A cysteine protease of Babesia microti and its interaction with tick cystatins.

Babesiosis is a tick-borne protozoonosis caused by Babesia, which can cause fever, hemolytic anemia, hemoglobinuria, and even death. Babesia microti is a parasite found in rodents and can be pathogenic to humans. In this study, the full-length cDNA of a B. microti cysteine protease (BmCYP) was expressed and the recombinant rBmCYP protein analyzed and characterized. BmCYP is encoded by an ORF of 1.3 kb, with a predicted molecular weight of 50 kDa and a theoretical pI of 8.5. The amino acid sequence of BmCYP exhibits an identity of 32.9 to 35.2% with cysteine proteases of Babesia ovis, Babesia bovis, and Theileria, respectively. The results of the proteinase assays show that rBmCYP has cysteine protease enzymatic activity. In addition, we demonstrate that tick cystatins rRhcyst-1 and rRhcyst-2 were able to effectively inhibit the activity of rBmCYP; the inhibition rates were 57.2% and 30.9%, respectively. Tick cystatins Rhcyst-1 and Rhcyst-2 were differentially expressed in ticks that fed on Babesia-infected mice relative to non-infected control ticks. Our results suggest that BmCYP is a functional enzyme with cysteine protease enzymatic activity and may be involved in tick-B. microti interactions.

Vibrational Spectroscopic Based Approach for Diagnosing Babesia bovis Infection.

Babesia bovis parasites present a serious and significant health concern for the beef and dairy industries in many parts of the world. Difficulties associated with the current diagnostic techniques include the following: they are prone to human error (microscopy) or expensive and time-consuming (polymerase chain reaction) to perform. Little is known about the biochemical changes in blood that are associated with Babesia infections. The discovery of new biomarkers will lead to improved diagnostic outcomes for the cattle industry. Vibrational spectroscopic technologies can record a chemical snapshot of the entire organism and the surrounding cell thereby providing a phenotype of the organism and the host infected cell. Here, we demonstrate the applicability of vibrational spectroscopic imaging techniques including Atomic Force Microscopy Infrared (AFM-IR) and confocal Raman microscopy to discover new biomarkers for B. bovis infections. Furthermore, we applied Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) to detect B. bovis in red blood cells (RBCs). Based on changes in the IR spectral bands, with ATR-FTIR in combination with Partial Least Squares-Discriminant Analysis we were able to discriminate infected samples from controls with a sensitivity and specificity of 92.0% and 91.7%, respectively, in less than 2 min, excluding sample extraction and preparation. The proposed method utilized a lysis approach to remove hemoglobin from the suspension of infected and uninfected cells, which significantly increased the sensitivity and specificity compared to measurements performed on intact infected red blood cells (intact infected RBC, 77.3% and 79.2%). This work represents a holistic spectroscopic study from the level of the single infected RBC using AFM-IR and confocal Raman to the detection of the parasite in a cell population using ATR-FTIR for a babesiosis diagnostic.

The application of the HyPer fluorescent sensor in the real-time detection of H2O2 in Babesia bovis merozoites in vitro.

In recent years, genetically encoded fluorescent probes have allowed a dramatic advancement in time-lapse imaging, enabling this imaging modality to be used to investigate intracellular events in several apicomplexan parasite species. In this study, we constructed a plasmid vector to stably express a genetically encoded H2O2 sensor probe called HyPer in Babesia bovis. The HyPer-transfected parasite population was successfully developed and subjected to a time-lapse imaging analysis under in vitro culture conditions. HyPer was capable of sensing an increasing H2O2 concentration in the parasite cells which was induced by the administration of paraquat as a superoxide donor. HyPer fluorescence co-staining with MitoTracker Red indicated the mitochondria as the major source of reactive oxygen species (ROS) in parasite cells. The fluctuating ROS dynamics in the parasite gliding toward, attaching to, and invading the target red blood cell was visualized and monitored in real time with the HyPer expressing parasite population. This is the first report to describe the application of the HyPer probe in an imaging analysis involving Babesia parasites. Hyper-expressing parasites can be widely utilized in studies to investigate the mechanisms of emergence and the reduction of oxidative stress, as well as the signal transduction in the parasite cells during host invasion and intercellular development.

Novel biotechnological platform based on baculovirus occlusion bodies carrying Babesia bovis small antigenic peptides for the design of a diagnostic enzyme-linked immunosorbent assay (ELISA).

Baculoviruses are large DNA virus of insects principally employed in recombinant protein expression. Its ability to form occlusion bodies (OBs), which are composed mainly of polyhedrin protein (POLH), makes them biotechnologically attractive, as these crystals (polyhedra) can incorporate foreign peptides and can be easily isolated. On the other hand, peptide microarrays allow rapid and inexpensive high-throughput serological screening of new candidates to be incorporated to OBs. To integrate these 2 biotechnological approaches, we worked on Babesia bovis, one of the causative agents of bovine babesiosis. Current molecular diagnosis of infection with B. bovis includes enzyme-linked immunosorbent assay (ELISA) techniques, which use merozoite lysate obtained from infected bovine erythrocytes. However, it is important to produce recombinant antigens that replace the use of crude antigens. Here, we describe a new biotechnological platform for the design of indirect ELISAs based on 5 antigenic peptides of 15 amino acid residues of B. bovis (ApBb), selected from a peptide microarray and expressed as a fusion to POLH. An Sf9POLHE44G packaging cell line infected with recombinant baculoviruses carrying POLH-ApBb fusions yielded higher levels of chimeric polyhedra, highlighting the advantage of a trans-contribution of a mutant copy of polyhedrin. Finally, the use of dissolved recombinant polyhedra as antigens was successful in an ELISA assay, as B. bovis-positive sera recognized the fusion POLH-ApBb. Thus, the use of this platform resulted in a promising alternative for molecular diagnosis of relevant infectious diseases.

Analysis of Stage-Specific Protein Expression during Babesia Bovis Development within Female Rhipicephalus Microplus.

Arthropod-borne protozoan pathogens have a complex life cycle that includes asexual reproduction of haploid stages in mammalian hosts and the development of diploid stages in invertebrate hosts. The ability of pathogens to invade, survive, and replicate within distinct cell types is required to maintain their life cycle. In this study, we describe a comparative proteomic analysis of a cattle pathogen, Babesia bovis, during its development within the mammalian and tick hosts with the goal of identifying cell-surface proteins expressed by B. bovis kinetes as potential targets for the development of a transmission blocking vaccine. To determine parasite tick-stage-specific cell-surface proteins, CyDye labeling was performed with B. bovis blood stages from the bovine host and kinetes from the tick vector. Cell-surface kinete-stage-specific proteins were identified using 2D difference in gel electrophoresis and analyzed by mass spectrometry. Ten proteins were identified as kinete-stage-specific, with orthologs found in closely related Apicomplexan pathogens. Transcriptional analysis revealed two genes were highly expressed by kinetes as compared with blood stages. Immunofluorescence using antibodies against the two proteins confirmed kinete-stage-specific expression. The identified cell-surface kinete proteins are potential candidates for the development of a B. bovis transmission blocking vaccine.

Iron superoxide dismutases in eukaryotic pathogens: new insights from Apicomplexa and Trypanosoma structures.

Prior studies have highlighted the potential of superoxide dismutases as drug targets in eukaryotic pathogens. This report presents the structures of three iron-dependent superoxide dismutases (FeSODs) from Trypanosoma cruzi, Leishmania major and Babesia bovis. Comparison with existing structures from Plasmodium and other trypanosome isoforms shows a very conserved overall fold with subtle differences. In particular, structural data suggest that B. bovis FeSOD may display similar resistance to peroxynitrite-mediated inactivation via an intramolecular electron-transfer pathway as previously described in T. cruzi FeSOD isoform B, thus providing valuable information for structure-based drug design. Furthermore, lysine-acetylation results in T. cruzi indicate that acetylation occurs at a position close to that responsible for the regulation of acetylation-mediated activity in the human enzyme.

Molecular characterization of a new Babesia bovis thrombospondin-related anonymous protein (BbTRAP2).

A gene encoding a Babesia bovis protein that shares significant degree of similarity to other apicomplexan thrombospondin-related anonymous proteins (TRAPs) was found in the genomic database and designated as BbTRAP2. Recombinant protein containing a conserved region of BbTRAP2 was produced in E. coli. A high antigenicity of recombinant BbTRAP2 (rBbTRAP2) was observed with field B. bovis-infected bovine sera collected from geographically different regions of the world. Moreover, antiserum against rBbTRAP2 specifically reacted with the authentic protein by Western blot analysis and an indirect fluorescent antibody test. Three bands corresponding to 104-, 76-, and 44-kDa proteins were identified in the parasite lysates and two bands of 76- and 44-kDa proteins were detected in the supernatant of cultivated parasites, indicating that BbTRAP2 was proteolytically processed and shed into the culture. Apical and surface localizations of BbTRAP2 were observed in the intracellular and extracellular parasites, respectively, by confocal laser microscopic examination. Moreover, native BbTRAP2 was precipitated by bovine erythrocytes, suggesting its role in the attachment to erythrocytes. Furthermore, the specific antibody to rBbTRAP2 inhibited the growth of B. bovis in a concentration-dependent manner. Consistently, pre-incubation of the free merozoites with the antibody to rBbTRAP2 resulted in an inhibition of the parasite invasion into host erythrocytes. Interestingly, the antibody to rBbTRAP2 was the most inhibitive for the parasite's growth as compared to those of a set of antisera produced against different recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), rhoptry-associated protein 1 C-terminal (BbRAP-1CT), and spherical body protein 1 (BbSBP-1). These results suggest that BbTRAP2 might be a potential candidate for development of a subunit vaccine against B. bovis infection.

Babesia bovis: lipids from virulent S2P and attenuated R1A strains trigger differential signalling and inflammatory responses in bovine macrophages.

The intra-erythrocytic protozoan Babesia bovis is an economically important pathogen that causes an acute and often fatal infection in adult cattle. Babesiosis limitation depends on the early activation of macrophages, essential cells of the host innate immunity, which can generate an inflammatory response mediated by cytokines and nitric oxide (NO). Herein, we demonstrate in bovine macrophages that lipids from B. bovis attenuated R1A strain (LA) produced a stronger NO release, an early TNFα mRNA induction and 2-fold higher IL-12p35 mRNA levels compared to the lipids of virulent S2P strain (LV). Neither LA nor LV induced anti-inflammatory IL-10. Regarding signalling pathways, we here report that LA induced a significant phosphorylation of p38 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) whereas LV only induced a reduced activation of ERK1/2. Besides, NF-κB was activated by LA and LV, but LA produced an early degradation of the inhibitor IκB. Interestingly, LV and the majority of its lipid fractions, exerted a significant inhibition of concanavalin A-induced peripheral blood mononuclear cell proliferation with respect to LA and its corresponding lipid fractions. In addition, we determined that animals infected with R1A developed a higher increase in IgM anti-phosphatidylcholine than those inoculated with S2P. Collectively, S2P lipids generated a decreased inflammatory response contributing to the evasion of innate immunity. Moreover, since R1A lipids induced a pro-inflammatory profile, we propose these molecules as good candidates for immunoprophylactic strategies against babesiosis.

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.

Cloning and characterization of peroxiredoxin in Babesia bovis.

We have identified the 2-Cys peroxiredoxin (Prx) from a bovine Babesia parasite, B. bovis. Prx is a recently described family of antioxidant enzymes that are highly conserved in eukaryotes and prokaryotes. B. bovis 2-Cys Prx (BbTPx-1) contained two conserved cysteine residues that corresponded to Cys47 and Cys170 of the yeast Prx and the amino acid sequences of two catalytic domains showed significant similarities to those of mammalian typical 2-Cys Prx. The antioxidant activity of the recombinant BbTPx-1 protein expressed in E. coli was demonstrated by a thiol mixed-function oxidation assay. Furthermore, we confirmed that BbTPx-1 was expressed in the cytoplasm of intra-erythrocytic B. bovis merozites. These results suggest that B. bovis likely uses TPx-1 as a way to reduce peroxides as a control of its intracellular redox balance so that it can live and grow in the host cell.

Merozoite surface antigen 2 proteins of Babesia bovis vaccine breakthrough isolates contain a unique hypervariable region composed of degenerate repeats.

The merozoite surface antigen 2 (MSA-2) proteins of Babesia bovis are members of the variable merozoite surface antigen (VMSA) family that have been implicated in erythrocyte invasion and are important targets for antibody-mediated blocking of invasion. Extensive sequence variation in another VMSA member, MSA-1, has been shown in all vaccine breakthrough isolates. To test the hypothesis that the msa-2 genes of vaccine breakthrough isolates would also encode a diverse set of proteins, the complete msa-2 locus was characterized from 12 Australian B. bovis strains and isolates, including two vaccine strains and eight vaccine breakthrough isolates, and compared to the loci in previously and newly characterized American strains. In contrast to American strains, the msa-2 loci of all Australian strains and isolates examined contain, in addition to msa-2c, only a solitary gene (designated msa-2a/b) closely related to American strain msa-2a and msa-2b. Nevertheless, the proteins encoded by these genes are quite diverse both between and within geographic regions and harbor evidence of genetic exchange among other VMSA family members, including msa-1. Moreover, all but one of the Australian breakthrough isolate MSA-2a/b proteins is markedly different from the vaccine strain from which immune escape occurred, consistent with their role in strain-specific protective immunity. The densest distribution of polymorphisms occurs in a hypervariable region (HVR) within the carboxy third of the molecule that is highly proline rich. Variation in length and content of the HVR is primarily attributable to differences in the order and number of degenerate nucleotide repeats encoding three motifs of unknown function.

Identification of a coronin-like protein in Babesia species.

The present study was designed to immunochemically identify a coronin-like protein in Babesia bovis, B. bigemina, B. divergens, and B. canis. A 2-kbp cDNA insert of B. bovis carried by plasmid BvN9 was sequenced by the dideoxichain-termination method on both strands. The cDNA insert contained a 1719-bp long open reading frame coding for a deduced protein sequence of 61.7 kDa. Sequence analysis using the PSI-BLAST program revealed about 30% protein sequence identity with a coronin-like protein of Plasmodium falciparum. The encoding sequence of the cDNA insert lacking 70 amino acids at the N-terminal was subcloned in frame into pGEX 4T-3 to produce a recombinant glutathione S-transferase (GST)-pBv fusion protein. Polyclonal antibodies prepared in rabbits immunized with the purified GST-fusion protein recognized a Babesia-specific component of approximately 60 kDa by immunoprecipitation with [35S]methionine-labeled parasites. However, two molecules with relative sizes of 60 and 70 kDa were recognized in Babesia-infected erythrocyte extracts by immunobloting analysis. The 70-kDa component was apparently of host erythrocyte origin. In an indirect fluorescent antibody test, the rabbit serum strongly reacted with the merozoite stage of the four Babesia species, but also, although weakly, with the host erythrocyte. A cosedimentation assay performed with GST-pBv fusion protein and exogenous actin from rabbit liver showed that the GST-pBv fusion protein, but not the GST protein, was associated to actin. From these results, we conclude that the protein present in the four Babesia species analyzed here may be considered as a novel coronin-like, actin-binding protein.

Evaluation of an enzyme-linked immunosorbent assay with recombinant rhoptry-associated protein 1 antigen against Babesia bovis for the detection of specific antibodies in cattle.

The gene encoding Babesia bovis rhoptry-associated protein 1 (RAP-1) was used to develop an enzyme-linked immunosorbent assay (ELISA) to measure specific antibodies against B. bovis. The B. bovis RAP-1 gene was subcloned into a baculovirus transfer vector, and the RAP-1 protein was expressed in insect cells infected with a recombinant baculovirus. The recombinant B. bovis RAP-1 of 65 kDa was detected with anti-RAP-1 mouse serum by Western blotting, and this recombinant RAP-1 was used as an antigen in the ELISA. The ELISA was able to differentiate between B. bovis-infected sera and B. bigemina-infected sera or noninfected normal bovine sera. The results demonstrate that the recombinant RAP-1 expressed in insect cells might be a useful antigen for the detection of antibodies to B. bovis.

Phosphatidylcholine formation is the predominant lipid biosynthetic event in the hemoparasite Babesia bovis.

This work examines the lipid composition and metabolism of bovine red blood cells infected by apicomplexan Babesia parasites, organisms closely related to Plasmodium sp. We found that erythrocytes infected with Babesia bovis (i-RBC) accumulate lipids and show striking increases in phosphatidylcholine, phosphatidic acid, diacylglycerol and cholesteryl esters as compared to uninfected erythrocytes cultured under the same conditions (n-RBC). A similar pattern was observed in cultures of erythrocytes infected with Babesia bigemina. The lipid profile of purified B. bovis merozoites showed that phosphatidylcholine is the most abundant phospholipid in this parasite (31.8% +/- 2.8 of total phospholipid), markedly differing from bovine n-RBC, in which it is only a minor component (4.8% +/- 0.6). B. bovis cultures incorporate radiolabeled choline into complex lipids, especially phosphatidylcholine, with minor amounts recovered in sphingomyelin and lysophosphatidylcholine. When [14C] stearate was used as precursor, the labeling pattern again gave the highest incorporation into phosphatidylcholine, with lesser incorporation in sphingomyelin, phosphatidylinositol, phosphatidylethanolamine and phosphatidic acid. Diacylglycerol and small amounts of cholesteryl esters were the only labeled neutral lipids found. B. bovis also incorporates [3H] myo-inositol into phosphatidylinositol. Parallel incubations with n-RBC as a control yielded no incorporation into either polar or neutral lipids with any precursor. These results indicate that the lipid changes observed in i-RBC can be explained on the basis of the lipid biosynthetic activities of the babesial parasite. Gas chromatography-mass spectrometry (GC-MS) analysis of fatty acid methyl esters from phospholipids of i-RBC and n-RBC showed the same qualitative composition in both. However, i-RBC had higher ratios of saturated to unsaturated fatty acids and B. bovis cultures did not desaturate [14C] stearate. Cholesterol was the only sterol detected by GC-MS. Phospholipase A2 treatment of i-RBC and n-RBC revealed no enhanced hemolytic effects in i-RBC, suggesting that the erythrocyte membrane phospholipid composition is essentially unaltered by the parasite. Labeling of i-RBC or n-RBC with [125I] Bolton-Hunter resulted in an enhanced phosphatidylserine labeling in i-RBC. This study provides the first data on B. bovis lipid constitution and biosynthesis. They show that phosphatidylcholine formation is the main biosynthetic process in these cells. The striking differences in the contents of phosphatidylcholine between host erythrocytes and the parasite suggests that it may be a useful target for both chemotherapy and immunoprophylaxis against bovine babesiosis.

Production and characterization of a panel of monoclonal antibodies for the identification of antigens of Babesia bovis.

A panel of monoclonal antibodies was produced and characterized by an indirect fluorescent antibody test (IFAT), an enzyme-linked immunosorbent assay (ELISA) and Western blotting with the aim of identifying antigens of Babesia bovis. After fusion, the resultant hybrids were selected by the IFAT, cloned, maintained in culture in vitro, and cryopreserved in liquid nitrogen. Ten clones producing monoclonal antibodies were found to react against the entire merozoites, three reacted on the surface of the merozoites, and one clone reacted against the polar region of the merozoites. All monoclonal antibodies reacted in ELISA, with the optical density varying from 0.368 to 0.502 (cut off = 0.022). The bands recognized by the monoclonal antibodies in Western blotting had molecular weights ranging from 162 to 19 kDa. Four clones recognized a single band of 73 kDa, and another four did not react in Western blotting.

Structure, sequence, and transcriptional analysis of the Babesia bovis rap-1 multigene locus.

The complexity of multigene families encoding rhoptry proteins and the generation of new variants in these families are constraints to development of vaccines incorporating rhoptry proteins. For example, the Babesia bigemina rhoptry associated protein (rap)-1 locus is composed of tandemly arranged genes including four polymorphic rap-1a genes and two classes of divergent genes, rap-1b and rap-1c. B. bigemina rap-1 polymorphism reflects recombination and gene conversion and results in multiple RAP-1 proteins with unique B- and T-cell epitopes. Is this complex locus structure and recombination a required feature of the rap-1 gene family among Babesia species? We addressed this question by analysis of the rap-1 locus in B. bovis. Sequence analysis of an 11 kb genomic clone representing the B. burn rap-1 locus revealed only two identical and continuous rap-1a gene copies, rap 1a-1 and rap-1a-2, located in a similar head to tail orientation. Using the conserved ig gene as a marker for the 3' boundary of the rap-1 locus, we conclude that divergent rap-1b and rap-1c genes, present in B. bigemina, are not similarly cis-linked to the B. bovis rap-1 locus. Analysis of the rap-1a genes 1 and 2 from each of multiple B. bovis strains from North and South America demonstrated RAP-1 size conservation with very limited amino acid sequence variation. The results suggest that the simple two gene arrangement in the B. bovis rap-1 gene family was generated by gene duplication and, in contrast to the B. bigemina rap-1 locus, both genes evolved together using homogenization mechanisms with point mutation as the single mechanism for gene variation. Three discontinuous non-rap-1 genes are closely cis-linked to the B. bovis rap-1 locus and the presence of multiple introns in these genes may limit rap-1 gene variation due to unequal crossing over. The different mechanisms likely involved in the evolution of the rap-1 family in B. bigemina versus B. bovis are reflected in the marked structural and antigenic polymorphism in the B. bigemina RAP-1 molecules as compared with the essentially monomorphic RAP-1 in B. bovis.

Babesia bovis host cell recognition proteins.

Babesia bovis enters host erythrocytes by invagination but nothing is known of the proteins involved. By means of metabolic labelling, differential centrifugation in oil and salt elution, a number of babesial proteins have been shown to bind to bovine erythrocytes. Strong binding is evidenced only by a 38/19 kDa pair. Preliminary experiments indicate that these two proteins also bind to human erythrocytes, although apparently to a lesser extent.