Epidrugs: alternative chemotherapy targeting Theileria annulata schizont stage parasites.

The growing emergence of resistance to current anti-theilerial agents necessitates the exploration of alternative approaches to drug discovery. This study evaluated the antiparasitic efficacy of 148 compounds derived from an epigenetic inhibitor library against the schizont stage of a Theileria annulata-infected cell line. Initial screening at a concentration of 10 µM identified 27 compounds exhibiting promising anti-theilerial activity. Further investigation, including determination of the 50% inhibitory concentration (IC50) and host cell cytotoxicity assay, highlighted seven highly effective compounds (SAHA, BVT-948, Trichostatin A, Methylstat, Plumbagin, Ryuvidine, and TCE-5003) against T. annulata-infected cells. Analysis of the active compounds revealed their inhibitory action against various human targets, such as HDAC (SAHA and Trichostatin A), SET domain (Ryuvidine), PRMT (BVT-948 and TCE-5003), histone demethylase (Methylstat), and ROS/apoptosis inducer (Plumbagin). We identified gene orthologs of these targets in Theileria and conducted molecular docking studies, demonstrating effective binding of the compounds with their respective targets in the parasite, supported by in vitro data. Additionally, we performed in silico ADME/T predictions, which indicated potential mutagenic and hepatotoxic effects of Plumbagin, Methylstat, and TCE-5003, rendering them unsuitable for drug development. Conversely, SAHA, Trichostatin A, and BVT-948 showed promising characteristics and may represent potential candidates for future development as chemotherapeutic agents against tropical theileriosis. These findings provide valuable insights into the search for novel anti-theilerial drugs and offer a basis for further research in this area.IMPORTANCETheileria annulata is a protozoan parasite responsible for tropical theileriosis, a devastating disease affecting cattle. Traditional chemotherapy has limitations, and the study explores the potential of epidrugs as an alternative treatment approach. Epidrugs are compounds that modify gene expression without altering the underlying DNA sequence, offering a novel way to combat parasitic infections. This research is pivotal as it addresses the urgent need for innovative therapies against T. annulata, contributing to the development of more effective and targeted treatments for infected livestock. Successful implementation of epidrugs could not only enhance the well-being of cattle but also have broader implications for the control of parasitic diseases, showcasing the paper's significance in advancing veterinary science and improving livestock health globally.

Screening and identification of Theileria annulata subtelomere-encoded variable secreted protein-950454 (SVSP454) interacting proteins from bovine B cells.

BACKGROUND: Theileria annulata is a protozoan parasite that can infect and transform bovine B cells, macrophages, and dendritic cells. The mechanism of the transformation is still not well understood, and some parasite molecules have been identified, which contribute to cell proliferation by regulating host signaling pathways. Subtelomeric variable secreted proteins (SVSPs) of Theileria might affect the host cell phenotype, but its function is still not clear. Therefore, in the present study, we explored the interactions of SVSP454 with host cell proteins to investigate the molecular mechanism of T. annulata interaction with host cells. METHODS: The transcription level of an SVSP protein from T. annulata, SVSP454, was analyzed between different life stages and transformed cell passages using qRT-PCR. Then, SVSP454 was used as a bait to screen its interacting proteins from the bovine B cell cDNA library using a yeast two-hybrid (Y2H) system. The potential interacting proteins of host cells with SVSP454 were further identified by using a coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays. RESULTS: SVSP454 was transcribed in all three life stages of T. annulata but had the highest transcription during the schizont stage. However, the transcription level of SVSP454 continuously decreased as the cultures passaged. Two proteins, Bos Taurus coiled-coil domain 181 (CCDC181) and Bos Taurus mitochondrial ribosomal protein L30 (MRPL30), were screened. The proteins CCDC181 and MRPL30 of the host were further identified to directly interact with SVSP454. CONCLUSION: In the present study, SVSP454 was used as a bait plasmid, and its prey proteins CCDC181 and MRPL30 were screened out by using a Y2H system. Then, we demonstrated that SVSP454 directly interacted with both CCDC181 and MRPL30 by Co-IP and BiFC assays. Therefore, we speculate that SVSP454-CCDC181/SVSP454MRPL30 is an interacting axis that regulates the microtubule network and translation process of the host by some vital signaling molecules. Identification of the interaction of SVSP454 with CCDC181 and MRPL30 will help illustrate the transformation mechanisms induced by T. annulata.

Development of an indirect ELISA based on the recombinant Spm2 protein for detection of tropical theileriosis.

Tropical theileriosis, caused by Theileria annulata, is distributed worldwide and causes great economic losses in dairy. The reliable diagnostic method is critical for prevention and control of the disease. In this study, a sporozoite and macroschizont gene 2 (spm2) protein from T. annulata was used to develop an indirect ELISA for tropical theileriosis. Specificity test showed that there were no cross-reactions with antibodies raised against other bovine piroplasm species using ELISA or western blotting. The specificity and sensitivity were 98.4% and 98.7%, respectively, with a threshold of 35.5% of the specific mean antibody rate (AbR). Furthermore, a total of 196 field sera samples collected from Xinjiang and Gansu provinces were detected by the spm2 ELISA and IFA. The results obtained with the spm2 ELISA and IFA in this study had the moderate agreement. The average positive rates of T. annulata sera samples detected in the present study were close to the prevalence of previous reports in these endemic areas. This indicated that the Spm2 ELISA could be used as a reliable diagnostic tool for serological survey of T. annulata infection in areas where Theileria parva is not present.

Isolation and purification of glycosylphosphatidylinositols (GPIs) in the schizont stage of Theileria annulata and determination of antibody response to GPI anchors in vaccinated and infected animals.

BACKGROUND: Tropical theileriosis is widely distributed from North Africa to East Asia. It is a tick-borne disease caused by Theileria annulata, an obligate two-host intracellular protozoan parasite of cattle. Theileria annulata use leukocytes and red blood cells for completion of the life-cycle in mammalian hosts. The stage of Theileria annulata in monocytes and B lymphocytes of cattle is an important step in pathogenicity and diagnosis of the disease. Glycosylphosphatidylinositols (GPIs) are a distinct class of glycolipid structures found in eukaryotic cells and are implicated in several biological functions. GPIs are particularly abundant in protozoan parasites, where they are found as free glycolipids or attached to proteins in the plasma membrane. RESULTS: In this study we first isolated and purified schizonts of Theileria annulata from infected leukocytes in Theileria annulata vaccine cell line (S15) by aerolysin-percoll technique. Then, the free GPIs of schizont stage and isolated GPI from cell membrane glycoproteins were purified by high performance liquid chromatography (HPLC) and confirmed by gas chromatography-mass spectrometry (GC-MS). Furthermore, enzyme linked immunosorbent assay (ELISA) on the serum samples obtained from naturally infected, as well as Theileria annulata-vaccinated animals, confirmed a significant (P < 0.01) high level of anti-GPI antibody in their serum. CONCLUSIONS: The results presented in this study show, to our knowledge for the first time, the isolation of GPI from the schizont stage of Theileria annulata and demonstrate the presence of anti-GPI antibody in the serum of naturally infected as well as vaccinated animals. This finding is likely to be valuable in studies aimed at the evaluation of chemically structures of GPIs in the schizont stage of Theileria annulata and also for pathogenicity and immunogenicity studies with the aim to develop GPI-based therapies or vaccines.

No change in mRNA expression of immune-related genes in peripheral blood mononuclear cells challenged with Theileria annulata in Murrah buffalo (Bubalus bubalis).

Water buffaloes (Bubalus bubalis) act as carrier to Theileria annulata and show less clinical sign of tropical theileriosis as compared to indigenous and exotic cattle. Differential expression of immune-related genes such as major histocompatibility complex, class II, DQ alpha 1 (MHC-DQα), signal-regulatory protein alpha (SIRPA), prion protein (PRNP), Toll-like receptor 10 (TLR10), c-musculoaponeurotic fibrosarcoma oncogene homolog (cMAF) and V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) genes influence host resistance to this disease in exotic, crossbred and indigenous cattle. In the present study we examined the differential mRNA expression of the abovesaid immune-related genes in response to T. annulata infection in buffaloes. Peripheral blood mononuclear cells (PBMCs) harvested from blood samples of buffaloes were challenged with ground-up tick supernatant carrying T. annulata sporozoites in vitro. After 48h of in vitro challenge qPCR was employed to measure the relative mRNA expression of MHC-DQα, SIRPA, PRNP, TLR10, cMAF and MAFB genes in infected and control PBMCs. In the current study, the selected genes showed no change in mRNA expression after T.annulata infection which indicates that they have little role in providing host resistance to theileriosis in buffaloes.

Proteomic analysis of the Theileria annulata schizont.

The apicomplexan parasite, Theileria annulata, is the causative agent of tropical theileriosis, a devastating lymphoproliferative disease of cattle. The schizont stage transforms bovine leukocytes and provides an intriguing model to study host/pathogen interactions. The genome of T. annulata has been sequenced and transcriptomic data are rapidly accumulating. In contrast, little is known about the proteome of the schizont, the pathogenic, transforming life cycle stage of the parasite. Using one-dimensional (1-D) gel LC-MS/MS, a proteomic analysis of purified T. annulata schizonts was carried out. In whole parasite lysates, 645 proteins were identified. Proteins with transmembrane domains (TMDs) were under-represented and no proteins with more than four TMDs could be detected. To tackle this problem, Triton X-114 treatment was applied, which facilitates the extraction of membrane proteins, followed by 1-D gel LC-MS/MS. This resulted in the identification of an additional 153 proteins. Half of those had one or more TMD and 30 proteins with more than four TMDs were identified. This demonstrates that Triton X-114 treatment can provide a valuable additional tool for the identification of new membrane proteins in proteomic studies. With two exceptions, all proteins involved in glycolysis and the citric acid cycle were identified. For at least 29% of identified proteins, the corresponding transcripts were not present in the existing expressed sequence tag databases. The proteomics data were integrated into the publicly accessible database resource at EuPathDB (www.eupathdb.org) so that mass spectrometry-based protein expression evidence for T. annulata can be queried alongside transcriptional and other genomics data available for these parasites.

A role for tertiary structure in the generation of antigenic diversity and molecular association of the Tams1 polypeptide in Theileria annulata.

The major merozoite-piroplasm surface antigen (mMPSA) of Theileria annulata, Tams1, is known to be antigenically diverse. The possession of variable N-linked glycosylation sites and removal of monoclonal antibody 5E1 reactivity by mild periodate treatment suggested, previously, that divergent epitopes may be conferred by secondary modification. This study has shown that monoclonal antibody 5E1 and polyspecific antisera raised against the native protein react against divergent amino acid epitopes that are dependent on a molecular conformation that is sensitive to periodate. Therefore, no experimental evidence exists to confirm the sequence prediction that Tams1 undergoes N-linked glycosylation. Data is also presented indicating that the conformation of the antigen results in presentation of divergent regions on the external surface of the molecule, while conserved regions are more likely to be internal and hidden. In addition, non-reducing SDS-PAGE analysis demonstrated that Tams1 can undergo molecular association to form homo-dimers, trimers and multimers. The potential influence of tertiary structure and inter-molecular association on Tams1 diversity and function is discussed.

Study of Theileria annulata population structure during bovine infection and following transmission to ticks.

Tams1 is the polymorphic immunodominant merozoite-piroplasm surface protein of Theileria annulata. Evidence for selection of divergent forms of Tams1 has been obtained recently. This study was performed to address whether selection takes place during persistent infection of the bovine host or during passage through the Hyalomma tick vector. Four calves were infected with a T. annulata isolate representing multiple parasite genotypes. The development of the parasite population was analysed by denaturing gradient gel electrophoresis (DGGE) using the Tams1 gene as a marker. In addition, the parasitaemia was measured by a semi-quantitative reverse line blot hybridization assay in order to correlate Tams1 variation to changes in parasitaemia. It was found that both parasitaemia and parasite population displayed limited variation during persistent infection. Ticks were allowed to acquire T. annulata during 2 periods of the bovine infection. Tams1 alleles detected in ticks fed during acute infection were identical to the population in the bovine host. However, ticks fed during the carrier status acquired parasites showing a single Tams1 isotype that represented, in several cases, a minor population in the bovine host at the time of infestation. Although only a limited number of ticks were studied, these preliminary data suggest that specific parasite genotypes may be selected during tick transmission from a carrier animal.

Detection of Theileria annulata in cattle and vector ticks by PCR using the Tams1 gene sequences.

A Polymerase Chain Reaction (PCR) and Southern blot hybridization for the detection of Theileria annulata are described. The PCR used primers amplifying a 785 base-pair fragment of the T. annulata gene which encodes the 30 kDa major merozoite surface antigen, Tams1. The sensitivity of the PCR in bovine blood was 1 piroplasm in 1 microl of blood. T. buffeli, T. parva, Babesia bigemina, B. bovis and B. divergens were not detected. The PCR detected down to 1 infected acinus/tick in resting and partially fed adult Hyalomma anatolicum anatolicum ticks and was negative for T. lestoquardi and T. equi, which are transmitted by this tick but are not infective to cattle. The specificity of the PCR was checked using 30 stocks of T. annulata, all of which were detected. Three stocks of T. lestoquardi, 4 of T. equi and 1 each of T. buffeli, T. parva, B. bigemina, B. bovis and B. divergens were used to ascertain there were no cross-reactions. A nested PCR using separate primers for the first reaction and the same primers for the second reaction detected T. annulata to the same sensitivity and specificity in saponin-extracted DNA samples stored for long periods at -20 degrees C.

Macroschizonts of Theileria annulata as vaccine and diagnostic tools.

T. annulata-infected cells present infection-associated peptides. These peptides represent target molecules of the cytotoxic acting cells. Their preparation and characterization may help to develop a sub-unit vaccine. Our studies show that macroschizont-infected bovine cells can be used as parasite antigen in serology for the detection of parasite-specific antibodies in serum of infected animals. Primers derived from the macroschizont of T. annulata can be used as molecular tools for the detection of parasite DNA in blood samples of carrier cattle.