are transcribed. Then, rRNA transcript expression profiles were compared by analyzing cDNA libraries generated from total RNA extracted from in vitro cultured parasites, B. bovis infected cattle, R. microplus larvae and egg sources. The 18s and ITS-2 expression profiles indicate that rRNA unit B is almost exclusively expressed in cultured parasites while units A, B, and C are co-transcribed in the in vivo total RNA sources. Collectively, the data indicate that differential transcription of rRNA occurs in B.bovis, depending on the life stage of the parasite and on the environment.

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.