Identification of benzamide inhibitors of histone deacetylase 1 from Babesia and Theileria species via high-throughput virtual screening and molecular dynamics simulations.

Theileria and Babesia species are eukaryotic protozoan parasites classified under the order Piroplasmida of the phylum Apicomplexa. Tick vectors transmit these microorganisms in tropical and subtropical regions to a wide range of animals, including ruminants, causing fatal and life-threatening diseases such as bovine babesiosis and theileriosis. Resistance to commercially available drugs requires the search for new drug candidates. Histone deacetylase (HDAC) has a potential to be utilized as a drug target; therefore, it may be considered as an effective alternative. Previous studies revealed that HDAC inhibitors, identified for human use, show promising anti-parasitic effects. We have herein focused on the class I HDAC enzyme, HDAC1, of the Babesia and Theileria species to discover potential benzamide inhibitors by following a streamlined workflow of computer-aided drug design methodology. Molecular docking and molecular dynamics simulations revealed that benzamide derivatives stably interacted with the HDAC1 active site in both parasites as hypothesized. Furthermore, specific residue insertions at the entry point of the active site cleft of parasitic HDAC1 could enable ways to design parasite-specific drugs without adversely affecting host enzymes.

Secreted parasite Pin1 isomerase stabilizes host PKM2 to reprogram host cell metabolism.

Metabolic reprogramming is an important feature of host-pathogen interactions and a hallmark of tumorigenesis. The intracellular apicomplexa parasite Theileria induces a Warburg-like effect in host leukocytes by hijacking signaling machineries, epigenetic regulators and transcriptional programs to create a transformed cell state. The molecular mechanisms underlying host cell transformation are unclear. Here we show that a parasite-encoded prolyl-isomerase, TaPin1, stabilizes host pyruvate kinase isoform M2 (PKM2) leading to HIF-1α-dependent regulation of metabolic enzymes, glucose uptake and transformed phenotypes in parasite-infected cells. Our results provide a direct molecular link between the secreted parasite TaPin1 protein and host gene expression programs. This study demonstrates the importance of prolyl isomerization in the parasite manipulation of host metabolism.

Simultaneous detection of Theileria annulata and Theileria orientalis infections using recombinase polymerase amplification.

Theileriosis is a disease of domesticated animals in tropical and subtropical countries and causes significant reductions in livestock productivity. The arid region of Punjab in Pakistan is notorious for the presence of the vector tick (Acari: Ixodidae) and tick-borne diseases, such as theileriosis and babesiosis. The distribution of Theileria annulata and T. orientalis in the Chakwal district of Punjab was determined by developing a multiplex recombinase polymerase amplification (RPA) assay as a scientific basis for formulating control strategies for bovine theileriosis. Specificity was evaluated using DNA from related piroplasm species, while analytical sensitivity was calculated using a long fragment of the enolase gene. A total of 188 blood samples were collected on FTA cards (Whatman®) from tick-infested asymptomatic breeds of cattle (Bos indicus, Bos taurus, and Bos indicus × Bos taurus) in the study district. Finally, infections with of T. annulata and T. orientalis were detected using the multiplex RPA and compared with the conventional multiplex polymerase chain reaction (PCR). The multiplex RPA specifically amplified 282-bp and 229-bp fragments of the enolase gene from T. annulata and T. orientalis and had no cross-reaction with other piroplasm species. It was determined that 45 (23.9%) and 5 (2.6%) out of 188 blood samples were positive for T. annulata and T. orientalis, respectively, when examined using RPA. Multiplex PCR detection indicated that 32 (17.0%) and 3 (1.6%) blood samples were positive for T. annulata and T. orientalis, respectively. In the present study, a specific RPA method was developed for simultaneous differentiation and detection of T. annulata and T. orientalis infections and used for the first time for the detection of the two bovine Theileria infections.

Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation.

Infectious agents develop intricate mechanisms to interact with host cell pathways and hijack their genetic and epigenetic machinery to change host cell phenotypic states. Among the Apicomplexa phylum of obligate intracellular parasites, which cause veterinary and human diseases, Theileria is the only genus that transforms its mammalian host cells. Theileria infection of bovine leukocytes induces proliferative and invasive phenotypes associated with activated signalling pathways, notably JNK and AP-1 (ref. 2). The transformed phenotypes are reversed by treatment with the theilericidal drug buparvaquone. We used comparative genomics to identify a homologue of the peptidyl-prolyl isomerase PIN1 in T. annulata (TaPIN1) that is secreted into the host cell and modulates oncogenic signalling pathways. Here we show that TaPIN1 is a bona fide prolyl isomerase and that it interacts with the host ubiquitin ligase FBW7, leading to its degradation and subsequent stabilization of c-JUN, which promotes transformation. We performed in vitro and in silico analysis and in vivo zebrafish xenograft experiments to demonstrate that TaPIN1 is directly inhibited by the anti-parasite drug buparvaquone (and other known PIN1 inhibitors) and is mutated in a drug-resistant strain. Prolyl isomerization is thus a conserved mechanism that is important in cancer and is used by Theileria parasites to manipulate host oncogenic signalling.

Identification of papain-like cysteine proteases from the bovine piroplasm Babesia bigemina and evolutionary relationship of piroplasms C1 family of cysteine proteases.

Papain-like cysteine proteases have been shown to have essential roles in parasitic protozoa and are under study as promising drug targets. Five genes were identified by sequence similarity search to be homologous to the cysteine protease family in the ongoing Babesia bigemina genome sequencing project database and were compared with the annotated genes from the complete bovine piroplasm genomes of Babesia bovis, Theileria annulata, and Theileria parva. Multiple genome alignments and sequence analysis were used to evaluate the molecular evolution events that occurred in the C1 family of cysteine proteases in these piroplasms of veterinary importance. BbiCPL1, one of the newly identified cysteine protease genes in the B. bigemina genome was expressed in Escherichia coli and shows activity against peptide substrates. Considerable differences were observed in the cysteine protease family between Babesia and Theileria genera, and this may partially explain the diverse infection mechanisms of these tick-borne diseases.

Alteration of host cell phenotype by Theileria annulata and Theileria parva: mining for manipulators in the parasite genomes.

The apicomplexan parasites Theileria annulata and Theileria parva cause severe lymphoproliferative disorders in cattle. Disease pathogenesis is linked to the ability of the parasite to transform the infected host cell (leukocyte) and induce uncontrolled proliferation. It is known that transformation involves parasite dependent perturbation of leukocyte signal transduction pathways that regulate apoptosis, division and gene expression, and there is evidence for the translocation of Theileria DNA binding proteins to the host cell nucleus. However, the parasite factors responsible for the inhibition of host cell apoptosis, or induction of host cell proliferation are unknown. The recent derivation of the complete genome sequence for both T. annulata and T. parva has provided a wealth of information that can be searched to identify molecules with the potential to subvert host cell regulatory pathways. This review summarizes current knowledge of the mechanisms used by Theileria parasites to transform the host cell, and highlights recent work that has mined the Theileria genomes to identify candidate manipulators of host cell phenotype.

Theileria orientalis: cloning a cDNA encoding a protein similar to thiol protease with haemoglobin-binding activity.

A gene encoding a protein (Tocp1) from Theileria orientalis was isolated from a cDNA library and the deduced amino acid sequence of Tocp1 has 476 amino acids. The primary structure of Tocp1 is similar to eukaryotic thiol proteases (EC 3.4.22.-), but no enzymatic activity was observed with the substitution of essential cysteine at the cysteine active site for glycine. Southern blot analysis showed that multiple genes similar to Tocp1 were present in the parasite genome. Sequence analysis of the genome of the parasite showed that there are at least five different genes similar to Tocp1. Tocp1 transcripts were detected in the T. orientalis piroplasma by Northern blot analysis. Western blot analysis showed that Tocp1 was expressed in the piroplasm of T. orientalis. To address the role of Tocp1 in the life cycle of T. orientalis, Tocp1 was expressed using pET32 expression system. Binding affinity to haemoglobin was demonstrated by enzyme-linked immunosorbent assay.

Molecular phylogenetic study of Theileria sp. (Thung Song) based on the thymidylate synthetase gene.

Theileria type Thung Song is an indigenous hemoparasite of dairy cattle from the south of Thailand. It has previously been classified using the analysis of a comparative set of small subunit ribosomal RNA nucleotide sequences. However, the classification of this parasite is still questionable since the Theileria type Thung Song was located as the intermediate parasite between pathogenic and benign groups. We use the thymidylate synthetase gene (TS) as an alternative for the rapid molecular phylogenetic tree construction of benign Theileria type Thung Song, Theileria sergenti and Theileria buffeli with Babesia bovis as an out-group. The partial nucleotide sequences were determined using PCR, cloning and dideoxy sequencing. The TS nucleotide sequence data were aligned and analyzed by distance and maximum likelihood methods to construct the phylogenetic trees. Bootstrap analysis was used to test the strength of the different phylogenetic reconstructions. All tree-building methods gave similar results. This study shows that T. sergenti and T. buffeli are closely related whereas Theileria type Thung Song is more distantly related.

Cloning of a cysteine proteinase gene of Theileria sergenti.

A cDNA encoding cysteine proteinase of Theileria sergenti was isolated from a piroplasm cDNA library and its nucleotide sequence was determined. The gene encodes a polypeptide of 402 amino acids with predicted molecular mass of 46.4 kDa. Analysis of the predicted amino acid sequence revealed a number of features common to known cysteine proteinases. Southern blot analysis showed that the cysteine proteinase gene was likely to be a single copy per genome.

The isolation and characterization of genomic and cDNA clones coding for a cdc2-related kinase (ThCRK2) from the bovine protozoan parasite Theileria.

The tick-transmitted protozoan parasites Theileria annulata and Theileria parva are important intracellular pathogens of domestic cattle in tropical and subtropical regions. Proliferative phases take place within both lymphocytes and erythrocytes. The lymphocyte is stimulated to enter the cell cycle by the parasite and the multinucleate parasite can establish a state in which karyokinesis and cytokinesis occur in phase with the host cell. The link between parasite nuclear division and cytokinesis is altered during the formation of merozoites (a non-dividing, invasive, extracellular stage). These features imply a high degree of control over parasite nuclear division and cytokinesis. Two different approaches have been used to identify clones from both species which are extremely highly conserved homologues. These encode a cdc2-related kinase which is > 60% identical to eukaryotic cyclin-dependent kinases of the p34cdc2/p32CDK2 subfamily. There is typical conservation of kinase domains, implying an in vivo protein kinase activity for the polypeptide. The PSTAIRE region, implicated in cyclin binding, is well conserved suggesting that ThCRK2 will bind cyclin molecules closely related to the eukaryotic A/B-type cyclins. However, there is divergence in certain key motifs potentially associated with binding of molecules that regulate the activity of the kinase. Expression patterns of RNA and protein indicate that ThCRK2 is likely to function in all dividing stages of the parasite and, taken together, the results point to a central role in the regulation of nuclear division.

Detection and characterization of Theileria sergenti proteinases.

The lysate of Theileria sergenti piroplasms was tested for proteinases using sodium dodecyl sulfate-polyacrylamide gel electrophoresis in which substrate was included in gel matrix. Six proteinases of molecular weight 330, 125, 98, 94, 67 and 58 kilodalton (kDa) were detected. From the results of the Triton X-114 phase partition, 330, 125 and 58 kDa proteinases were partitioned into aqueous phase, which indicated that they were not associated with parasite membranes. All these three enzymes were classified into metalloproteinase family because of their sensitivities to metal-ion chelating compounds, ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline. On the other hand, 98 and 94 kDa proteinases were membrane-associated metalloproteinases which were preferentially inhibited by 1,10-phenanthroline. Another metalloproteinase of 67 kDa which was inhibited by EDTA and 1,10-phenanthroline was not associated with parasite membranes. Proteinases of 98 and 94 kDa degraded heat-denatured hemoglobin.