Integration of DNA Repair, Antigenic Variation, Cytoadhesion, and Chance in Babesia Survival: A Perspective.

Apicomplexan parasites live in hostile environments in which they are challenged chemically and their hosts attempt in many ways to kill them. In response, the parasites have evolved multiple mechanisms that take advantage of these challenges to enhance their survival. Perhaps the most impressive example is the evolutionary co-option of DNA repair mechanisms by the parasites as a means to rapidly manipulate the structure, antigenicity, and expression of the products of specific multigene families. The purpose of variant proteins that mediate cytoadhesion has long been thought to be primarily the avoidance of splenic clearance. Based upon known biology, I present an alternative perspective in which it is survival of the oxidative environment within which Babesia spp. parasites live that has driven integration of DNA repair, antigenic variation, and cytoadhesion, and speculate on how genome organization affects that integration. This perspective has ramifications for the development of parasite control strategies.

Babesia bovis Rad51 ortholog influences switching of ves genes but is not essential for segmental gene conversion in antigenic variation.

The tick-borne apicomplexan parasite, Babesia bovis, a highly persistent bovine pathogen, expresses VESA1 proteins on the infected erythrocyte surface to mediate cytoadhesion. The cytoadhesion ligand, VESA1, which protects the parasite from splenic passage, is itself protected from a host immune response by rapid antigenic variation. B. bovis relies upon segmental gene conversion (SGC) as a major mechanism to vary VESA1 structure. Gene conversion has been considered a form of homologous recombination (HR), a process for which Rad51 proteins are considered pivotal components. This could make BbRad51 a choice target for development of inhibitors that both interfere with parasite genome integrity and disrupt HR-dependent antigenic variation. Previously, we knocked out the Bbrad51 gene from the B. bovis haploid genome, resulting in a phenotype of sensitivity to methylmethane sulfonate (MMS) and apparent loss of HR-dependent integration of exogenous DNA. In a further characterization of BbRad51, we demonstrate here that ΔBbrad51 parasites are not more sensitive than wild-type to DNA damage induced by γ-irradiation, and repair their genome with similar kinetics. To assess the need for BbRad51 in SGC, RT-PCR was used to observe alterations to a highly variant region of ves1α transcripts over time. Mapping of these amplicons to the genome revealed a significant reduction of in situ transcriptional switching (isTS) among ves loci, but not cessation. By combining existing pipelines for analysis of the amplicons, we demonstrate that SGC continues unabated in ΔBbrad51 parasites, albeit at an overall reduced rate, and a reduction in SGC tract lengths was observed. By contrast, no differences were observed in the lengths of homologous sequences at which recombination occurred. These results indicate that, whereas BbRad51 is not essential to babesial antigenic variation, it influences epigenetic control of ves loci, and its absence significantly reduces successful variation. These results necessitate a reconsideration of the likely enzymatic mechanism(s) underlying SGC and suggest the existence of additional targets for development of small molecule inhibitors.

Disruption of VirB6 Paralogs in Anaplasma phagocytophilum Attenuates Its Growth.

Many pathogenic bacteria translocate virulence factors into their eukaryotic hosts by means of type IV secretion systems (T4SS) spanning the inner and outer membranes. Genes encoding components of these systems have been identified within the order Rickettsiales based upon their sequence similarities to other prototypical systems. Anaplasma phagocytophilum strains are obligate intracellular, tick-borne bacteria that are members of this order. The organization of these components at the genomic level was determined in several Anaplasma phagocytophilum strains, showing overall conservation, with the exceptions of the virB2 and virB6 genes. The virB6 loci are characterized by the presence of four virB6 copies (virB6-1 through virB6-4) arranged in tandem within a gene cluster known as the sodB-virB operon. Interestingly, the virB6-4 gene varies significantly in length among different strains due to extensive tandem repeats at the 3' end. To gain an understanding of how these enigmatic virB6 genes function in A. phagocytophilum, we investigated their expression in infected human and tick cells. Our results show that these genes are expressed by A. phagocytophilum replicating in both cell types and that VirB6-3 and VirB6-4 proteins are surface exposed. Analysis of an A. phagocytophilum mutant carrying the Himar1 transposon within the virB6-4 gene demonstrated that the insertion not only disrupted its expression but also exerted a polar effect on the sodB-virB operon. Moreover, the altered expression of genes within this operon was associated with the attenuated in vitro growth of A. phagocytophilum in human and tick cells, indicating the importance of these genes in the physiology of this obligate intracellular bacterium in such different environments.IMPORTANCE Knowledge of the T4SS is derived from model systems, such as Agrobacterium tumefaciens The structure of the T4SS in Rickettsiales differs from the classical arrangement. These differences include missing and duplicated components with structural alterations. Particularly, two sequenced virB6-4 genes encode unusual C-terminal structural extensions resulting in proteins of 4,322 (GenBank accession number AGR79286.1) and 9,935 (GenBank accession number ANC34101.1) amino acids. To understand how the T4SS is used in A. phagocytophilum, we describe the expression of the virB6 paralogs and explore their role as the bacteria replicate within its host cell. Conclusions about the importance of these paralogs for colonization of human and tick cells are supported by the deficient phenotype of an A. phagocytophilum mutant isolated from a sequence-defined transposon insertion library.

A symbiotic bacterium of shipworms produces a compound with broad spectrum anti-apicomplexan activity.

Apicomplexan parasites cause severe disease in both humans and their domesticated animals. Since these parasites readily develop drug resistance, development of new, effective drugs to treat infection caused by these parasites is an ongoing challenge for the medical and veterinary communities. We hypothesized that invertebrate-bacterial symbioses might be a rich source of anti-apicomplexan compounds because invertebrates are susceptible to infections with gregarines, parasites that are ancestral to all apicomplexans. We chose to explore the therapeutic potential of shipworm symbiotic bacteria as they are bona fide symbionts, are easily grown in axenic culture and have genomes rich in secondary metabolite loci [1,2]. Two strains of the shipworm symbiotic bacterium, Teredinibacter turnerae, were screened for activity against Toxoplasma gondii and one strain, T7901, exhibited activity against intracellular stages of the parasite. Bioassay-guided fractionation identified tartrolon E (trtE) as the source of the activity. TrtE has an EC50 of 3 nM against T. gondii, acts directly on the parasite itself and kills the parasites after two hours of treatment. TrtE exhibits nanomolar to picomolar level activity against Cryptosporidium, Plasmodium, Babesia, Theileria, and Sarcocystis; parasites representing all branches of the apicomplexan phylogenetic tree. The compound also proved effective against Cryptosporidium parvum infection in neonatal mice, indicating that trtE may be a potential lead compound for preclinical development. Identification of a promising new compound after such limited screening strongly encourages further mining of invertebrate symbionts for new anti-parasitic therapeutics.

Knockout of Babesia bovis rad51 ortholog and its complementation by expression from the BbACc3 artificial chromosome platform.

Babesia bovis establishes persistent infections of long duration in cattle, despite the development of effective anti-disease immunity. One mechanism used by the parasite to achieve persistence is rapid antigenic variation of the VESA1 cytoadhesion ligand through segmental gene conversion (SGC), a phenomenon thought to be a form of homologous recombination (HR). To begin investigation of the enzymatic basis for SGC we initially identified and knocked out the Bbrad51 gene encoding the B. bovis Rad51 ortholog. BbRad51 was found to be non-essential for in vitro growth of asexual-stage parasites. However, its loss resulted in hypersensitivity to methylmethane sulfonate (MMS) and an apparent defect in HR. This defect rendered attempts to complement the knockout phenotype by reinsertion of the Bbrad51 gene into the genome unsuccessful. To circumvent this difficulty, we constructed an artificial chromosome, BbACc3, into which the complete Bbrad51 locus was inserted, for expression of BbRad51 under regulation by autologous elements. Maintenance of BbACc3 makes use of centromeric sequences from chromosome 3 and telomeric ends from chromosome 1 of the B. bovis C9.1 line. A selection cassette employing human dihydrofolate reductase enables recovery of transformants by selection with pyrimethamine. We demonstrate that the BbACc3 platform is stably maintained once established, assembles nucleosomes to form native chromatin, and expands in telomere length over time. Significantly, the MMS-sensitivity phenotype observed in the absence of Bbrad51 was successfully complemented at essentially normal levels. We provide cautionary evidence, however, that in HR-competent parasites BbACc3 can recombine with native chromosomes, potentially resulting in crossover. We propose that, under certain circumstances this platform can provide a useful alternative for the genetic manipulation of this group of parasites, particularly when regulated gene expression under the control of autologous elements may be important.

Variable and Variant Protein Multigene Families in Babesia bovis Persistence.

Cattle infected with Babesia bovis face a bifurcated fate: Either die of the severe acute infection, or survive and carry for many years a highly persistent but generally asymptomatic infection. In this review, the author describes known and potential contributions of three variable or highly variant multigene-encoded families of proteins to persistence in the bovine host, and the mechanisms by which variability arises among these families. Ramifications arising from this variability are discussed.

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.

Extensive Shared Chemosensitivity between Malaria and Babesiosis Blood-Stage Parasites.

The apicomplexan parasites that cause malaria and babesiosis invade and proliferate within erythrocytes. To assess the potential for common antiparasitic treatments, we measured the sensitivities of multiple species of Plasmodium and Babesia parasites to the chemically diverse collection of antimalarial compounds in the Malaria Box library. We observed that these parasites share sensitivities to a large fraction of the same inhibitors and we identified compounds with strong babesiacidal activity.

Shared elements of host-targeting pathways among apicomplexan parasites of differing lifestyles.

Apicomplexans are a diverse group of obligate parasites occupying different intracellular niches that require modification to meet the needs of the parasite. To efficiently manipulate their environment, apicomplexans translocate numerous parasite proteins into the host cell. Whereas some parasites remain contained within a parasitophorous vacuole membrane (PVM) throughout their developmental cycle, others do not, a difference that affects the machinery needed for protein export. A signal-mediated pathway for protein export into the host cell has been characterized in Plasmodium parasites, which maintain the PVM. Here, we functionally demonstrate an analogous host-targeting pathway involving organellar staging prior to secretion in the related bovine parasite, Babesia bovis, a parasite that destroys the PVM shortly after invasion. Taking into account recent identification of a similar signal-mediated pathway in the coccidian parasite Toxoplasma gondii, we suggest a model in which this conserved pathway has evolved in multiple steps from signal-mediated trafficking to specific secretory organelles for controlled secretion to a complex protein translocation process across the PVM.

The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic innovation underlying host-parasite interaction.

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.

Unusual chromatin structure associated with monoparalogous transcription of the Babesia bovis ves multigene family.

Rapid antigenic variation in Babesia bovis involves the variant erythrocyte surface antigen-1 (VESA1), a heterodimeric protein with subunits encoded by two branches of the ves multigene family. The ves1α and ves1ß gene pair encoding VESA1a and 1b, respectively, are transcribed in a monoparalogous manner from a single locus of active ves transcription (LAT), just one of many quasi-palindromic ves loci. To determine whether this organization plays a role in transcriptional regulation, chromatin structure was first assessed. Limited treatment of isolated nuclei with micrococcal nuclease to assay nucleosomal patterning revealed a periodicity of 156-159 bp in both bulk chromatin and specific gene coding regions. This pattern also was maintained in the intergenic regions (IGr) of non-transcribed ves genes. In contrast, the LAT IGr adopts a unique pattern, yielding an apparent cluster of five closely-spaced hypersensitive sites flanked by regions of reduced nucleosomal occupancy. ves loci fall into three patterns of overall sensitivity to micrococcal nuclease or DNase I digestion, with only the LAT being consistently very sensitive. Non-transcribed ves genes are inconsistent in their sensitivity to the two enzymatic probes. Non-linear DNA structure in chromatin was investigated to determine whether unique structure arising as a result of the quasi-palindromic nature of the LAT may effect transcriptional control. The in vitro capacity of ves IGr sequences to adopt stable higher-order DNA structure is demonstrated here, but the presence of such structure in vivo was not supported. Based upon these results a working model is proposed for the chromatin structural remodeling responsible for the sequential expression of ves multigene family members from divergently-organized loci.

Outbreak of equine piroplasmosis in Florida.

CASE DESCRIPTION: A 7-year-old Quarter Horse gelding was hospitalized in Ocala, Fla, because of lethargy, fever, anorexia, and swelling of distal aspects of the limbs. A tentative diagnosis of equine piroplasmosis (EP) was made on the basis of examination of a blood smear. The case was reported to the Florida State Veterinarian, and infection with Babesia equi was confirmed. The subsequent investigation included quarantine and testing of potentially exposed horses for B equi and Babesia caballi infections, tick surveillance, and owner-agent interviews. CLINICAL FINDINGS: 210 horses on 25 premises were tested for infection with EP pathogens. Twenty B equi-infected horses on 7 premises were identified; no horses tested positive for B caballi. Seven horses, including the index case, had clinical findings consistent with EP Dermacentor variabilis was considered the only potential tick vector for B equi collected, and all D variabilis specimens tested negative for Babesia organisms via PCR assay. Results of the epidemiological investigation suggested that B equi was spread by use of shared needles and possibly blood transfusions. All horses that tested positive were involved in nonsanctioned Quarter Horse racing, and management practices were thought to pose substantial risk of transmission of blood-borne pathogens. TREATMENT AND OUTCOME: Final outcome of B equi-infected horses was euthanasia, death from undetermined causes, or shipment to a US federal research facility. CLINICAL RELEVANCE: This investigation highlights the importance of collaboration between private veterinary practitioners, state veterinary diagnostic laboratories, and regulatory officials in the recognition, containment, and eradication of foreign animal disease.

Characterization of the unusual bidirectional ves promoters driving VESA1 expression and associated with antigenic variation in Babesia bovis.

Rapid clonal antigenic variation in Babesia bovis involves the variant erythrocyte surface antigen-1 (VESA1) protein expressed on the infected-erythrocyte surface. Because of the significance of this heterodimeric protein for demonstrated mechanisms of parasite survival and virulence, there is a need to understand how expression of the ves multigene family encoding this protein is controlled. As an initial step toward this goal, we present here initial characterization of the ves promoter driving transcription of VESA1a and -1b subunits. A series of transfection constructs containing various sequence elements from the in vivo locus of active ves transcription (LAT) were used to drive expression of the firefly luciferase gene in a dual luciferase-normalized assay. The results of this approach reveal the presence of two bidirectional promoter activities within the 434-bp intergenic region (IGr), influenced by putative regulatory sequences embedded within the flanking ves1α and ves1ß genes. Repressor-like effects on the apposing gene were observed for intron 1 of both ves1α and ves1ß. This effect is apparently not dependent upon intronic promoter activity and acts only in cis. The expression of genes within the ves family is likely modulated by local elements embedded within ves coding sequences outside the intergenic promoter region in concert with chromatin modifications. These results provide a framework to help us begin to understand gene regulation during antigenic variation in B. bovis.

The Babesia bovis VESA1 virulence factor subunit 1b is encoded by the 1beta branch of the ves multigene family.

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.

varDB: common ground for a shifting landscape.

Antigenic variation is a phylogenetically widespread phenomenon thought to lead to survival benefits for the pathogen. Although governed by genetic mechanisms, antigenic variation is ultimately manifested in variant proteins. The varDB database is an attempt to gain an overview of common structures and functions of variant proteins related to enhanced survival. varDB provides a wealth of sequence data and several tools to facilitate their analysis, but current limitations preclude achievement of its full promise. A critique of this database and how it could serve the scientific community is provided here.

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

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.

Dynamics of anemia progression and recovery in Babesia bigemina infection is unrelated to initiating parasite burden.

To be informative, immunization-and-challenge experiments in support of vaccine development rely on host responses that enable distinctions to be made in the responses of immunized and non-immunized animals to infectious challenge. It is therefore important that animals be challenged with standardized infectious doses that allow such distinctions to be made. We report here the results of a challenge titration experiment in which cattle were challenged with Babesia bigemina, at dosages ranging over six orders of magnitude. No significant dose-dependent differences were observed in the maximum fever attained, duration of fever, minimum hematocrit reached, kinetics with which anemia developed or was resolved, or animal weight gain. Significant differences were noted only in the length of time post-infection required to initiate fever, reach maximum fever, and attain maximum reduction in hematocrit. These results suggest that, in the absence of further supporting evidence, it is not possible to conclude any direct anti-parasite effects from reductions in maximum fever or hematocrit drop during B. bigemina immunization-and-challenge experiments. However, lengthening of the time to reduction in hematocrit may be a useful indicator of overt suppression of the challenge inoculum.

Evaluation of cattle inoculated with Babesia bovis clones adhesive in vitro to bovine brain endothelial cells.

A comparative assessment of the virulence of Babesia bovis clones that adhere or not to bovine brain endothelial cells was done using two clones of B. bovis: (1) a clone phenotypically characterized as virulent (2F8) and (2) a clone of reduced virulence (RAD). Of these subpopulations, we selected those that had adhesive characteristics (a) or nonadhesive characteristics (na) in cultured endothelial cells. Twenty Holstein cattle, 12 months of age or older, were used in this study, and these cattle were randomly assigned to five groups of four animals each. The clones and their respective subpopulations were inoculated via intramuscular injection at a 0.5 x 10(7) infected erythrocyte dosage. Group A was inoculated with aRAD, group B with naRAD, group C with a2F8, group D with na2F8, and group E remained as a control. All inoculated animals showed a decrease in the packed cell volume (PCV), with group D showing the largest decrease (39.53%) and longest time (7 days) with rectal temperature above 39.5 degrees C. Babesia was observed in stained blood smears from only six cattle. While the four parasite subpopulations were pathogenic, significant differences were not noted among them, despite that the subpopulations considered to be virulent caused the greatest reduction in PCV per individual.

Antigenic variation as an exploitable weakness of babesial parasites.

Babesia bovis and its bovine host interact in many ways, resulting in a range of disease and infection phenotypes. Host responses to the parasite elicit or select for a variety of responses on the part of the parasite, the full range of which is not yet known. One well-established phenomenon, thought to aid parasite survival by evasion of host adaptive immune responses, is the sequential expansion of antigenically variant populations during an infection, a phenomenon referred to as "antigenic variation". Antigenic variation in B. bovis, like that in the human malarial parasite, Plasmodium falciparum, is intimately linked to a second survival mechanism, cytoadhesion. In cytoadhesion, mature parasite-containing erythrocytes bind to the capillary and post-capillary venous endothelium through parasite-derived ligands. The reliance of these parasites on both functions, and on their linkage, may provide opportunities to develop anti-babesial and, perhaps, anti-malarial protection strategies. The development of inhibitors of DNA metabolism in B. bovis may be used to abrogate the process of antigenic variation, whereas small molecular mimics may provide the means to vaccinate against a wide range of variants or to prevent the surface export of variant antigen ligands. In this article, aspects of antigenic variation and cytoadhesion in bovine babesiosis are explored, with a discussion of opportunities for prophylactic or therapeutic intervention in these intertwined processes.