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.

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.

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.

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.

[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.

SMYD3 promotes cancer invasion by epigenetic upregulation of the metalloproteinase MMP-9.

Upregulation of the matrix metalloproteinase (MMP)-9 plays a central role in tumor progression and metastasis by stimulating cell migration, tumor invasion, and angiogenesis. To gain insights into MMP-9 expression, we investigated its epigenetic control in a reversible model of cancer that is initiated by infection with intracellular Theileria parasites. Gene induction by parasite infection was associated with trimethylation of histone H3K4 (H3K4me3) at the MMP-9 promoter. Notably, we found that the H3K4 methyltransferase SMYD3 was the only histone methyltransferase upregulated upon infection. SMYD3 is overexpressed in many types of cancer cells, but its contributions to malignant pathophysiology are unclear. We found that overexpression of SMYD3 was sufficient to induce MMP-9 expression in transformed leukocytes and fibrosarcoma cells and that proinflammatory phorbol esters further enhanced this effect. Furthermore, SMYD3 was sufficient to increase cell migration associated with MMP-9 expression. In contrast, RNA interference-mediated knockdown of SMYD3 decreased H3K4me3 modification of the MMP-9 promoter, reduced MMP-9 expression, and reduced tumor cell proliferation. Furthermore, SMYD3 knockdown also reduced cellular invasion in a zebrafish xenograft model of cancer. Together, our results define SMYD3 as an important new regulator of MMP-9 transcription, and they provide a molecular link between SMYD3 overexpression and metastatic cancer progression.

TGF-b2 induction regulates invasiveness of Theileria-transformed leukocytes and disease susceptibility.

Theileria parasites invade and transform bovine leukocytes causing either East Coast fever (T. parva), or tropical theileriosis (T. annulata). Susceptible animals usually die within weeks of infection, but indigenous infected cattle show markedly reduced pathology, suggesting that host genetic factors may cause disease susceptibility. Attenuated live vaccines are widely used to control tropical theileriosis and attenuation is associated with reduced invasiveness of infected macrophages in vitro. Disease pathogenesis is therefore linked to aggressive invasiveness, rather than uncontrolled proliferation of Theileria-infected leukocytes. We show that the invasive potential of Theileria-transformed leukocytes involves TGF-b signalling. Attenuated live vaccine lines express reduced TGF-b2 and their invasiveness can be rescued with exogenous TGF-b. Importantly, infected macrophages from disease susceptible Holstein-Friesian (HF) cows express more TGF-b2 and traverse Matrigel with great efficiency compared to those from disease-resistant Sahiwal cattle. Thus, TGF-b2 levels correlate with disease susceptibility. Using fluorescence and time-lapse video microscopy we show that Theileria-infected, disease-susceptible HF macrophages exhibit increased actin dynamics in their lamellipodia and podosomal adhesion structures and develop more membrane blebs. TGF-b2-associated invasiveness in HF macrophages has a transcription-independent element that relies on cytoskeleton remodelling via activation of Rho kinase (ROCK). We propose that a TGF-b autocrine loop confers an amoeboid-like motility on Theileria-infected leukocytes, which combines with MMP-dependent motility to drive invasiveness and virulence.

JunD protects the liver from ischemia/reperfusion injury by dampening AP-1 transcriptional activation.

The AP-1 transcription factor modulates a wide range of cellular processes, including cellular proliferation, programmed cell death, and survival. JunD is a major component of the AP-1 complex following liver ischemia/reperfusion (I/R) injury; however, its precise function in this setting remains unclear. We investigated the functional significance of JunD in regulating AP-1 transcription following partial lobar I/R injury to the liver, as well as the downstream consequences for hepatocellular remodeling. Our findings demonstrate that JunD plays a protective role, reducing I/R injury to the liver by suppressing acute transcriptional activation of AP-1. In the absence of JunD, c-Jun phosphorylation and AP-1 activation in response to I/R injury were elevated, and this correlated with increased caspase activation, injury, and alterations in hepatocyte proliferation. The expression of dominant negative JNK1 inhibited c-Jun phosphorylation, AP-1 activation, and hepatic injury following I/R in JunD-/- mice but, paradoxically, led to an enhancement of AP-1 activation and liver injury in JunD+/- littermates. Enhanced JunD/JNK1-dependent liver injury correlated with the acute induction of diphenylene iodonium-sensitive NADPH-dependent superoxide production by the liver following I/R. In this context, dominant negative JNK1 expression elevated both Nox2 and Nox4 mRNA levels in the liver in a JunD-dependent manner. These findings suggest that JunD counterbalances JNK1 activation and the downstream redox-dependent hepatic injury that results from I/R, and may do so by regulating NADPH oxidases.

Deciphering AP-1 function in tumorigenesis: fra-ternizing on target promoters.

Multi-gene families of transcription factors pose a formidable challenge to molecular and functional analysis. Dissecting distinct functions for individual family members requires a combination of approaches in different cellular and animal models. The AP-1 transcription factor complex serves as a paradigm for understanding the dynamics of transcriptional regulation. Knockout, knockdown and transgenic strategies, inducible alleles, mutational analysis, chemical genetics, etc.; researchers have applied all the tricks of the trade to understand how AP-1 works. AP-1 refers to a mixture of dimers formed between members of the Jun, Fos and ATF families. The complexity of the AP-1 biological functions reflects the wide combinatorial diversity of its components. AP-1 has been linked to cancer and neoplastic transformation ever since the first jun and fos genes were cloned as cellular homologues of viral oncogenes twenty years ago. Because of the oncogenic or tumor suppressive activity exhibited by distinct Jun and Fos nuclear proteins depending on the cell context and the genetic background of the tumor, the AP-1 complex has been called a "double-edged sword" in tumorigenesis. The cumulating results over the last decade are finally leading to the identification of specific functions for individual AP-1 components and their contribution to neoplastic disease. Here, we focus on the Fra-1 protein in tumorigenesis, which offers an illustrative example of this helter-skelter voyage.

Dimer composition and promoter context contribute to functional cooperation between AP-1 and NFAT.

The transcription factors activator protein 1 (AP-1) and nuclear factor of activated T-cells (NFAT) cooperate to induce the expression of cytokines during the immune response. While much is known about the signaling pathways and physical interactions between NFAT and AP-1 dimers following lymphocyte activation, few studies have addressed the role of AP-1 composition in modulating NFAT:AP-1-dependent transcription. We examined the function of specific AP-1 complexes using "tethered" AP-1 dimers with defined composition. We found that NFAT can functionally cooperate with all AP-1 dimers tested. Noteworthy, Jun approximately Jun-containing dimers, which are relatively inactive when tested on an AP-1-dependent promoter, are effective co-activators of an NFAT:AP-1-dependent promoter. Interestingly, specific AP-1 dimer combinations behave differently when tested on interleukin 2 (IL2) and interleukin 4 (IL4) gene regulatory regions. Moreover, the requirement for NFAT to activate each of the promoters is different. Our results suggest that higher NFAT levels are necessary to activate the IL4 promoter. Hence changes in AP-1 composition and the level of participating NFAT proteins can differentially influence cytokine gene expression, resulting in biological consequences for the modulation and dynamics of the immune response.

Fra-1 promotes growth and survival in RAS-transformed thyroid cells by controlling cyclin A transcription.

Fra-1 is frequently overexpressed in epithelial cancers and implicated in invasiveness. We previously showed that Fra-1 plays crucial roles in RAS transformation in rat thyroid cells and mouse fibroblasts. Here, we report a novel role for Fra-1 as a regulator of mitotic progression in RAS-transformed thyroid cells. Fra-1 expression and phosphorylation are regulated during the cell cycle, peaking at G2/M. Knockdown of Fra-1 caused a proliferative block and apoptosis. Although most Fra-1-knockdown cells accumulated in G2, a fraction of cells entering M-phase underwent abortive cell division and exhibited hallmarks of genomic instability (micronuclei, lagging chromosomes and anaphase bridges). Furthermore, we established a link between Fra-1 and the cell-cycle machinery by identifying cyclin A as a novel transcriptional target of Fra-1. During the cell cycle, Fra-1 was recruited to the cyclin A gene (ccna2) promoter, binding to previously unidentified AP-1 sites and the CRE. Fra-1 also induced the expression of JunB, which in turn interacts with the cyclin A promoter. Hence, Fra-1 induction is important in thyroid tumorigenesis, critically regulating cyclin expression and cell-cycle progression.

JunD is a profibrogenic transcription factor regulated by Jun N-terminal kinase-independent phosphorylation.

JunD is implicated in the regulation of hepatic stellate cell (HSC) activation and liver fibrosis via its transcriptional regulation of the tissue inhibitor of metalloproteinases-1 (TIMP-1) gene. In the present study we found in vivo evidence of a role for JunD in fibrogenesis. Expression of JunD was demonstrated in alpha-SMA-positive activated HSCs of fibrotic rodents and human livers. The junD-/- mice were protected from carbon tetrachloride-induced fibrosis. The livers of injured junD-/- mice displayed significantly reduced formation of fibrotic crosslinked collagen and a smaller number of alpha-SMA-positive HSCs compared with those of wild-type (wt) mice. Hepatic TIMP-1 mRNA expression in injured junD-/- mice was 78% lower and in culture activated junD-/- HSCs was 50%-80% lower than that in wt mice. In examining the signal transduction mechanisms that regulate JunD-dependent TIMP-1 expression, we found a role for phosphorylation of the Ser100 residue of JunD but ruled out JNK as a mediator of this event, suggesting ERK1/2 is utilized. In conclusion, a signaling pathway for the development of fibrosis involves the regulation of TIMP-1 expression by phosphorylated JunD.

Lack of JunD promotes pressure overload-induced apoptosis, hypertrophic growth, and angiogenesis in the heart.

BACKGROUND: The Jun family of activator protein 1 (AP-1) transcription factors (c-Jun, JunB, and JunD) is involved in fundamental biological processes such as proliferation, apoptosis, tumor angiogenesis, and hypertrophy. The role of individual AP-1 transcription factors in the stressed heart is not clear. In the present study we analyzed the role of JunD in survival, hypertrophy, and angiogenesis in the pressure-overloaded mouse heart after thoracic aortic constriction. METHODS AND RESULTS: Mice lacking JunD (knockout [KO]) showed increased mortality and enhanced cardiomyocyte apoptosis and fibrosis associated with increased levels of hypoxia-induced factor-1alpha, vascular endothelial growth factor (VEGF), p53, and Bax protein and reduced levels of Bcl-2 protein after 7 days of severe pressure overload compared with wild-type (WT) siblings. Cardiomyocyte hypertrophy in surviving KO mice was enhanced compared with that in WT mice. Chronic moderate pressure overload for 12 weeks caused enhanced left ventricular hypertrophy in KO mice, and survival and interstitial fibrosis were comparable with WT mice. Cardiac function, 12 weeks after operation, was comparable among shams and pressure-overloaded mice of both genotypes. In addition, KO mice exposed to chronic pressure overload showed higher cardiac capillary density associated with increased protein levels of VEGF. CONCLUSIONS: Thus, JunD limits cardiomyocyte hypertrophy and protects the pressure-overloaded heart from cardiac apoptosis. These beneficial effects of JunD, however, are associated with antiangiogenic properties.