Theileria parasites sequester host eIF5A to escape elimination by host-mediated autophagy.

Intracellular pathogens develop elaborate mechanisms to survive within the hostile environments of host cells. Theileria parasites infect bovine leukocytes and cause devastating diseases in cattle in developing countries. Theileria spp. have evolved sophisticated strategies to hijack host leukocytes, inducing proliferative and invasive phenotypes characteristic of cell transformation. Intracellular Theileria parasites secrete proteins into the host cell and recruit host proteins to induce oncogenic signaling for parasite survival. It is unknown how Theileria parasites evade host cell defense mechanisms, such as autophagy, to survive within host cells. Here, we show that Theileria annulata parasites sequester the host eIF5A protein to their surface to escape elimination by autophagic processes. We identified a small-molecule compound that reduces parasite load by inducing autophagic flux in host leukocytes, thereby uncoupling Theileria parasite survival from host cell survival. We took a chemical genetics approach to show that this compound induced host autophagy mechanisms and the formation of autophagic structures via AMPK activation and the release of the host protein eIF5A which is sequestered at the parasite surface. The sequestration of host eIF5A to the parasite surface offers a strategy to escape elimination by autophagic mechanisms. These results show how intracellular pathogens can avoid host defense mechanisms and identify a new anti-Theileria drug that induces autophagy to target parasite removal.

Trifloxystrobin blocks the growth of Theileria parasites and is a promising drug to treat Buparvaquone resistance.

Theileria parasites are responsible for devastating cattle diseases, causing major economic losses across Africa and Asia. Theileria spp. stand apart from other apicomplexa parasites by their ability to transform host leukocytes into immortalized, hyperproliferating, invasive cells that rapidly kill infected animals. The emergence of resistance to the theilericidal drug Buparvaquone raises the need for new anti-Theileria drugs. We developed a microscopy-based screen to reposition drugs from the open-access Medicines for Malaria Venture (MMV) Pathogen Box. We show that Trifloxystrobin (MMV688754) selectively kills lymphocytes or macrophages infected with Theileria annulata or Theileria parva parasites. Trifloxystrobin treatment reduced parasite load in vitro as effectively as Buparvaquone, with similar effects on host gene expression, cell proliferation and cell cycle. Trifloxystrobin also inhibited parasite differentiation to merozoites (merogony). Trifloxystrobin inhibition of parasite survival is independent of the parasite TaPin1 prolyl isomerase pathway. Furthermore, modeling studies predicted that Trifloxystrobin and Buparvaquone could interact distinctly with parasite Cytochrome B and we show that Trifloxystrobin was still effective against Buparvaquone-resistant cells harboring TaCytB mutations. Our study suggests that Trifloxystrobin could provide an effective alternative to Buparvaquone treatment and represents a promising candidate for future drug development against Theileria spp.

Putative SET-domain methyltransferases in Cryptosporidium parvum and histone methylation during infection.

Cryptosporidium parvum is a leading cause of diarrhoeal illness worldwide being a significant threat to young children and immunocompromised patients, but the pathogenesis caused by this parasite remains poorly understood. C. parvum was recently linked with oncogenesis. Notably, the mechanisms of gene expression regulation are unexplored in Cryptosporidium and little is known about how the parasite impact host genome regulation. Here, we investigated potential histone lysine methylation, a dynamic epigenetic modification, during the life cycle of the parasite. We identified SET-domain containing proteins, putative lysine methyltransferases (KMTs), in the C. parvum genome and classified them phylogenetically into distinct subfamilies (namely CpSET1, CpSET2, CpSET8, CpKMTox and CpAKMT). Our structural analysis further characterized CpSET1, CpSET2 and CpSET8 as histone lysine methyltransferases (HKMTs). The expression of the CpSET genes varies considerably during the parasite life cycle and specific methyl-lysine antibodies showed dynamic changes in parasite histone methylation during development (CpSET1:H3K4; CpSET2:H3K36; CpSET8:H4K20). We investigated the impact of C. parvum infection on the host histone lysine methylation. Remarkably, parasite infection led to a considerable decrease in host H3K36me3 and H3K27me3 levels, highlighting the potential of the parasite to exploit the host epigenetic regulation to its advantage. This is the first study to describe epigenetic mechanisms occurring throughout the parasite life cycle and during the host-parasite interaction. A better understanding of histone methylation in both parasite and host genomes may highlight novel infection control strategies.

Dynamic methylation of histone H3K18 in differentiating Theileria parasites.

Lysine methylation on histone tails impacts genome regulation and cell fate determination in many developmental processes. Apicomplexa intracellular parasites cause major diseases and they have developed complex life cycles with fine-tuned differentiation events. Yet, apicomplexa genomes have few transcription factors and little is known about their epigenetic control systems. Tick-borne Theileria apicomplexa species have relatively small, compact genomes and a remarkable ability to transform leucocytes in their bovine hosts. Here we report enriched H3 lysine 18 monomethylation (H3K18me1) on the gene bodies of repressed genes in Theileria macroschizonts. Differentiation to merozoites (merogony) leads to decreased H3K18me1 in parasite nuclei. Pharmacological manipulation of H3K18 acetylation or methylation impacted parasite differentiation and expression of stage-specific genes. Finally, we identify a parasite SET-domain methyltransferase (TaSETup1) that can methylate H3K18 and represses gene expression. Thus, H3K18me1 emerges as an important epigenetic mark which controls gene expression and stage differentiation in Theileria parasites.

Cell geometry and the cytoskeleton impact the nucleo-cytoplasmic localisation of the SMYD3 methyltransferase.

Mechanical cues from the cellular microenvironment are converted into biochemical signals controlling diverse cell behaviours, including growth and differentiation. But it is still unclear how mechanotransduction ultimately affects nuclear readouts, genome function and transcriptional programs. Key signaling pathways and transcription factors can be activated, and can relocalize to the nucleus, upon mechanosensing. Here, we tested the hypothesis that epigenetic regulators, such as methyltransferase enzymes, might also contribute to mechanotransduction. We found that the SMYD3 lysine methyltransferase is spatially redistributed dependent on cell geometry (cell shape and aspect ratio) in murine myoblasts. Specifically, elongated rectangles were less permissive than square shapes to SMYD3 nuclear accumulation, via reduced nuclear import. Notably, SMYD3 has both nuclear and cytoplasmic substrates. The distribution of SMYD3 in response to cell geometry correlated with cytoplasmic and nuclear lysine tri-methylation (Kme3) levels, but not Kme2. Moreover, drugs targeting cytoskeletal acto-myosin induced nuclear accumulation of Smyd3. We also observed that square vs rectangular geometry impacted the nuclear-cytoplasmic relocalisation of several mechano-sensitive proteins, notably YAP/TAZ proteins and the SETDB1 methyltransferase. Thus, mechanical cues from cellular geometric shapes are transduced by a combination of transcription factors and epigenetic regulators shuttling between the cell nucleus and cytoplasm. A mechanosensitive epigenetic machinery could potentially affect differentiation programs and cellular memory.

The clever strategies used by intracellular parasites to hijack host gene expression.

Intracellular pathogens need to develop sophisticated mechanisms to survive and thrive in the hostile environment within host cells. Unicellular, eukaryotic parasites from the Apicomplexa phylum have become masters of manipulating their host cells, exploiting signaling, and metabolic pathways to hijack host gene expression to their own advantage. These intracellular parasites have developed a wide range of strategies that affect transcriptional machineries and epigenetic events in the host cell nucleus. In recent years, many laboratories have risen to the challenge of studying the epigenetics of host-pathogen interactions with the hope that unraveling the complexity of the mechanisms involved will provide important insights into parasitism and provide clues to fight infection. In this review, we survey some of these many strategies that Apicomplexan parasites employ to hijack their hosts, including inducing epigenetic enzymes, secreting epigenators into host cells, sequestering host signaling proteins, and co-opting non-coding RNAs to change gene and protein expression. We cite selected examples from the literature on Apicomplexa parasites (including Toxoplasma, Theileria, and Cryptosporidium) to highlight the success of these parasitic processes. We marvel at the effectiveness of the strategies that these pathogens have evolved and wonder what mysteries lie ahead in exploring the epigenetics of host-parasite interactions.

Mutations in the TaPIN1 peptidyl prolyl isomerase gene in Theileria annulata parasites isolated in Sudan.

The tick-borne parasite Theileria annulata is the causative agent of tropical theileriosis or Mediterranean theileriosis. Infection of bovine leukocytes by the obligate intracellular parasites induces proliferative and invasive phenotypes associated with activated signaling pathways. The transformed phenotypes of infected cells are reversible by treatment with the theilericidal drug buparvaquone. Recent reports of resistance to buparvaquone in Africa and Asia highlight the need to investigate the mechanisms and prevalence of drug resistance. We screened 67 T. annulata isolates from Sudan to investigate mutations in the T. annulata prolyl isomerase I gene (TaPIN1). The secreted TaPin1 interacts with host proteins to induce pathways driving oncogenic transformation and metabolic reprogramming. We found an Alanine-to-Proline mutation at position 53 (A53P) in the catalytic loop that was previously found in Tunisian drug-resistant samples. This is the first study reporting independent confirmation of the A53P mutation in geographically isolated samples. We found several additional mutations in the predicted N-terminal signal peptide that might affect TaPin1 processing or targeting. We found that many parasites also share mutations in both the TaPIN1 and the cytochrome b genes, suggesting that these two genes represent important biomarkers to follow the spread of resistance in Africa, the Middle East and Asia.

The SMYD3 methyltransferase promotes myogenesis by activating the myogenin regulatory network.

The coordinated expression of myogenic regulatory factors, including MyoD and myogenin, orchestrates the steps of skeletal muscle development, from myoblast proliferation and cell-cycle exit, to myoblast fusion and myotubes maturation. Yet, it remains unclear how key transcription factors and epigenetic enzymes cooperate to guide myogenic differentiation. Proteins of the SMYD (SET and MYND domain-containing) methyltransferase family participate in cardiac and skeletal myogenesis during development in zebrafish, Drosophila and mice. Here, we show that the mammalian SMYD3 methyltransferase coordinates skeletal muscle differentiation in vitro. Overexpression of SMYD3 in myoblasts promoted muscle differentiation and myoblasts fusion. Conversely, silencing of endogenous SMYD3 or its pharmacological inhibition impaired muscle differentiation. Genome-wide transcriptomic analysis of murine myoblasts, with silenced or overexpressed SMYD3, revealed that SMYD3 impacts skeletal muscle differentiation by targeting the key muscle regulatory factor myogenin. The role of SMYD3 in the regulation of skeletal muscle differentiation and myotube formation, partially via the myogenin transcriptional network, highlights the importance of methyltransferases in mammalian myogenesis.

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.

[Parasites and cancer: is there a causal link?] / Parasites et cancer : existe-t-il un lien ?

Over 20 % of cancers have infectious origins, including well-known examples of microbes such as viruses (HPV, EBV) and bacteria (H. pylori). The contribution of intracellular eukaryotic parasites to cancer etiology is largely unexplored. Epidemiological and clinical reports indicate that eukaryotic protozoan, such as intracellular apicomplexan that cause diseases of medical or economic importance, can be linked to various cancers: Theileria and Cryptosporidium induce host cell transformation while Plasmodium was linked epidemiologically to the "African lymphoma belt" over fifty years ago. These intracellular eukaryotic parasites hijack cellular pathways to manipulate the host cell epigenome, cellular machinery, signaling pathways and epigenetic programs and marks, such as methylation and acetylation, for their own benefit. In doing so, they tinker with the same pathways as those deregulated during cancer onset. Here we discuss how epidemiological evidence linking eukaryotic intracellular parasites to cancer onset are further strengthened by recent mechanistic studies in three apicomplexan parasites.

Host-parasite interactions: an intimate epigenetic relationship.

The epigenetics of host-pathogen interactions is emerging as an interesting angle from which to study how parasites have evolved sophisticated strategies to manipulate host gene transcription and protein expression. In this review, we discuss the application of an operational framework to investigate the host cell signalling pathways that are induced by intracellular parasites and the epigenomic consequences in the host nucleus. To illustrate this conceptual approach, we have focused on examples from two eukaryotic intracellular parasites of the apicomplexa phylum: Theileria and Toxoplasma. We review recent findings on intracellular parasitism strategies for hijacking host nuclear functions and discuss how we might think of the parasite and its proteome as an intracellular epigenator.

[Pin1: a multi-talented peptidyl prolyl cis-trans isomerase and a promising therapeutic target for human cancers]. / Pin1: une peptidyl-prolyl cis-trans isomérase multifonctionnelle et une cible anticancéreuse prometteuse.

Post-translational modifications are critical to modulate protein function. A post-translational mechanism, peptidyl prolyl cis-trans isomerisation, plays a key role in protein regulation. Pin1 is a ubiquitous peptidyl prolyl cis-trans isomerase conserved from Archae to Human. This enzyme binds and isomerizes phospho-serine/threonine-proline motifs. This process can induce conformational change in protein targets and modulates their activity, cellular localization, phosphorylation state, stability and/or protein-protein interactions. Pin1 activity regulates proteins involved in cell proliferation, pluripotency or cellular invasion. Pin1 is overexpressed in several human cancers and contributes to tumorigenesis. Its inactivation constitutes a promising therapeutic strategy.

What's the damage? The impact of pathogens on pathways that maintain host genome integrity.

Maintaining genome integrity and transmission of intact genomes is critical for cellular, organismal, and species survival. Cells can detect damaged DNA, activate checkpoints, and either enable DNA repair or trigger apoptosis to eliminate the damaged cell. Aberrations in these mechanisms lead to somatic mutations and genetic instability, which are hallmarks of cancer. Considering the long history of host-microbe coevolution, an impact of microbial infection on host genome integrity is not unexpected, and emerging links between microbial infections and oncogenesis further reinforce this idea. In this review, we compare strategies employed by viruses, bacteria, and parasites to alter, subvert, or otherwise manipulate host DNA damage and repair pathways. We highlight how microbes contribute to tumorigenesis by directly inducing DNA damage, inactivating checkpoint controls, or manipulating repair processes. We also discuss indirect effects resulting from inflammatory responses, changes in cellular metabolism, nuclear architecture, and epigenome integrity, and the associated evolutionary tradeoffs.

[How does the apicomplexan parasite Theileria control host cell identity?]. / Comment le parasite Apicomplexe Theileria manipule-t-il l'identité cellulaire de son hôte bovin ?⋆.

Infectious agents, like bacteria or virus, are responsible for a large number of pathologies in mammals. Microbes have developed mechanisms for interacting with host cell pathways and hijacking cellular machinery to change the phenotypic state. In this review, we focus on an interesting apicomplexan parasite called Theileria. Infection by the tick-transmitted T. annulata parasite causes Tropical Theileriosis in North Africa and Asia, and the related T. parva parasite causes East Coast Fever in Sub-Saharan Africa. This parasite is the only eukaryote known to induce the transformation of its mammalian host cells. Indeed, T. annulata and T. parva infect bovine leukocytes leading to transforming phenotypes, which partially mirror human lymphoma pathologies. Theileria infection causes hyperproliferation, invasiveness and escape from apoptosis, presumably through the manipulation of host cellular pathways. Several host-signaling mechanisms have been implicated. Here we describe the mechanisms involved in parasite-induced transformation phenotypes.

OncomiR addiction is generated by a miR-155 feedback loop in Theileria-transformed leukocytes.

The intracellular parasite Theileria is the only eukaryote known to transform its mammalian host cells. We investigated the host mechanisms involved in parasite-induced transformation phenotypes. Tumour progression is a multistep process, yet 'oncogene addiction' implies that cancer cell growth and survival can be impaired by inactivating a single gene, offering a rationale for targeted molecular therapies. Furthermore, feedback loops often act as key regulatory hubs in tumorigenesis. We searched for microRNAs involved in addiction to regulatory loops in leukocytes infected with Theileria parasites. We show that Theileria transformation involves induction of the host bovine oncomiR miR-155, via the c-Jun transcription factor and AP-1 activity. We identified a novel miR-155 target, DET1, an evolutionarily-conserved factor involved in c-Jun ubiquitination. We show that miR-155 expression led to repression of DET1 protein, causing stabilization of c-Jun and driving the promoter activity of the BIC transcript containing miR-155. This positive feedback loop is critical to maintain the growth and survival of Theileria-infected leukocytes; transformation is reversed by inhibiting AP-1 activity or miR-155 expression. This is the first demonstration that Theileria parasites induce the expression of host non-coding RNAs and highlights the importance of a novel feedback loop in maintaining the proliferative phenotypes induced upon parasite infection. Hence, parasite infection drives epigenetic rewiring of the regulatory circuitry of host leukocytes, placing miR-155 at the crossroads between infection, regulatory circuits and transformation.

The methylation landscape of tumour metastasis.

The metastatic cascade which leads to the death of cancer patients results from a multi-step process of tumour progression caused by genetic and epigenetic alterations in key regulatory molecules. It is, therefore, crucial to improve our understanding of the regulation of genes controlling the metastatic process to identify predictive biomarkers and to develop more effective therapies to treat advanced disease. The study of epigenetic mechanisms of gene regulation offers a novel approach for innovative diagnosis and treatment of cancer patients. Recent discoveries provide compelling evidence that the methylation landscape (changes in both DNA methylation and histone post-translational modifications) is profoundly altered in cancer cells and contributes to the altered expression of genes regulating tumour phenotypes. However, the impact of methylation events specifically on the advanced metastatic process is poorly understood compared with the initial oncogenic events. Moreover, the characterisation of a large number of histone-modifying enzymes has revealed their active roles in cancer progression, via the regulation of specific target genes controlling different metastatic phenotypes. Here, we discuss two main methylating events (DNA methylation and histone-tail methylation) involved in oncogenesis and metastasis formation. The potential reversibility of these molecular events makes them promising biomarkers of metastatic potential and potential therapeutic targets.