Evolution des génomes microsporidiens et mécanisme d'adaptation moléculaire chez les parasites intracellulaires obligatoires

Microsporidia are obligate intracellular parasites characterized by a unique invasive mechanism, and, in some species, by a highly compact nuclear genome. To improve our knowledge of gene expression in these opportunistic pathogens, experiments on transcriptional regulation mechanisms were undertaken. In the microsporidia Encephalitozoon cuniculi (a mammalian parasite with a genome size of 2.9 Mb), we demonstrated a polycistronic gene organization. At the time of transcription, the transcripts are randomly processed leading to an mRNA population composed of monocistronic and polycistronic mRNA. To study the structure of microsporidian genomes and their adaptation capacities, the sequencing of Brachiola algerae genome (an insect and mammalian microsporidia) has been initiated in collaboration with Genoscope. Thirty chromosome bands from 160 to 2240 kb were identified, giving a haploid genome size estimated of 23 Mb. The study of the chromosomal distribution of the 16S rRNA unit showed the presence of 16S copy on at least 9 chromosome bands. The first analysis of this AT rich genome (more than 75%) revealed the presence of different kinds of transposable elements as well as genes encoding for enzymes involved in the RNA interference process. We further demonstrated that two Anopheles gambiae cellular lineages Sua4.0 (haemocytic cell) and Mos 55 (embryonic cells) can be infected by B. algerae providing a model to study in vitro the interaction between An. gambiae and its pathogens. The successful use of the RNA interference in these two types of cells will contribute to a better understanding of the adaptation mechanisms of pathogens like P. falciparum and B. algerae.Les microsporidies sont des parasites intracellulaires obligatoires caractérisés par un mode d'infestation original, et pour certaines espèces, par un très fort degré de compaction du génome nucléaire. Des travaux portant sur les mécanismes de régulation transcriptionnelle chez Encephalitozoon cuniculi montrent qu'une organisation polycistronique des gènes est présente. Une maturation aléatoire des messagers conduit à la formation d'une population d'ARNm comprenant des ARNm monocistroniques et polycistroniques. Afin d'améliorer nos connaissances sur la structure des génomes microsporidiens et leur capacité d'adaptation, le sequençage du génome de Brachiola algerae a été initié : 30 bandes d'ADN chromosomiques ont pu être visualisées (taille du génome estimée à 23Mb). Les premières données de séquence ont révélées la présence de plusieurs classes d'éléments transposables ainsi que des gènes codant des enzymes impliquées dans le processus d'ARN interférenc

Evolution des génomes microsporidiens et mécanisme d'adaptation moléculaire chez les parasites intracellulaires obligatoires

Les microsporidies sont des parasites intracellulaires obligatoires caractérisés par un mode d'infestation original, et pour certaines espèces, par un très fort degré de compaction du génome nucléaire. Des travaux portant sur les mécanismes de régulation transcriptionnelle chez Encephalitozoon cuniculi montrent qu'une organisation polycistronique des gènes est présente. Une maturation aléatoire des messagers conduit à la formation d'une population d'ARNm comprenant des ARNm monocistroniques et polycistroniques. Afin d'améliorer nos connaissances sur la structure des génomes microsporidiens et leur capacité d'adaptation, le sequençage du génome de Brachiola algerae a été initié : 30 bandes d'ADN chromosomiques ont pu être visualisées (taille du génome estimée à 23Mb). Les premières données de séquence ont révélées la présence de plusieurs classes d'éléments transposables ainsi que des gènes codant des enzymes impliquées dans le processus d'ARN interférenceCLERMONT FD-BCIU Sci.et Tech. (630142101) / SudocSudocFranceF

Identification of two new polar tube proteins related to polar tube protein 2 in the microsporidian Antonospora locustae.

International audienceMicrosporidia are obligate intracellular eukaryotic parasites with a broad host spectrum characterized by a unique and highly sophisticated invasion apparatus, the polar tube (PT). In a previous study, two PT proteins, named AlPTP1 (50 kDa) and AlPTP2 (35 kDa), were identified in Antonospora locustae, an orthoptera parasite that is used as a biological control agent against locusts. Antibodies raised against AlPTP2 cross-reacted with a band migrating at ~70 kDa, suggesting that this 70-kDa antigen is closely related to AlPTP2. A blastp search against the A. locustae genome database allowed the identification of two further PTP2-like proteins named AlPTP2b (568 aa) and AlPTP2c (599 aa). Both proteins are characterized by a specific serine- and glycine-rich N-terminal extension with elastomeric structural features and share a common C-terminal end conserved with AlPTP2 (~88% identity for the last 250 aa). MS analysis of the 70-kDa band revealed the presence of AlPTP2b. Specific anti-AlPTP2b antibodies labelled the extruded PTs of the A. locustae spores, confirming that this antigen is a PT component. Finally, we showed that several PTP2-like proteins are also present in other phylogenetically related insect microsporidia, including Anncaliia algerae and Paranosema grylli

In vitro propagation of the microsporidian pathogen Brachiola algerae and studies of its chromosome and ribosomal DNA organization in the context of the complete genome sequencing project

International audienceBrachiola algerae has a broad host spectrum from human to mosquitoes. The successful infection of two mosquito cell lines (Mos55: embryonic cells and Sua 4.0: hemocyte-like cells) and a human cell line (HFF) highlights the efficient adaptive capacity of this microsporidian pathogen. The molecular karyotype of this microsporidian species was determined in the context of the B. algerae genome sequencing project, showing that its haploid genome consists of 30 chromosomal-sized DNAs ranging from 160 to 2240 kbp giving an estimated genome size of 23 Mbp. A contig of 12,269 bp including the DNA sequence of the B. algerae ribosomal transcription unit has been built from initial genomic sequences and the secondary structure of the large subunit rRNA constructed. The data obtained indicate that B. algerae should be an excellent parasitic model to understand genome evolution in relation to infectious capacity

Comparative genomics of microsporidian genomes reveals a minimal non-coding RNA set and new insights for transcription in minimal eukaryotic genomes

Microsporidia are ubiquitous intracellular pathogens whose opportunistic nature led to their increased recognition with the rise of the AIDS pandemic. As the RNA world was largely unexplored in this parasitic lineage, we developed a dedicated in silico methodology to carry out exhaustive identification of ncRNAs across the Encephalitozoon and Nosema genera. Thus, the previously missing U1 small nuclear RNA (snRNA) and small nucleolar RNAs (snoRNAs) targeting only the LSU rRNA were highlighted and were further validated using 5' and 3'RACE-PCR experiments. Overall, the 15 ncRNAs that were found shared between Encephalitozoon and Nosema spp. may represent the minimal core set required for parasitic life. Interestingly, the systematic presence of a CCC-or GGG-like motif in 5' of all ncRNA and mRNA gene transcripts regardless of the RNA polymerase involved suggests that the RNA polymerase machineries in microsporidia species could use common factors. Our data provide additional insights in accordance with the simplification processes observed in these reduce genomes and underline the usefulness of sequencing closely related species to help identify highly divergent ncRNAs in these parasites

Identification and localization of polar tube proteins in the extruded polar tube of the microsporidian Anncaliia algerae

Abstract Microsporidia are obligate intracellular parasites able to infect a wide range of hosts from invertebrates to vertebrates. The success of their invasion process is based on an original organelle, the polar tube, which is suddenly extruded from the spore to inoculate the sporoplasm into the host cytoplasm. The polar tube is mainly composed of proteins named polar tube proteins (PTPs). A comparative analysis allowed us to identify genes coding for 5 PTPs (PTP1 to PTP5) in the genome of the microsporidian Anncaliia algerae . While PTP1 and PTP2 are found on the whole polar tube, PTP3 is present in a large part of the extruded polar tube except at its end-terminal part. On the contrary, PTP4 is specifically detected at the end-terminal part of the polar tube. To complete PTPs repertoire, sequential sporal protein extractions were done with high concentration of reducing agents. In addition, a method to purify polar tubes was developed. Mass spectrometry analysis conducted on both samples led to the identification of a PTP3-like protein (PTP3b), and a new PTP (PTP7) only found at the extremity of the polar tube. The specific localization of PTPs asks the question of their roles in cell invasion processes used by A. algerae

The Prediction and Validation of Small CDSs Expand the Gene Repertoire of the Smallest Known Eukaryotic Genomes.

The proper prediction of the gene catalogue of an organism is essential to obtain a representative snapshot of its overall lifestyle, especially when it is not amenable to culturing. Microsporidia are obligate intracellular, sometimes hard to culture, eukaryotic parasites known to infect members of every animal phylum. To date, sequencing and annotation of microsporidian genomes have revealed a poor gene complement with highly reduced gene sizes. In the present paper, we investigated whether such gene sizes may have induced biases for the methodologies used for genome annotation, with an emphasis on small coding sequence (CDS) gene prediction. Using better delineated intergenic regions from four Encephalitozoon genomes, we predicted de novo new small CDSs with sizes ranging from 78 to 255 bp (median 168) and corroborated these predictions by RACE-PCR experiments in Encephalitozoon cuniculi. Most of the newly found genes are present in other distantly related microsporidian species, suggesting their biological relevance. The present study provides a better framework for annotating microsporidian genomes and to train and evaluate new computational methods dedicated at detecting ultra-small genes in various organisms

Draft genome sequence of the anaerobic intestinal parasite Blastocystis subtype 4 (ST4)

International audienceBlastocystis is a highly prevalent anaerobic eukaryotic intestinal parasite found in the intestinal tract of human and various animals. Although the role of Blastocystis as human pathogen remains unclear, it can cause acute or chronic digestive disorders and some studies have suggested an association with irritable bowel syndrome. Seventeen subtypes (ST1-ST17), among which the first nine are found in human, have been identified based on the gene coding for the small-subunit ribosomal RNA. We have previously sequenced the first whole genome of Blastocystis ST7 (Denoeud et al., 2011). It consists of a 18.8 Mb nuclear genome with more than 6000 genes and a circular genome of 29 Kbp located within mitochondria-like organelles (MLO). Here we report the sequencing and annotation of the genome of a Blastocystis ST4 isolate. Genome sequencing was done with the Illumina HiSeq 2000 system generating more than 43 millions of 100-bp paired-end reads. The sequence reads were de novo assembled using the IDBA-ud algorithm. In total, 3996 scaffolds higher than 200 bp were obtained, with a scaffold N50 determined to be 20,431 bp. The draft genome sequence of Blastocystis ST4 has a total of 13.36 Mbp. As expected, assembly also provided a circular genome of ~27 kb in size corresponding to the whole MLO genome sequence. Genes were predicted using the Maker gene annotation pipeline. A first run of Maker was performed using the ab initio predictor Augustus trained with 413 genes manually designed from the ST4 scaffolds, the ~6000 annotated genes of the ST7 genome and available ESTs data from both ST7 and ST1. Genes determined from this first run were then used to train another ab initio gene prediction program called SNAP. A second run of Maker similar to the first run and including the newly trained gene predictor SNAP was finally performed. This led to significant improvements in gene prediction accuracy with a final annotation set of 6046 genes. The same pipeline was also used to complete and correct the previous annotation of the ST7 genome (Denoeud et al., 2011). Gene functions (for ST4 predicted genes) were annotated by Blast2GO and blastP analyses with NCBI, Swissprot/Uniprot and KEGG databases. Finally OrthoMCL was applied to compare both ST4 and ST7 genomes. This led to the identification of new candidate genes, in particular some potential virulence factors that may be involved in the physiopathology of this parasite. The sequencing of other ST genomes is under progress and should be very helpful for a better understanding of the genetic diversity, pathogenesis, metabolic potential and genome evolution of this neglected human parasite