A locus conferring tolerance to Theileria infection in African cattle.

East Coast fever, a tick-borne cattle disease caused by the Theileria parva parasite, is among the biggest natural killers of cattle in East Africa, leading to over 1 million deaths annually. Here we report on the genetic analysis of a cohort of Bos indicus (Boran) cattle demonstrating heritable tolerance to infection with T. parva (h2 = 0.65, s.e. 0.57). Through a linkage analysis we identify a 6 Mb genomic region on bovine chromosome 15 that is significantly associated with survival outcome following T. parva exposure. Testing this locus in an independent cohort of animals replicates this association with survival following T. parva infection. A stop gained variant in a paralogue of the FAF1 gene in this region was found to be highly associated with survival across both related and unrelated animals, with only one of the 20 homozygote carriers (T/T) of this change succumbing to the disease in contrast to 44 out of 97 animals homozygote for the reference allele (C/C). Consequently, we present a genetic locus linked to tolerance of one of Africa's most important cattle diseases, raising the promise of marker-assisted selection for cattle that are less susceptible to infection by T. parva.

miR-126-5p by direct targeting of JNK-interacting protein-2 (JIP-2) plays a key role in Theileria-infected macrophage virulence.

Theileria annulata is an apicomplexan parasite that infects and transforms bovine macrophages that disseminate throughout the animal causing a leukaemia-like disease called tropical theileriosis. Using deep RNAseq of T. annulata-infected B cells and macrophages we identify a set of microRNAs induced by infection, whose expression diminishes upon loss of the hyper-disseminating phenotype of virulent transformed macrophages. We describe how infection-induced upregulation of miR-126-5p ablates JIP-2 expression to release cytosolic JNK to translocate to the nucleus and trans-activate AP-1-driven transcription of mmp9 to promote tumour dissemination. In non-disseminating attenuated macrophages miR-126-5p levels drop, JIP-2 levels increase, JNK1 is retained in the cytosol leading to decreased c-Jun phosphorylation and dampened AP-1-driven mmp9 transcription. We show that variation in miR-126-5p levels depends on the tyrosine phosphorylation status of AGO2 that is regulated by Grb2-recruitment of PTP1B. In attenuated macrophages Grb2 levels drop resulting in less PTP1B recruitment, greater AGO2 phosphorylation, less miR-126-5p associated with AGO2 and a consequent rise in JIP-2 levels. Changes in miR-126-5p levels therefore, underpin both the virulent hyper-dissemination and the attenuated dissemination of T. annulata-infected macrophages.

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.

Repurposing Auranofin and Evaluation of a New Gold(I) Compound for the Search of Treatment of Human and Cattle Parasitic Diseases: From Protozoa to Helminth Infections.

Neglected parasitic diseases remain a major public health issue worldwide, especially in tropical and subtropical areas. Human parasite diversity is very large, ranging from protozoa to worms. In most cases, more effective and new drugs are urgently needed. Previous studies indicated that the gold(I) drug auranofin (Ridaura®) is effective against several parasites. Among new gold(I) complexes, the phosphole-containing gold(I) complex {1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl (abbreviated as GoPI) is an irreversible inhibitor of both purified human glutathione and thioredoxin reductases. GoPI-sugar is a novel 1-thio-ß-d-glucopyranose 2,3,4,6-tetraacetato-S-derivative that is a chimera of the structures of GoPI and auranofin, designed to improve stability and bioavailability of GoPI. These metal-ligand complexes are of particular interest because of their combined abilities to irreversibly target the essential dithiol/selenol catalytic pair of selenium-dependent thioredoxin reductase activity, and to kill cells from breast and brain tumors. In this work, screening of various parasites-protozoans, trematodes, and nematodes-was undertaken to determine the in vitro killing activity of GoPI-sugar compared to auranofin. GoPI-sugar was found to efficiently kill intramacrophagic Leishmania donovani amastigotes and adult filarial and trematode worms.

Cell penetrating peptides to dissect host-pathogen protein-protein interactions in Theileria-transformed leukocytes.

One powerful application of cell penetrating peptides is the delivery into cells of molecules that function as specific competitors or inhibitors of protein-protein interactions. Ablating defined protein-protein interactions is a refined way to explore their contribution to a particular cellular phenotype in a given disease context. Cell-penetrating peptides can be synthetically constrained through various chemical modifications that stabilize a given structural fold with the potential to improve competitive binding to specific targets. Theileria-transformed leukocytes display high PKA activity, but PKA is an enzyme that plays key roles in multiple cellular processes; consequently genetic ablation of kinase activity gives rise to a myriad of confounding phenotypes. By contrast, ablation of a specific kinase-substrate interaction has the potential to give more refined information and we illustrate this here by describing how surgically ablating PKA interactions with BAD gives precise information on the type of glycolysis performed by Theileria-transformed leukocytes. In addition, we provide two other examples of how ablating specific protein-protein interactions in Theileria-infected leukocytes leads to precise phenotypes and argue that constrained penetrating peptides have great therapeutic potential to combat infectious diseases in general.

TSC-22 Promotes Interleukin-2-Deprivation Induced Apoptosis in T-Lymphocytes.

Originally described as a TGF-ß-inducible gene, tsc-22 (Transforming growth factor-beta Stimulated Clone 22) encodes a transcriptional regulator affecting biological processes such as cell growth, differentiation, or apoptosis. Along with GILZ (Glucocorticoid-Induced Leucine Zipper), TSC-22 belongs to the evolutionary conserved TSC-22 Domain family. We previously showed that, in T-lymphocytes, GILZ expression was induced upon IL-2 withdrawal, delaying apoptosis through down-regulation of the pro-apoptotic protein BIM expression. The aim of this work was then to elucidate the respective roles of GILZ and TSC-22 upon IL-2 deprivation-induced apoptosis. We report here that these two highly homologous genes are concomitantly expressed in most human tissues and in primary T-lymphocytes and that expression of TSC-22 promotes T-lymphocytes apoptosis by inhibiting GILZ functions. Indeed, we demonstrated that TSC-22 expression in the murine lymphoid CTLL-2 cell line promoted IL-2 deprivation-induced apoptosis. BIM expression and caspases-9 and -3 activities were markedly increased in TSC-22 expressing clones compared to control clones. Analysis of GILZ expression revealed that TSC-22 prevented the induction of the GILZ protein upon IL-2 deprivation, by inhibiting gilz mRNA transcription. These results suggested that TSC-22 could counteract the protective effect of GILZ on IL-2-deprivation-induced apoptosis. Moreover, TSC-22-induced inhibition of GILZ expression was also found in CTLL-2 cells treated with glucocorticoids or TGF-ß. In the human NKL cell line deprived of IL-2, TSC-22 showed the same effect and thus may represent a potent repressor of GILZ expression in IL-2-dependent cells, independently of the cell type, or the stimulus, leading to an increase of IL-2-deprived T-cells apoptosis. J. Cell. Biochem. 117: 1855-1868, 2016. © 2016 Wiley Periodicals, Inc.

[TSC-22D proteins: new regulators of cell homeostasis?]. / Les protéines de la famille TSC-22D - De nouveaux régulateurs de l'homéostasie cellulaire ?

The GILZ (glucocorticoid-induced leucine zipper) protein has first been identified as a glucocorticoid-responsive gene and is now presented as a major regulator of inflammation. Expanding literature documents a role for GILZ as a mediator of the immuno-modulatory and anti-inflammatory effects of glucocorticoids, mainly through interference with key signal transduction pathways such as nuclear factor-kappa B (NF-kB) or activated protein-1 (AP-1). The TSC-22 (TGF-ß-stimulated clone-22) protein is described as an apoptosis modulator and as a new tumor suppressor gene. GILZ and TSC-22, characterized by the presence of a leucine zipper domain and a TSC-box, belong to the TSC-22D (TSC-22 domain) family of proteins which comprises today 18 members. Functions of these proteins suggest that this family plays a major role in cell homeostasis and in the regulation of the immune system.

Glucocorticoid-induced leucine zipper (GILZ) promotes the nuclear exclusion of FOXO3 in a Crm1-dependent manner.

GILZ (glucocorticoid-induced leucine zipper) is an ubiquitous protein whose expression is induced by glucocorticoids in lymphoid cells. We previously showed that GILZ expression is rapidly induced upon interleukin 2 deprivation in T-cells, protecting cells from apoptosis induced by forkhead box subgroup O3 (FOXO3). The aim of this work is to elucidate the molecular mechanism of FOXO factor inhibition by GILZ. We show in the myeloid cell line HL-60 and the lymphoid CTLL-2 T-cell line that GILZ down-regulates the expression of p27(KIP1) and Bim, two FOXO targets involved in cell cycle regulation and apoptosis, respectively. GILZ inhibits FOXO1, FOXO3, and FOXO4 transcriptional activities measured with natural or synthetic FOXO-responsive promoters in HL-60 cells. This inhibitory effect is independent of protein kinase B and IkappaB kinase phosphorylation sites. GILZ does not hinder FOXO3 DNA-binding activity and does not physically interact with FOXO3. However, using fluorescence microscopy, we observe that GILZ expression provokes a Crm-1-dependent nuclear exclusion of FOXO3 leading to its relocalization to the cytoplasm. Moreover, GILZ exclusive cytoplasmic localization is a prerequisite for FOXO3 inhibition and relocalization. We propose that GILZ is a general inhibitor of FOXO factors acting through an original mechanism by preventing them from reaching target genes within the nucleus.

Inherited Tolerance in Cattle to the Apicomplexan Protozoan Theileria parva is Associated with Decreased Proliferation of Parasite-Infected Lymphocytes.

Theileria parva is the causative agent of East Coast fever and Corridor disease, which are fatal, economically important diseases of cattle in eastern, central and southern Africa. Improved methods of control of the diseases are urgently required. The parasite transforms host lymphocytes, resulting in a rapid, clonal expansion of infected cells. Resistance to the disease has long been reported in cattle from T. parva-endemic areas. We reveal here that first- and second-generation descendants of a single Bos indicus bull survived severe challenge with T. parva, (overall survival rate 57.3% compared to 8.7% for unrelated animals) in a series of five field studies. Tolerant cattle displayed a delayed and less severe parasitosis and febrile response than unrelated animals. The in vitro proliferation of cells from surviving cattle was much reduced compared to those from animals that succumbed to infection. Additionally, some pro-inflammatory cytokines such as IL1ß, IL6, TNFα or TGFß which are usually strongly expressed in susceptible animals and are known to regulate cell growth or motility, remain low in tolerant animals. This correlates with the reduced proliferation and less severe clinical reactions observed in tolerant cattle. The results show for the first time that the inherited tolerance to T. parva is associated with decreased proliferation of infected lymphocytes. The results are discussed in terms of whether the reduced proliferation is the result of a perturbation of the transformation mechanism induced in infected cells or is due to an innate immune response present in the tolerant cattle.

Clinical Evaluation of Corridor Disease in Bos indicus (Boran) Cattle Naturally Infected With Buffalo-Derived Theileria parva.

Corridor disease (CD) is a fatal condition of cattle caused by buffalo-derived Theileria parva. Unlike the related condition, East Coast fever, which results from infection with cattle-derived T. parva, CD has not been extensively studied. We describe in detail the clinical and laboratory findings in cattle naturally infected with buffalo-derived T. parva. Forty-six cattle were exposed to buffalo-derived T. parva under field conditions at the Ol Pejeta Conservancy, Kenya, between 2013 and 2018. The first signs of disease observed in all animals were nasal discharge (mean day of onset was 9 days post-exposure), enlarged lymph nodes (10 days post-exposure), and pyrexia (13.7 days post-exposure). Coughing and labored breathing were observed in more than 50% of animals (14 days post-exposure). Less commonly observed signs, corneal edema (22%) and diarrhea (11%), were observed later in the disease progression (19 days post-exposure). All infections were considered clinically severe, and 42 animals succumbed to infection. The mean time to death across all studies was 18.4 days. The mean time from onset of clinical signs to death was 9 days and from pyrexia to death was 4.8 days, indicating a relatively short duration of clinical illness. There were significant relationships between days to death and the days to first temperature (chi2 = 4.00, p = 0.046), and days to peak temperature (chi2 = 25.81, p = 0.001), animals with earlier onset pyrexia died sooner. These clinical indicators may be useful for assessing the severity of disease in the future. All infections were confirmed by the presence of macroschizonts in lymph node biopsies (mean time to parasitosis was 11 days). Piroplasms were detected in the blood of two animals (4%) and 20 (43%) animals seroconverted. In this study, we demonstrate the successful approach to an experimental field study for CD in cattle. We also describe the clinical progression of CD in naturally infected cattle, including the onset and severity of clinical signs and pathology. Laboratory diagnoses based on examination of blood samples are unreliable, and alternatives may not be available to cattle keepers. The rapid development of CD requires recognition of the clinical signs, which may be useful for early diagnosis of the disease and effective intervention for affected animals.

Inverted CD4+/CD8+ T cell ratio in Boran (Bos indicus) cattle.

The CD4+/CD8+ ratio is used as a marker of the immune regulation of T cell balance. When the ratio in peripheral blood is less than 1, this is considered an indication of immune suppression in an individual. Previous work on bovine Peripheral Blood Mononuclear Cells (PBMC) has consistently reported a ratio ≥1 as seen in other mammalian hosts, i.e. higher circulating CD4+ cell numbers than CD8+ cell numbers. However, a consistent inverted CD4+/CD8+ ratio (<1) was observed in Boran cattle, an African Bos indicus breed. The T cell populations were characterized in Boran cattle (n = 52), revealing higher percentages of circulating CD8+ cells (31.9 % average) than CD4+ cells (19.1 % average), thus resulting in the inversion of the expected T cell homeostasis in these animals. The results show that this inversion is not an effect of age or relatedness of the cattle, rather, it was shared by almost all Boran cattle used in this study. Despite this inversion being a feature shared by both males and females, the female cattle had significantly higher CD4+/CD8+ ratios than the male Boran. This paper describes the characteristics of the T cell fractions in the study animals and compares the findings to those of other Boran cattle in Kenya, and four other cattle breeds representing African indicine, African taurine, Brazilian indicine and European taurine cattle. We demonstrate that the consistent observation of inverted CD4+/CD8+ cell ratio was restricted to the Boran.

Apicomplexan autophagy and modulation of autophagy in parasite-infected host cells.

Apicomplexan parasites are responsible for a number of important human pathologies. Obviously, as Eukaryotes they share a number of cellular features and pathways with their respective host cells. One of them is autophagy, a process involved in the degradation of the cell's own components. These intracellular parasites nonetheless seem to present a number of original features compared to their very evolutionarily distant host cells. In mammals and other metazoans, autophagy has been identified as an important contributor to the defence against microbial pathogens. Thus, host autophagy also likely plays a key role in the control of apicomplexan parasites, although its potential manipulation and subversion by intracellular parasites creates a complex interplay in the regulation of host and parasite autophagy. In this mini-review, we summarise current knowledge on autophagy in both parasites and their host cells, in the context of infection by three Apicomplexa: Plasmodium, Toxoplasma, and Theileria.

From Christian de Duve to Yoshinori Ohsumi: More to autophagy than just dining at home.

Christian de Duve first coined the expression "autophagy" during his seminal work on the discovery of lysosomes, which led to him being awarded the Nobel Prize in Physiology or Medicine in 1974. The term was adopted to distinguish degradation of intracellular components from the uptake and degradation of extracellular substances that he called "heterophagy". Studies until the 1990s were largely observational/morphological-based until in 1993 Yoshinori Oshumi described a genetic screen in yeast undergoing nitrogen deprivation that led to the isolation of autophagy-defective mutants now better known as ATG (AuTophaGy-related) genes. The screen identified mutants that fell into 15 complementation groups implying that at least 15 genes were involved in the regulation of autophagy in yeast undergoing nutrient deprivation, but today, 41 yeast ATG genes have been described and many (though not all) have orthologues in humans. Attempts to identify the genetic basis of autophagy led to an explosion in its research and it's not surprising that in 2016 Yoshinori Oshumi was awarded the Nobel Prize in Physiology or Medicine. Our aim here is not to exhaustively review the ever-expanding autophagy literature (>60 papers per week), but to celebrate Yoshinori Oshumi's Nobel Prize by highlighting just a few aspects that are not normally extensively covered. In an accompanying mini-review we address the role of autophagy in early-diverging eukaryote parasites that like yeast, lack lysosomes and so use a digestive vacuole to degrade autophagosome cargo and also discuss how parasitized host cells react to infection by subverting regulation of autophagy.

Vanin-1 inactivation antagonizes the development of adrenocortical neoplasia in Sf-1 transgenic mice.

SF-1 (NR5A1) overexpression can induce adrenocortical tumor formation in transgenic mice and is associated with more severe prognosis in patients with adrenocortical cancer. In this study we have identified Vanin-1 (Vnn1), a SF-1 target gene, as a novel modulator of the tumorigenic effect of Sf-1 overexpression in the adrenal cortex. Vanin-1 is endowed with pantetheinase activity, releasing cysteamine in tissues and regulating cell response to oxidative stress by modulating the production of glutathione. Sf-1 transgenic mice developed adrenocortical neoplastic lesions (both dysplastic and nodular) with a frequency increasing with age. Genetic ablation of the Vnn1 gene in Sf-1 transgenic mice significantly reduced the severity of neoplastic lesions in the adrenal cortex. This effect could be reversed by treatment of Sf-1 transgenic/Vnn1 null mice with cysteamine. These data show that alteration of the mechanisms controlling intracellular redox and detoxification mechanisms is relevant to the pathogenesis of adrenocortical neoplasia induced by SF-1 overexpression.

Beyond steroidogenesis: novel target genes for SF-1 discovered by genomics.

Steroidogenic Factor-1 (SF-1) is a nuclear receptor transcription factor that has an essential role in the development of adrenal glands and gonads and in the regulation of steroidogenic gene expression. Recent studies using genomic approaches have revealed that SF-1 also has an important role in regulating proliferation of adrenocortical cells and have revealed its role in the control of a variety of biological processes as diverse as angiogenesis, adhesion to the extracellular matrix, cytoskeleton dynamics, transcriptional and post-transcriptional regulation of gene expression and apoptosis in the adrenal cortex. The identification of the complete set of SF-1 target genes will be of great importance to open new avenues for therapeutic intervention in adrenal diseases.

Genetics and genomics of childhood adrenocortical tumors.

Adrenocortical tumors in children are usually diagnosed because of signs of virilization and their prognosis is poor. They possess several distinct pathological features compared to adrenocortical tumors in adults and have an exceptional prevalence in southern Brazil, where they are nearly invariably linked to the presence of a germline specific TP53 (R337H) mutation. Other important factors in childhood adrenocortical tumor pathogenesis are overexpression of the Steroidogenic Factor-1 transcription factor and imprinting defects in the 11p15 genomic region, causing overexpression of Insulin-like Growth Factor-2. Genomic studies have revealed the prognostic relevance of the expression of some Major Histocompatibility Complex genes and the deregulation of the Insulin-like Growth Factor/mammalian Target Of Rapamycin pathway by microRNAs in these tumors. Our hope is that these findings will constitute the basis for the development of novel therapies that will be more active against these tumors and less toxic for the patients.