Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata

Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (β-actin), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.     

Stochastic induction of Theileria annulata merogony in vitro by chloramphenicol

Theileria annulata inhabits the cytoplasm of bovine leukocytes where it can be found as a multinucleated schizont. The schizont is the pathogenic stage of the life cycle and by interfering with host signalling pathways, it induces unlimited host cell proliferation and protection against apoptosis. In the infected animal, the schizont differentiates to the merozoite life cycle stage in a process called merogony. This takes place within the host leukocyte, resulting in the production of merozoites that are subsequently released by leukocyte lysis. In established cultures of T. annulata-transformed cells, merogony does not spontaneously occur, but the process can be activated by a shift in temperature. In this study we show that chloramphenicol induces schizont differentiation in proliferating T. annulata-transformed cells. We demonstrate that chloramphenicol-induced merogony is inherently asynchronous and has a quantitative basis. The process is accompanied by the down-regulation of schizont-specific surface proteins, de novo expression of merozoite-specific markers such as Tamr1 and Tams1 and the morphological hallmarks of merogony. Chloramphenicol-induced parasite differentiation was found to be associated with diminished proliferation potential and extensive morphological changes of the host cell, including increased numbers of pseudopodia. Significantly, chloramphenicol treatment can accelerate merogony induced by elevated temperature, supporting postulation that the differentiation event is a stochastic process that can be manipulated to alter the outcome of parasitic infection.     

Lymphocytes and Macrophages Are Infected by Theileria equi,but T Cells and B Cells Are Not Required to Establish Infection In Vivo

Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence.     

Exosomes Secreted by Toxoplasma gondii-Infected L6 Cells: Their Effects on Host Cell Proliferation and Cell Cycle Changes

Toxoplasma gondii infection induces alteration of the host cell cycle and cell proliferation. These changes are not only seen in directly invaded host cells but also in neighboring cells. We tried to identify whether this alteration can be mediated by exosomes secreted by T. gondii-infected host cells. L6 cells, a rat myoblast cell line, and RH strain of T. gondii were selected for this study. L6 cells were infected with or without T. gondii to isolate exosomes. The cellular growth patterns were identified by cell counting with trypan blue under confocal microscopy, and cell cycle changes were investigated by flow cytometry. L6 cells infected with T. gondii showed decreased proliferation compared to uninfected L6 cells and revealed a tendency to stay at S or G2/M cell phase. The treatment of exosomes isolated from T. gondii-infected cells showed attenuation of cell proliferation and slight enhancement of S phase in L6 cells. The cell cycle alteration was not as obvious as reduction of the cell proliferation by the exosome treatment. These changes were transient and disappeared at 48 hr after the exosome treatment. Microarray analysis and web-based tools indicated that various exosomal miRNAs were crucial for the regulation of target genes related to cell proliferation. Collectively, our study demonstrated that the exosomes originating from T. gondii could change the host cell proliferation and alter the host cell cycle.     

Cell Cycle-Dependent Phosphorylation of Theileria annulata Schizont Surface Proteins

The invasion of Theileria sporozoites into bovine leukocytes is rapidly followed by the destruction of the surrounding host cell membrane, allowing the parasite to establish its niche within the host cell cytoplasm. Theileria infection induces host cell transformation, characterised by increased host cell proliferation and invasiveness, and the activation of anti-apoptotic genes. This process is strictly dependent on the presence of a viable parasite. Several host cell kinases, including PI3-K, JNK, CK2 and Src-family kinases, are constitutively activated in Theileria-infected cells and contribute to the transformed phenotype. Although a number of host cell molecules, including IkB kinase and polo-like kinase 1 (Plk1), are recruited to the schizont surface, very little is known about the schizont molecules involved in host-parasite interactions. In this study we used immunofluorescence to detect phosphorylated threonine (p-Thr), serine (p-Ser) and threonine-proline (p-Thr-Pro) epitopes on the schizont during host cell cycle progression, revealing extensive schizont phosphorylation during host cell interphase. Furthermore, we established a quick protocol to isolate schizonts from infected macrophages following synchronisation in S-phase or mitosis, and used mass spectrometry to detect phosphorylated schizont proteins. In total, 65 phosphorylated Theileria proteins were detected, 15 of which are potentially secreted or expressed on the surface of the schizont and thus may be targets for host cell kinases. In particular, we describe the cell cycle-dependent phosphorylation of two T. annulata surface proteins, TaSP and p104, both of which are highly phosphorylated during host cell S-phase. TaSP and p104 are involved in mediating interactions between the parasite and the host cell cytoskeleton, which is crucial for the persistence of the parasite within the dividing host cell and the maintenance of the transformed state.     

Characterisation of infection associated microRNA and protein cargo in extracellular vesicles of Theileria annulata infected leukocytes

The protozoan parasites Theileria annulata and Theileria parva are unique amongst intracellular eukaryotic pathogens as they induce a transformation‐like phenotype in their bovine host cell. T. annulata causes tropical theileriosis, which is frequently fatal, with infected leukocytes becoming metastatic and forming foci in multiple organs resulting in destruction of the lymphoid system. Exosomes, a subset of extracellular vesicles (EV), are critical in metastatic progression in many cancers. Here, we characterised the cargo of EV from a control bovine lymphosarcoma cell line (BL20) and BL20 infected with T. annulata (TBL20) by comparative mass spectrometry and microRNA (miRNA) profiling (data available via ProteomeXchange, identifier PXD010713 and NCBI GEO, accession number GSE118456, respectively). Ingenuity pathway analysis that many infection‐associated proteins essential to migration and extracellular matrix digestion were upregulated in EV from TBL20 cells compared with BL20 controls. An altered repertoire of host miRNA, many with known roles in tumour and/or infection biology, was also observed. Focusing on the tumour suppressor miRNA, bta‐miR‐181a and bta‐miR‐181b, we identified putative messenger RNA targets and confirmed the interaction of bta‐miR181a with ICAM‐1. We propose that EV and their miRNA cargo play an important role in the manipulation of the host cell phenotype and the pathobiology of Theileria infection.     

A PKA survival pathway inhibited by DPT-PKI, a new specific cell permeable PKA inhibitor, is induced by T. annulata in parasitized B-lymphocytes

T. annulata, an intracellular pathogenic parasite of the Aplicomplexa protozoan family infects bovine B-lymphocytes and macrophages. Parasitized cells that become transformed survive and proliferate independently of exogenous growth factors. In the present study, we used the isogenic non parasitized BL3 and parasitized TBL3 B cell lines, as a model to evaluate the contribution of two-major PI3-K- and PKA-dependent anti-apoptotic pathways in the survival of T. annulata parasitized B lymphocytes. We found that T. annulata increases PKA activity, induces over-expression of the catalytic subunit and down-regulates the pro-survival phosphorylation state of Akt/PKB. Consistent with a role of PKA activation in survival, two pharmacological inhibitors H89 and KT5720 ablate PKA-dependent survival of parasitized cells. To specifically inhibit PKA pro-survival pathways we linked the DPTsh1 peptide shuttle sequence to PKI_5–24 and we generated DPT-PKI, a cell permeable PKI. DPT-PKI specifically inhibited PKA activity in bovine cell extracts and, as expected, also inhibited the PKA-dependent survival of T. annulata parasitized TBL3 cells. Thus, parasite-dependent constitutive activation of PKA in TBL3 cells generates an anti-apoptotic pathway that can protect T. annulata-infected B cells from apoptosis. These results also indicate that DPT-PKI could be a powerful tool to inhibit PKA pathways in other cell types.     

Up-regulation of hyaluronan receptors in Toxoplasma gondii-infected monocytic cells

The apicomplexan, obligate intracellular parasite Toxoplasma gondii orally infects humans and animals. The parasites cross the intestinal epithelium, invade leukocytes in the general circulation and then disseminate into the peripheral organs. The mechanism of extravasation of the infected leukocytes, however, remains poorly understood. It is known that adhesion of leukocytes to extracellular matrix (ECM) is an important factor in extravasation, and CD44 and ICAM-1 on the leukocyte surface are known receptors for hyaluronan (HA), an ECM component. In this study, we demonstrated up-regulation of CD44 and ICAM-1 expression on the surface of T. gondii-infected human monocytic THP-1 cells and fresh isolated human monocyte. T. gondii-infected THP-1 cells adhered more efficiently to immobilized HA than did non-infected cells. T. gondii-infected monocytes in the general circulation might preferentially adhere to the ECM and migrate out from blood vessels, so transporting parasites into the peripheral organs.     

Population diversity and multiplicity of infection in Theileria annulata

The tick-borne apicomplexan parasite Theileria annulata is endemic in many sub-tropical countries and causes the bovine disease tropical theileriosis. Although the parasite is known to be highly diverse, detailed information is lacking on the genetic structure of natural populations and levels of multiplicity of infection in the cattle host. With the widespread deployment of live attenuated vaccines and the emergence of drug-resistant parasites in the field, it is vital to appreciate the factors which shape genetic diversity of the parasite both within individual hosts and in the wider population. This study addresses these issues and represents an extensive genetic analysis of T. annulata populations in two endemic countries utilising a high-throughput adaptation of a micro- and mini-satellite genotyping system. Parasite material was collected from infected cattle in defined regions of Turkey and Tunisia to allow a variety of analyses to be conducted. All animals (n = 305) were found to harbour multiple parasite genotypes and only two isolates shared an identical predominant multi-locus profile. A modelling approach was used to demonstrate that host age, location and vaccination status play a measurable role in determining multiplicity of infection in an individual animal. Age was shown to positively correlate with multiplicity of infection and while positive vaccination status exerted a similar effect, it was shown to be due not simply to the presence of the immunising genotype. Importantly, no direct evidence was found for the immunising genotype spreading or recombining within the local parasite community. Genetic analysis confirmed the tentative conclusion of a previous study that the parasite population appears to be, in general, panmictic. Nevertheless, evidence supporting linkage disequilibrium and a departure from panmixia was uncovered in some localities and a number of explanations for these findings are advanced.     

Theileria highjacks JNK2 into a complex with the macroschizont GPI (GlycosylPhosphatidylInositol)‐anchored surface protein p104

Constitutive c‐Jun N‐terminal kinase (JNK) activity characterizes bovine T and B cells infected with Theileria parva, and B cells and macrophages infected with Theileria annulata. Here, we show that T. annulata infection of macrophages manipulates JNK activation by recruiting JNK2 and not JNK1 to the parasite surface, whereas JNK1 is found predominantly in the host cell nucleus. At the parasite's surface, JNK2 forms a complex with p104, a GPI‐(GlycosylPhosphatidylInositol)‐anchor T. annulata plasma membrane protein. Sequestration of JNK2 depended on Protein Kinase‐A (PKA)‐mediated phosphorylation of a JNK‐binding motif common to T. parva and a cell penetrating peptide harbouring the conserved p104 JNK‐binding motif competitively ablated binding, whereupon liberated JNK2 became ubiquitinated and degraded. Cytosolic sequestration of JNK2 suppressed small mitochondrial ARF‐mediated autophagy, whereas it sustained nuclear JNK1 levels, c‐Jun phosphorylation, and matrigel traversal. Therefore, T. annulata sequestration of JNK2 contributes to both survival and dissemination of Theileria‐transformed macrophages.     

Intracellular Theileria annulata Promote Invasive Cell Motility through Kinase Regulation of the Host Actin Cytoskeleton

The intracellular, protozoan Theileria species parasites are the only eukaryotes known to transform another eukaryotic cell. One consequence of this parasite-dependent transformation is the acquisition of motile and invasive properties of parasitized cells in vitro and their metastatic dissemination in the animal, which causes East Coast Fever (T. parva) or Tropical Theileriosis (T. annulata). These motile and invasive properties of infected host cells are enabled by parasite-dependent, poorly understood F-actin dynamics that control host cell membrane protrusions. Herein, we dissected functional and structural alterations that cause acquired motility and invasiveness of T. annulata-infected cells, to understand the molecular basis driving cell dissemination in Tropical Theileriosis. We found that chronic induction of TNFα by the parasite contributes to motility and invasiveness of parasitized host cells. We show that TNFα does so by specifically targeting expression and function of the host proto-oncogenic ser/thr kinase MAP4K4. Blocking either TNFα secretion or MAP4K4 expression dampens the formation of polar, F-actin-rich invasion structures and impairs cell motility in 3D. We identified the F-actin binding ERM family proteins as MAP4K4 downstream effectors in this process because TNFα-induced ERM activation and cell invasiveness are sensitive to MAP4K4 depletion. MAP4K4 expression in infected cells is induced by TNFα-JNK signalling and maintained by the inhibition of translational repression, whereby both effects are parasite dependent. Thus, parasite-induced TNFα promotes invasive motility of infected cells through the activation of MAP4K4, an evolutionary conserved kinase that controls cytoskeleton dynamics and cell motility. Hence, MAP4K4 couples inflammatory signaling to morphodynamic processes and cell motility, a process exploited by the intracellular Theileria parasite to increase its host cell''s dissemination capabilities.     

Theileria annulata: cross-reactions between a cell culture schizont antigen and antigens of East African species in the indirect fluorescent antibody test

A schizont antigen for the indirect fluorescent antibody test was prepared from an in vitro culture suspension of lymphoid cells infected with Theileria annulata macroschizonts. Two cattle recovered from T. annulata infection showed marked rises in antibody titer to this schizont antigen, with peak titers of 1:40,960 and 1:2560. Using sera from these recovered cattle, T. annulata cell culture schizont antigen was shown to cross-react markedly with Theileria parva and Theileria lawrencei cell culture schizonts and with Theileria mutans piroplasms in the indirect fluorescent antibody test. In contrast, using high-titer antisera to T. parva, T. lawrencei, and T. mutans, serological cross-reactions with T. annulata schizonts were only detected with T. parva and T. lawrencei antisera, and in both instances these were minimal. The value of the indirect fluorescent antibody test in the differential diagnosis of Theileria species pathogenic for cattle is discussed.     

A Bovine Lymphosarcoma Cell Line Infected with Theileria annulata Exhibits an Irreversible Reconfiguration of Host Cell Gene Expression

Theileria annulata, an intracellular parasite of bovine lymphoid cells, induces substantial phenotypic alterations to its host cell including continuous proliferation, cytoskeletal changes and resistance to apoptosis. While parasite induced modulation of host cell signal transduction pathways and NFκB activation are established, there remains considerable speculation on the complexities of the parasite directed control mechanisms that govern these radical changes to the host cell. Our objectives in this study were to provide a comprehensive analysis of the global changes to host cell gene expression with emphasis on those that result from direct intervention by the parasite. By using comparative microarray analysis of an uninfected bovine cell line and its Theileria infected counterpart, in conjunction with use of the specific parasitacidal agent, buparvaquone, we have identified a large number of host cell gene expression changes that result from parasite infection. Our results indicate that the viable parasite can irreversibly modify the transformed phenotype of a bovine cell line. Fifty percent of genes with altered expression failed to show a reversible response to parasite death, a possible contributing factor to initiation of host cell apoptosis. The genes that did show an early predicted response to loss of parasite viability highlighted a sub-group of genes that are likely to be under direct control by parasite infection. Network and pathway analysis demonstrated that this sub-group is significantly enriched for genes involved in regulation of chromatin modification and gene expression. The results provide evidence that the Theileria parasite has the regulatory capacity to generate widespread change to host cell gene expression in a complex and largely irreversible manner.     

Toxoplasma gondii inhibits granzyme B-mediated apoptosis by the inhibition of granzyme B function in host cells

Host defense to the apicomplexan parasite Toxoplasma gondii is critically dependent on CD8⁺ T cells, whose effector functions include the induction of apoptosis in target cells following the secretion of granzyme proteases. Here we demonstrate that T. gondii induces resistance of host cells to apoptosis induced by recombinant granzyme B. Granzyme B induction of caspase-independent cytochrome c release was blocked in T. gondii-infected cells. Prevention of apoptosis could not be attributed to altered expression of the Bcl-2 family of apoptotic regulatory proteins, but was instead associated with reduced granzyme B-mediated, caspase-independent cleavage of procaspase 3 to the p20 form in T. gondii-infected cells, as well as reduced granzyme B-mediated cleavage of the artificial granzyme B substrate, GranToxiLux. The reduction in granzyme B proteolytic function in T. gondii-infected cells could not be attributed to altered granzyme B uptake or reduced trafficking of granzyme B to the cytosol, implying a T. gondii-mediated inhibition of granzyme B activity. Apoptosis and GranToxiLux cleavage were similarly inhibited in T. gondii-infected cells exposed to the natural killer-like cell line YT-1. The endogenous granzyme B inhibitor PI-9 was not up-regulated in infected cells. We believe these findings represent the first demonstration of granzyme B inhibition by a cellular pathogen and indicate a new modality for host cell protection by T. gondii that may contribute to parasite immune evasion.     

α2-3 Sialic acid glycoconjugate loss and its effect on infection with Toxoplasma parasites

Recognition of sialylated glycoconjugates is important for host cell invasion by Apicomplexan parasites. Toxoplasma gondii parasites penetrate host cells via interactions between their microneme proteins and sialylated glycoconjugates on the surface of host cells. However, the role played by sialic acids during infection with T. gondii is not well understood. Here, we focused on the role of α2-3 sialic acid linkages as they appear to be widely expressed in vertebrates. Removal of α2-3 sialic acid linkages on macrophages by neuraminidase treatment did not influence the rate of infection or growth of T. gondii, nor did it affect phagocytosis in vitro. Sialyltransferase ST3Gal-I deficient mice (ST3Gal-I^−/− mice) lost α2-3 sialic acid linkages in macrophages and spleen cells. The numbers of T. gondii-infected CD11b⁺ cells in peritoneal cavities of the infected ST3Gal-I^−/− mice were relatively lower than those of the infected wild type animals. In addition, CD8⁺ T cell populations and numbers in the spleens and peritoneal cavities of the ST3Gal-I^−/− mice were significantly lower than those in the wild type animals before and after the T. gondii infection. ST3Gal-I^−/− mice had severe liver damage and reduced survival rates following peritoneal infection with T. gondii. Furthermore, adoptive transfer of immune CD8⁺ cells from wild type mice to ST3Gal-I^−/− mice increased their survival during infection with T. gondii. Our data show that parasite invasion via α2-3 sialic acid linkages might not contribute on host survival and indicate the impact that loss of α2-3 sialic acid linkages has on CD8⁺ T cell populations, which are necessary for effective immune responses against infection with T. gondii.