Jean-Yves Coppée

Advances in high-throughput sequencing have led to the discovery of widespread transcription of natural antisense transcripts (NATs) in a large number of organisms, where these transcripts have been shown to play important roles in the regulation of gene expression. Likewise, the existence of NATs has been observed in Plasmodium but our understanding towards their genome-wide distribution remains incomplete due to the limited depth and uncertainties in the level of strand specificity of previous datasets. To gain insights into the genome-wide distribution of NATs in P. falciparum, we performed RNA-ligation based strand-specific RNA sequencing at unprecedented depth. Our data indicate that 78.3% of the genome is transcribed during blood-stage development. Moreover, our analysis reveals significant levels of antisense transcription from at least 24% of protein-coding genes and that while expression levels of NATs change during the intraerythrocytic developmental cycle (IDC), they do not correlate with the corresponding mRNA levels. Interestingly, antisense transcription is not evenly distributed across coding regions (CDSs) but strongly clustered towards the 3′-end of CDSs. Furthermore, for a significant subset of NATs, transcript levels correlate with mRNA levels of neighboring genes. Finally, we were able to identify the polyadenylation sites (PASs) for a subset of NATs, demonstrating that at least some NATs are polyadenylated. We also mapped the PASs of 3443 coding genes, yielding an average 3′ untranslated region length of 523 bp. Our strand-specific analysis of the P. falciparum transcriptome expands and strengthens the existing body of evidence that antisense transcription is a substantial phenomenon in P. falciparum. For a subset of neighboring genes we find that sense and antisense transcript levels are intricately linked while other NATs appear to be regulated independently of mRNA transcription. Our deep strand-specific dataset will provide a valuable resource for the precise determination of expression levels as it separates sense from antisense transcript levels, which we find to often significantly differ. In addition, the extensive novel data on 3′ UTR length will allow others to perform searches for regulatory motifs in the UTRs and help understand post-translational regulation in P. falciparum.

Background: Drug susceptible clinical isolates of Candida albicans frequently become highly tolerant to drugs during chemotherapy, with dreadful consequences to patient health. We used RNA sequencing (RNA-seq) to analyze the transcriptomes of a CDR (Candida Drug Resistance) strain and its isogenic drug sensitive counterpart. Results: RNA-seq unveiled differential expression of 228 genes including a) genes previously identified as involved in CDR, b) genes not previously associated to the CDR phenotype, and c) novel transcripts whose function as a gene is uncharacterized. In particular, we show for the first time that CDR acquisition is correlated with an overexpression of the transcription factor encoding gene CZF1. CZF1 null mutants were susceptible to many drugs, independently of known multidrug resistance mechanisms. We show that CZF1 acts as a repressor of β-glucan synthesis, thus negatively regulating cell wall integrity. Finally, our RNA-seq data allowed us to identify a new transcribed region, upstream of the TAC1 gene, which encodes the major CDR transcriptional regulator. Conclusion: Our results open new perspectives of the role of Czf1 and of our understanding of the transcriptional and post-transcriptional mechanisms that lead to the acquisition of drug resistance in C. albicans, with potential for future improvements of therapeutic strategies.

Brown algae of the genus Ectocarpus exhibit high levels of genetic diversity and variability in morphological and physiological characteristics. With the establishment of E. siliculosus as a model and the availability of a complete genome sequence, it is now of interest to analyze variability among different species, ecotypes, and strains of the genus Ectocarpus both at the genome and the transcriptome level.

Background: Arsenic is present in numerous ecosystems and microorganisms have developed various mechanisms to live in such hostile environments. Herminiimonas arsenicoxydans, a bacterium isolated from arsenic contaminated sludge, has acquired remarkable capabilities to cope with arsenic. In particular our previous studies have suggested the existence of a temporal induction of arsenite oxidase, a key enzyme in arsenic metabolism, in the presence of As(III). Results: Microarrays were designed to compare gene transcription profiles under a temporal As(III) exposure. Transcriptome kinetic analysis demonstrated the existence of two phases in arsenic response. The expression of approximatively 14% of the whole genome was significantly affected by an As(III) early stress and 4% by an As(III) late exposure. The early response was characterized by arsenic resistance, oxidative stress, chaperone synthesis and sulfur metabolism. The late response was characterized by arsenic metabolism and associated mechanisms such as phosphate transport and motility. The major metabolic changes were confirmed by chemical, transcriptional, physiological and biochemical experiments. These early and late responses were defined as general stress response and specific response to As(III), respectively. Conclusion: Gene expression patterns suggest that the exposure to As(III) induces an acute response to rapidly minimize the immediate effects of As(III). Upon a longer arsenic exposure, a broad metabolic response was induced. These data allowed to propose for the first time a kinetic model of the As(III) response in bacteria.

Background: Both the speciation and toxicity of arsenic are affected by bacterial transformations, i.e. oxidation, reduction or methylation. These transformations have a major impact on environmental contamination and more particularly on arsenic contamination of drinking water. Herminiimonas arsenicoxydans has been isolated from an arsenic- contaminated environment and has developed various mechanisms for coping with arsenic, including the oxidation of As(III) to As(V) as a detoxification mechanism. Results: In the present study, a differential transcriptome analysis was used to identify genes, including arsenite oxidase encoding genes, involved in the response of H. arsenicoxydans to As(III). To get insight into the molecular mechanisms of this enzyme activity, a Tn5 transposon mutagenesis was performed. Transposon insertions resulting in a lack of arsenite oxidase activity disrupted aoxR and aoxS genes, showing that the aox operon transcription is regulated by the AoxRS two-component system. Remarkably, transposon insertions were also identified in rpoN coding for the alternative N sigma factor (σ⁵⁴) of RNA polymerase and in dnaJ coding for the Hsp70 co-chaperone. Western blotting with anti-AoxB antibodies and quantitative RT-PCR experiments allowed us to demonstrate that the rpoN and dnaJ gene products are involved in the control of arsenite oxidase gene expression. Finally, the transcriptional start site of the aoxAB operon was determined using rapid amplification of cDNA ends (RACE) and a putative -12/-24 σ⁵⁴-dependent promoter motif was identified upstream of aoxAB coding sequences. Conclusion: These results reveal the existence of novel molecular regulatory processes governing arsenite oxidase expression in H. arsenicoxydans. These data are summarized in a model that functionally integrates arsenite oxidation in the adaptive response to As(III) in this microorganism.

A number of molecular tools have been developed to monitor the emergence and spread of anti-malarial drug resistance to Plasmodium falciparum. One of the major obstacles to the wider implementation of these tools is the absence of practical methods enabling high throughput analysis. Here a new Zip-code array is described, called FlexiChip, linked to a dedicated software program, which largely overcomes this problem.

Over the last years, significant progress has been made in the comprehension of the molecular mechanism of malaria resistance to drugs. Together with in vivo tests, the molecular monitoring is now part of the survey strategy of the Plasmodium sensitivity. Currently, DNA-microarray analysis allows the simultaneous study of many single nucleotide polymorphisms (SNP) of Plasmodium isolates. In December 2005, the International Federation of the Red Cross distributed two million three hundred thousand long-lasting insecticide nets to pregnant women and mothers of under five years children in the whole Niger. Then, Niger adopted artemisinin-based combination therapy as first-line treatment.

Background: In man, infection by the Gram-negative enteropathogen Yersinia pseudotuberculosis is usually limited to the terminal ileum. However, in immunocompromised patients, the microorganism may disseminate from the digestive tract and thus cause a systemic infection with septicemia. Results: To gain insight into the metabolic pathways and virulence factors expressed by the bacterium at the blood stage of pseudotuberculosis, we compared the overall gene transcription patterns (the transcriptome) of bacterial cells cultured in either human plasma or Luria-Bertani medium. The most marked plasma-triggered metabolic consequence in Y. pseudotuberculosis was the switch to high glucose consumption, which is reminiscent of the acetogenic pathway (known as "glucose overflow") in Escherichia coli. However, upregulation of the glyoxylate shunt enzymes suggests that (in contrast to E. coli) acetate may be further metabolized in Y. pseudotuberculosis. Our data also indicate that the bloodstream environment can regulate major virulence genes (positively or negatively); the yadA adhesin gene and most of the transcriptional units of the pYV-encoded type III secretion apparatus were found to be upregulated, whereas transcription of the pH6 antigen locus was strongly repressed. Conclusion: Our results suggest that plasma growth of Y. pseudotuberculosis is responsible for major transcriptional regulatory events and prompts key metabolic reorientations within the bacterium, which may in turn have an impact on virulence.

Background: Establishment of aspergillosis is depending upon the exit from dormancy and germination of the conidia of Aspergillus fumigatus in the lung. To gain an understanding of the molecular mechanisms underlying the early steps of conidial germination, we undertook a transcriptomic analysis using macroarrays constructed with PCR fragments from > 3,000 genes (around one third of the annotated A. fumigatus genome). Results: Major results of this analysis are the following: (i) conidia stored pre-packaged mRNAs transcripts (27% of genes have transcripts in the resting conidia; (ii) incubation at 37°C in a nutritive medium induced up- and down-regulation of genes: 19% of the total number of genes deposited on the array were up-regulated whereas 22% of the genes with pre-packaged mRNA in the resting conidia were down-regulated; (iii) most modifications were seen during the first 30 min of germination whereas very little modification of gene expression occurred during the following hour; (iv) one-year old conidia and one-week old conidia behaved similarly at transcriptional level. Conclusion: Transcriptomic data indicate that the exit from dormancy is associated with a shift from a fermentative metabolism to a respiratory metabolism as well as a trend toward immediate protein synthesis.

Background: Translation of the genome sequence of Plasmodium sp. into biologically relevant information relies on high through-put genomics technology which includes transcriptome analysis. However, few studies to date have used this powerful approach to explore transcriptome alterations of P. falciparum parasites exposed to antimalarial drugs. Results: The rapid action of artesunate allowed us to study dynamic changes of the parasite transcriptome in synchronous parasite cultures exposed to the drug for 90 minutes and 3 hours. Developmentally regulated genes were filtered out, leaving 398 genes which presented altered transcript levels reflecting drug-exposure. Few genes related to metabolic pathways, most encoded chaperones, transporters, kinases, Zn-finger proteins, transcription activating proteins, proteins involved in proteasome degradation, in oxidative stress and in cell cycle regulation. A positive bias was observed for over-expressed genes presenting a subtelomeric location, allelic polymorphism and encoding proteins with potential export sequences, which often belonged to subtelomeric multi-gene families. This pointed to the mobilization of processes shaping the interface between the parasite and its environment. In parallel, pathways were engaged which could lead to parasite death, such as interference with purine/pyrimidine metabolism, the mitochondrial electron transport chain, proteasome-dependent protein degradation or the integrity of the food vacuole. Conclusion: The high proportion of over-expressed genes encoding proteins exported from the parasite highlight the importance of extra-parasitic compartments as fields for exploration in drug research which, to date, has mostly focused on the parasite itself rather than on its intra and extra erythrocytic environment. Further work is needed to clarify which transcriptome alterations observed reflect a specific response to overcome artesunate toxicity or more general perturbations on the path to cellular death.