Influence de stress oxydatifs sur la biosynthèse de mycotoxines de Fusarium spp. Contaminantes de l'épi de Maïs.

Fusarium est un champignon responsable de la fusariose, maladie nécrosante observée chez de nombreuses céréales et le maïs. Certaines espèces fusariennes sont également productrices de mycotoxines, métabolites secondaires extrêmement stables dont les toxicités pour l'homme et les animaux sont largement décrites. Ces biosynthèses interviennent avant la récolte et entraînent la contamination des grains de céréales. A l'heure actuelle, il n'existe pas de procédé permettant d'éliminer les mycotoxines ni même de réduire leurs toxicités. Ainsi, limiter l'occurrence des mycotoxines sur les grains de céréales implique de limiter leurs productions au champ. Parmi les facteurs susceptibles de moduler les productions de toxines par Fusarium, la nature du substrat, dans notre cas les composés du grain de maïs, pourrait être déterminante. De façon plus précise, les molécules pro ou anti-oxydantes impliquées dans les mécanismes de défense de la plante en réponse à l'attaque par un pathogène sont-elles susceptibles d'influencer la biosynthèse de mycotoxines ? Le rôle central et ubiquitaire de H2O2 lors de la mise en place des mécanismes de défense de la plante nous a conduit à détailler les effets potentiels in vitro de H2O2 sur la production de toxines de la famille des trichothécènes de type B (TCTB) par Fusarium graminearum et Fusarium culmorum. Les effets d'autres composés pro- ou anti-oxydants impliqués ou non dans les mécanismes de défense de la plante ont également été testés. Après une phase de mise au point méthodologique, l'ensemble de nos résultats a permis de mettre en évidence l'existence d'un lien important entre la biosynthèse de TCTB et le métabolisme oxydatif fongique. De plus, il semblerait que la nature de ce lien dépende du type de TCTB considéré. Une approche transcriptomique locale sur des gènes Tri a permis de conclure à l'implication de régulations transcriptionnelles dans les modulations de la production de TCTB en conditions de stress oxydatifs. Une approche transcriptomique globale devrait, à terme, permettre d'identifier des voies métaboliques liées à celle de la production de toxines.Fusarium is a fungus that causes tissue necrosis on many cereals and corn. Several species can also produce mycotoxins, very stable secondary metabolites which toxicities for human beings and animals are widely illustrated. These biosynthesis take place before harvest and lead to kernel contamination. Nowadays, no process exists that could allow neither to remove the toxins nor to reduce their toxicities. Therefore, limiting toxins occurrences in kernels implies limiting their production in the field, before harvest. Among the factors liable to modulate toxins productions by Fusarium, the substrate composition, in our case corn kernel composition, could be decisive. More precisely, are pro- or anti-oxidant molecules involved in plant defence mechanisms against a pathogen attack likely to influence the biosynthesis of mycotoxins? The central and ubiquitous role of H2O2 when plant defence mechanisms are triggered leaded us to look in detail at possible effects of H2O2 on the production of toxins that belong to the type B trichothecenes family (TCTB) by Fusarium graminearum and Fusarium culmorum. Effects of other pro- or anti-oxidant compounds, involved or not in plant defence mechanisms, were tested as well. After a period of methods adjustments, our results allowed us to observe a link between TCTB biosynthesis and the oxidative metabolism of the fungus. Furthermore, this link seems to be different depending on the type of TCTB that is considered. A local transcriptomic approach on Tri genes allowed us to conclude to transcriptional regulations occurring when situations of oxidative stress occur. Forward, a total transcriptomic approach should let us identify other metabolic pathways linked to toxins biosynthesis

DNA-encoded nucleosome occupancy is associated with transcription levels in the human malaria parasite Plasmodium falciparum.

BackgroundIn eukaryotic organisms, packaging of DNA into nucleosomes controls gene expression by regulating access of the promoter to transcription factors. The human malaria parasite Plasmodium falciparum encodes relatively few transcription factors, while extensive nucleosome remodeling occurs during its replicative cycle in red blood cells. These observations point towards an important role of the nucleosome landscape in regulating gene expression. However, the relation between nucleosome positioning and transcriptional activity has thus far not been explored in detail in the parasite.ResultsHere, we analyzed nucleosome positioning in the asexual and sexual stages of the parasite's erythrocytic cycle using chromatin immunoprecipitation of MNase-digested chromatin, followed by next-generation sequencing. We observed a relatively open chromatin structure at the trophozoite and gametocyte stages, consistent with high levels of transcriptional activity in these stages. Nucleosome occupancy of genes and promoter regions were subsequently compared to steady-state mRNA expression levels. Transcript abundance showed a strong inverse correlation with nucleosome occupancy levels in promoter regions. In addition, AT-repeat sequences were strongly unfavorable for nucleosome binding in P. falciparum, and were overrepresented in promoters of highly expressed genes.ConclusionsThe connection between chromatin structure and gene expression in P. falciparum shares similarities with other eukaryotes. However, the remarkable nucleosome dynamics during the erythrocytic stages and the absence of a large variety of transcription factors may indicate that nucleosome binding and remodeling are critical regulators of transcript levels. Moreover, the strong dependency between chromatin structure and DNA sequence suggests that the P. falciparum genome may have been shaped by nucleosome binding preferences. Nucleosome remodeling mechanisms in this deadly parasite could thus provide potent novel anti-malarial targets

The multifunctional autophagy pathway in the human malaria parasite, Plasmodium falciparum.

Autophagy is a catabolic pathway typically induced by nutrient starvation to recycle amino acids, but can also function in removing damaged organelles. In addition, this pathway plays a key role in eukaryotic development. To date, not much is known about the role of autophagy in apicomplexan parasites and more specifically in the human malaria parasite Plasmodium falciparum. Comparative genomic analysis has uncovered some, but not all, orthologs of autophagy-related (ATG) genes in the malaria parasite genome. Here, using a genome-wide in silico analysis, we confirmed that ATG genes whose products are required for vesicle expansion and completion are present, while genes involved in induction of autophagy and cargo packaging are mostly absent. We subsequently focused on the molecular and cellular function of P. falciparum ATG8 (PfATG8), an autophagosome membrane marker and key component of the autophagy pathway, throughout the parasite asexual and sexual erythrocytic stages. In this context, we showed that PfATG8 has a distinct and atypical role in parasite development. PfATG8 localized in the apicoplast and in vesicles throughout the cytosol during parasite development. Immunofluorescence assays of PfATG8 in apicoplast-minus parasites suggest that PfATG8 is involved in apicoplast biogenesis. Furthermore, treatment of parasite cultures with bafilomycin A 1 and chloroquine, both lysosomotropic agents that inhibit autophagosome and lysosome fusion, resulted in dramatic morphological changes of the apicoplast, and parasite death. Furthermore, deep proteomic analysis of components associated with PfATG8 indicated that it may possibly be involved in ribophagy and piecemeal microautophagy of the nucleus. Collectively, our data revealed the importance and specificity of the autophagy pathway in the malaria parasite and offer potential novel therapeutic strategies

Exploratory analysis of genomic segmentations with Segtools

<p>Abstract</p> <p>Background</p> <p>As genome-wide experiments and annotations become more prevalent, researchers increasingly require tools to help interpret data at this scale. Many functional genomics experiments involve partitioning the genome into labeled segments, such that segments sharing the same label exhibit one or more biochemical or functional traits. For example, a collection of ChlP-seq experiments yields a compendium of peaks, each labeled with one or more associated DNA-binding proteins. Similarly, manually or automatically generated annotations of functional genomic elements, including <it>cis</it>-regulatory modules and protein-coding or RNA genes, can also be summarized as genomic segmentations.</p> <p>Results</p> <p>We present a software toolkit called <it>Segtools </it>that simplifies and automates the exploration of genomic segmentations. The software operates as a series of interacting tools, each of which provides one mode of summarization. These various tools can be pipelined and summarized in a single HTML page. We describe the Segtools toolkit and demonstrate its use in interpreting a collection of human histone modification data sets and <it>Plasmodium falciparum </it>local chromatin structure data sets.</p> <p>Conclusions</p> <p>Segtools provides a convenient, powerful means of interpreting a genomic segmentation.</p

Deciphering the Ubiquitin-Mediated Pathway in Apicomplexan Parasites: A Potential Strategy to Interfere with Parasite Virulence

Reversible modification of proteins through the attachment of ubiquitin or ubiquitin-like modifiers is an essential post-translational regulatory mechanism in eukaryotes. The conjugation of ubiquitin or ubiquitin-like proteins has been demonstrated to play roles in growth, adaptation and homeostasis in all eukaryotes, with perturbation of ubiquitin-mediated systems associated with the pathogenesis of many human diseases, including cancer and neurodegenerative disorders

An Apicoplast Localized Ubiquitylation System Is Required for the Import of Nuclear-encoded Plastid Proteins

Apicomplexan parasites are responsible for numerous important human diseases including toxoplasmosis, cryptosporidiosis, and most importantly malaria. There is a constant need for new antimalarials, and one of most keenly pursued drug targets is an ancient algal endosymbiont, the apicoplast. The apicoplast is essential for parasite survival, and several aspects of its metabolism and maintenance have been validated as targets of anti-parasitic drug treatment. Most apicoplast proteins are nuclear encoded and have to be imported into the organelle. Recently, a protein translocon typically required for endoplasmic reticulum associated protein degradation (ERAD) has been proposed to act in apicoplast protein import. Here, we show ubiquitylation to be a conserved and essential component of this process. We identify apicoplast localized ubiquitin activating, conjugating and ligating enzymes in Toxoplasma gondii and Plasmodium falciparum and observe biochemical activity by in vitro reconstitution. Using conditional gene ablation and complementation analysis we link this activity to apicoplast protein import and parasite survival. Our studies suggest ubiquitylation to be a mechanistic requirement of apicoplast protein import independent to the proteasomal degradation pathway.This work was funded by grants from the National Institutes of Health to BS (AI 64671) and funds provided by the University of California, Riverside to KLR. SA was supported by a predoctoral fellowship from the American Heart Association, and GGD by a C.J. Martin Overseas Fellowship from the Australian National Health and Medical Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript