Cimicifugin, a broad-spectrum inhibitor of Theileria annulata and Plasmodium falciparum CDK7.

Cyclin-dependent kinase 7 is an attractive therapeutic target for the treatment of cancers, and a previous report suggested that Plasmodium falciparum CDK7 is a potential drug target for developing new anti-malarial drugs. In this study, we aimed to characterize and evaluate the drug target potential of Theileria annulata CDK7. Theileria annulata is responsible for tropical theileriosis, which induces a phenotype similar to cancerous cells like immortalization, hyperproliferation, and dissemination. Virtual screening of the MyriaScreen II library predicted 14 compounds with high binding energies to the ATP-binding pocket of TaCDK7. Three compounds (cimicifugin, ST092793, and ST026925) of these 14 compounds were non-cytotoxic to the uninfected bovine cells (BoMac cells). Cimicifugin treatment led to the activation of the extrinsic apoptosis pathway and induced autophagy in T. annulata-infected cells. Furthermore, cimicifugin also inhibited the growth of P. falciparum, indicating that it has both anti-theilerial and anti-malarial activities and that TaCDK7 and PfCDK7 are promising drug targets.

Identification of a Novel Interaction between Theileria Prohibitin (TaPHB-1) and Bovine RuvB-Like AAA ATPase 1.

Theileriosis is a tick-borne disease caused by Theileria annulata, an intracellular parasite that belongs to the phylum Apicomplexa. The infective forms of the parasite to cattle are sporozoites that are introduced into the host when the infected ticks take a blood meal. The sporozoites selectively invade bovine B cells, macrophages, or monocytes, leading to their cellular transformation. The parasite factors involved in the host cell transformation are not well explored. In pursuit of this, we revisited the probable secretome of the parasite and, following a stringent downscaling criterion, have identified Theileria prohibitin (TaPHB-1) as one of factors secreted into the host cells. Interestingly, in infected cells, TaPHB-1 localized both on the parasite surface and in the host cytoplasm, and independent approaches such as coimmunoprecipitation, yeast two-hybrid screening (Y2H), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed RuvB-like AAA ATPase 1 (RUVBL-1) as one of its interacting partners. Further, the T. annulata infection does not affect the localization of bovine prohibitin. Mitigating the expression of bovine RUVBL-1 precluded the translocation of TaPHB-1 in the host cell cytoplasm without affecting the host cell viability. Taken together, we report for the first time a unique interaction of TaPHB-1 with bovine RUVBL-1 that is likely needed to cause cancer-like hallmarks during theileriosis. IMPORTANCE Theileria annulata is an apicomplexan parasite that causes tropical theileriosis in cattle. It is the only eukaryotic pathogen able to cause cellular transformation of host cells yielding a cancer-like phenotype. The parasite factors responsible for the transformation of the host cell are largely unknown. This study demonstrates for the first time the partial role of Theileria prohibitin (TaPHB-1) in maintaining the transformed state of the host cell and its interaction with RuvB-like AAA ATPase 1 (RUVBL-1) in a T. annulata-infected bovine cell line. Interestingly, the knockdown of bovine RUVBL-1 rendered the parasites metabolically inactive, implying that the identified interaction is critical for parasite survival. This study contributes to our understanding the Theileria-host interactions and offers scope for novel therapeutic interventions to control theileriosis.

Deciphering the kinome of Theileria annulata for identification of drug targets and anti-theilerial drug.

Tropical theileriosis is one of the major parasitic diseases of ruminants. It is a tick-borne disease caused by an apicomplexan parasite, Theileria annulata. In the infected cells, these parasites induce phenotypes similar to cancerous cells. Among the most critical changes induced by the parasite are immortalization, hyperproliferation, and dissemination. The proliferative signal in the T. annulata transformed cells are provided by different kinases such as mitogen-activated protein kinases, SRC family kinases, casein kinase-2, and phosphatidylinositide 3-kinase. Deregulation of protein kinases in cancer is also well known. Targeting protein kinases in a cancerous cell is one of the most common methods in cancer therapy. Here, we revisited the kinome of T. annulata and studied its evolutionary relationship with other piroplasms. This analysis revealed that T. annulata kinome encodes 54 protein kinases. Based on our analysis, 12 of these 54 kinases were identified for the first time in the T. annulata proteome. Three protein kinases, TA16570, TA09820, and TA07000, had <40% identity with Bos taurus and >40% identity with the previously identified potential drug targets present in the Therapeutic Target Database (TTD). These 3 proteins were predicted to be essential for the survival of T. annulata and were selected as drug targets. Screening these drug targets in the Protein Kinase Inhibitor Database (PKID) led to shortlisting of 5 drugs. Only Dabrafenib, out of these 5 drugs, could bind to the ATP binding site (in silico) of the Calcium Dependent Protein Kinase 3 of both T. annulata and Theileria parva. Further, dabrafenib could inhibit the proliferation of T. annulata infected bovine leucocytes in 6 days proliferation assay with the IC50 value of 0.66 µM. Also, this drug did not have a cytotoxic effect on bovine peripheral blood mononuclear cells. In summary, the analysis of T. annulata kinome led to the identification of dabrafenib as a potential drug for treating theileriosis.

Design of a multi-epitope protein as a subunit vaccine against lumpy skin disease using an immunoinformatics approach.

Lumpy skin disease (LSD) is a transboundary viral disease of cattle that causes substantial economic loss globally. There is no specific treatment and subunit vaccine for this disease to date. Reports of the global spread of this disease are worrisome. We designed a multi-epitope protein using an immunoinformatics approach in this study. We analyzed the proteome of LSDV and found 32 structural/surface proteins. Four of these 32 proteins were predicted as antigenic and non-homologous to bovine and highly conserved in 26 LSDV isolates. The predicted B-cell epitopes and CTL epitopes were stitched together with the help of an AAY linker leading to the formation of a multi-epitope protein. The in silico study revealed that the modeled subunit vaccine candidate and TLR4 receptor interact with high affinity. This interaction was also found to be stable using a molecular dynamics simulation study. Our study demonstrates a leap towards developing a subunit vaccine against LSD.

MMV560185 from pathogen box induces extrinsic pathway of apoptosis in Theileria annulata infected bovine leucocytes.

Tropical theileriosis is a lymphoproliferative disease caused by the intracellular schizonts of Theileria annulata, an apicomplexan parasite. It causes severe infection in cattle and the untreated cattle would possibly die within 3-4 weeks of infection. The chemotherapy for this disease is largely dependent on the use of hydroxynaphthoquinone, namely buparvaquone. There have been reports recently of the development of resistance against this drug in T. annulata. Hence, identification of new drug molecule(s) or repurposing of existing drug molecule(s) against T. annulata is quite important. Here, we present the screening of 400 compounds included in the open-access Pathogen box from Medicine for Malaria Venture (MMV) to discover the novel compounds with potential inhibitory activity against T. annulata infected bovine leucocytes. We identified two compounds, MMV000062 and MMV560185, with IC50 values of 2.97 µM and 3.07 µM, respectively. MMV000062 and MMV560185 were found non-toxic to BoMac cells with CC50 values 34 µM and > 100 µM, respectively. The therapeutic indices of these compounds, MMV000062 and MMV560185, were calculated as more than 33 and 11, respectively. Further, it was observed that the parasite-infected cells under long-term culture were unable to recover with these compounds. We further deciphered that MMV560185 kills the infected cell by activation of TNFR-1 mediated extrinsic pathway of the apoptosis. The phenotypic characteristics of apoptosis were confirmed by Transmission Electron Microscopy. Our results suggest that it may be possible to develop MMV560185 further for chemotherapeutics of tropical theilerosis.

Immuno-informatics Analysis to Identify Novel Vaccine Candidates and Design of a Multi-Epitope Based Vaccine Candidate Against Theileria parasites.

Theileriosis poses a serious threat to ruminants in tropical and subtropical countries. It is a tick-borne disease, caused by an apicomplexan parasite, Theileria. The high disease burden in animals causes huge economic losses to marginal farmers. Further, with increasing cases of resistance to commonly used drugs, it is highly desirable to develop better and cost-effective vaccines against theileriosis. The only available vaccine, live attenuated parasite vaccine, has many drawbacks and hence is unsuitable for controlling this disease. Immuno-informatics has emerged as a useful tool in down selection of potential molecules for vaccine development. In this study, we have used an immuno-informatics driven genome-wide screening strategy to identify potential vaccine targets containing important and effective dominant immunogens against Theileria. The proteome of Theileria annulata was screened for proteins with probability of plasma membrane localization or GPI anchor. The proteins non-homologous to the host (bovine) were selected and their antigenicity was analyzed. The B-cell epitopes were identified in the selected proteins and mapped in the modeled structure of the proteins. A total of 19 linear epitopes in 12 proteins, exposed in the extracellular space and having the potential to induce protective antibodies were obtained. Additionally, CTL epitopes which are peptides with 9-mer core sequence, were also identified, modeled and docked with bovine MHC-I structures. The CTL epitopes showing high binding energy with the bovine MHC-I were further engineered in silico to design a putative multi-epitope vaccine candidate against Theileria parasites. The docking studies and molecular dynamics studies with the predicted multi-epitope vaccine candidate and modeled bovine TLR4 exhibited strong binding energy, suggesting that the complex is stable and the putative multi-epitope vaccine candidate can be a potentially good candidate for vaccine development.