Transcriptomic analysis of the exit from dormancy of Aspergillus fumigatus conidia

<p>Abstract</p> <p>Background</p> <p>Establishment of aspergillosis is depending upon the exit from dormancy and germination of the conidia of <it>Aspergillus fumigatus </it>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 <it>A</it>. <it>fumigatus </it>genome).</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Microarray estimation of genomic inter-strain variability in the genus Ectocarpus (Phaeophyceae)

<p/> <p>Background</p> <p>Brown algae of the genus <it>Ectocarpus </it>exhibit high levels of genetic diversity and variability in morphological and physiological characteristics. With the establishment of <it>E. siliculosus </it>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 <it>Ectocarpus </it>both at the genome and the transcriptome level.</p> <p>Results</p> <p>We used an <it>E. siliculosus </it>gene expression microarray based on EST sequences from the genome-sequenced strain (reference strain) to carry out comparative genome hybridizations for five <it>Ectocarpus </it>strains: four <it>E. siliculosus </it>isolates (the male genome strain, a female strain used for outcrosses with the genome strain, a strain isolated from freshwater, and a highly copper-tolerant strain), as well as one strain of the sister species <it>E. fasciculatus</it>. Our results revealed significant genomic differences between ecotypes of the same species, and enable the selection of conserved probes for future microarray experiments with these strains. In the two closely related strains (a male and a female strain used for crosses), genomic differences were also detected, but concentrated in two smaller genomic regions, one of which corresponds to a viral insertion site.</p> <p>Conclusion</p> <p>The high variability between strains supports the concept of <it>E. siliculosus </it>as a complex of cryptic species. Moreover, our data suggest that several parts of the <it>Ectocarpus </it>genome may have evolved at different rates: high variability was detected particularly in transposable elements and fucoxanthin chlorophyll a/c binding proteins.</p

Endoplasmic Reticulum Stress-Sensing Mechanism Is Activated in Entamoeba histolytica upon Treatment with Nitric Oxide

The Endoplasmic Reticulum stores calcium and is a site of protein synthesis and modification. Changes in ER homeostasis lead to stress responses with an activation of the unfolded protein response (UPR). The Entamoeba histolytica endomembrane system is simple compared to those of higher eukaryotes, as a canonical ER is not observed. During amoebiasis, an infection of the human intestine and liver by E. histolytica, nitric oxide (NO) triggers an apoptotic-like event preceded by an impairment of energy production and a loss of important parasite pathogenic features. We address the question of how this ancient eukaryote responds to stress induced by immune components (i.e. NO) and whether stress leads to ER changes and subsequently to an UPR. Gene expression analysis suggested that NO triggers stress responses marked by (i) dramatic up-regulation of hsp genes although a bona fide UPR is absent; (ii) induction of DNA repair and redox gene expression and iii) up-regulation of glycolysis-related gene expression. Enzymology approaches demonstrate that NO directly inhibits glycolysis and enhance cysteine synthase activity. Using live imaging and confocal microscopy we found that NO dramatically provokes extensive ER fragmentation. ER fission in E. histolytica appears as a protective response against stress, as it has been recently proposed for neuron self-defense during neurologic disorders. Chronic ER stress is also involved in metabolic diseases including diabetes, where NO production reduces ER calcium levels and activates cell death. Our data highlighted unique cellular responses of interest to understand the mechanisms of parasite death during amoebiasis

Dynamic RNA profiling in Plasmodium falciparum synchronized blood stages exposed to lethal doses of artesunate

<p>Abstract</p> <p>Background</p> <p>Translation of the genome sequence of <it>Plasmodium sp</it>. 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 <it>P. falciparum </it>parasites exposed to antimalarial drugs.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Expansion of the SOS regulon of Vibrio cholerae through extensive transcriptome analysis and experimental validation

The SOS response is an almost ubiquitous response of cells to genotoxic stresses. The full complement of genes in the SOS regulon for Vibrio species has only been addressed through bioinformatic analyses predicting LexA binding box consensus and in vitro validation. Here, we perform whole transcriptome sequencing from Vibrio cholerae treated with mitomycin C as an SOS inducer to characterize the SOS regulon and other pathways affected by this treatment. Comprehensive transcriptional profiling allowed us to define the full landscape of promoters and transcripts active in V. cholerae. We performed extensive transcription start site (TSS) mapping as well as detection/quantification of the coding and non-coding RNA (ncRNA) repertoire in strain N16961. To improve TSS detection, we developed a new technique to treat RNA extracted from cells grown in various conditions. This allowed for identification of 3078 TSSs with an average 5'UTR of 116 nucleotides, and peak distribution between 16 and 64 nucleotides; as well as 629 ncRNAs. Mitomycin C treatment induced transcription of 737 genes and 28 ncRNAs at least 2 fold, while it repressed 231 genes and 17 ncRNAs. Data analysis revealed that in addition to the core genes known to integrate the SOS regulon, several metabolic pathways were induced. This study allowed for expansion of the Vibrio SOS regulon, as twelve genes (ubiEJB, tatABC, smpA, cep, VC0091, VC1190, VC1369-1370) were found to be co-induced with their adjacent canonical SOS regulon gene(s), through transcriptional read-through. Characterization of UV and mitomycin C susceptibility for mutants of these newly identified SOS regulon genes and other highly induced genes and ncRNAs confirmed their role in DNA damage rescue and protection. We show that genotoxic stress induces a pervasive transcriptional response, affecting almost 20% of the V. cholerae genes. We also demonstrate that the SOS regulon is larger than previously known, and its syntenic organization is conserved among Vibrio species. Furthermore, this specific co-localization is found in other γ-proteobacteria for genes recN-smpA and rmuC-tatABC, suggesting SOS regulon conservation in this phylum. Finally, we comment on the limitations of widespread NGS approaches for identification of all RNA species in bacteria

Growth of Yersinia pseudotuberculosis in human plasma: impacts on virulence and metabolic gene expression

<p>Abstract</p> <p>Background</p> <p>In man, infection by the Gram-negative enteropathogen <it>Yersinia pseudotuberculosis </it>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.</p> <p>Results</p> <p>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 <it>Y. pseudotuberculosis </it>was the switch to high glucose consumption, which is reminiscent of the acetogenic pathway (known as "glucose overflow") in <it>Escherichia coli</it>. However, upregulation of the glyoxylate shunt enzymes suggests that (in contrast to <it>E. coli</it>) acetate may be further metabolized in <it>Y. pseudotuberculosis</it>. Our data also indicate that the bloodstream environment can regulate major virulence genes (positively or negatively); the <it>yadA </it>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.</p> <p>Conclusion</p> <p>Our results suggest that plasma growth of <it>Y. pseudotuberculosis </it>is responsible for major transcriptional regulatory events and prompts key metabolic reorientations within the bacterium, which may in turn have an impact on virulence.</p

RNA Sequencing Reveals Widespread Transcription of Natural Antisense RNAs in Entamoeba Species.

Entamoeba is a genus of Amoebozoa that includes the intestine-colonizing pathogenic species Entamoeba histolytica. To understand the basis of gene regulation in E. histolytica from an evolutionary perspective, we have profiled the transcriptomes of its closely related species E. dispar, E. moshkovskii and E. invadens. Genome-wide identification of transcription start sites (TSS) and polyadenylation sites (PAS) revealed the similarities and differences of their gene regulatory sequences. In particular, we found the widespread initiation of antisense transcription from within the gene coding sequences is a common feature among all Entamoeba species. Interestingly, we observed the enrichment of antisense transcription in genes involved in several processes that are common to species infecting the human intestine, e.g., the metabolism of phospholipids. These results suggest a potentially conserved and compact gene regulatory system in Entamoeba

Role of the BAHD1 Chromatin-Repressive Complex in Placental Development and Regulation of Steroid Metabolism.

BAHD1 is a vertebrate protein that promotes heterochromatin formation and gene repression in association with several epigenetic regulators. However, its physiological roles remain unknown. Here, we demonstrate that ablation of the Bahd1 gene results in hypocholesterolemia, hypoglycemia and decreased body fat in mice. It also causes placental growth restriction with a drop of trophoblast glycogen cells, a reduction of fetal weight and a high neonatal mortality rate. By intersecting transcriptome data from murine Bahd1 knockout (KO) placentas at stages E16.5 and E18.5 of gestation, Bahd1-KO embryonic fibroblasts, and human cells stably expressing BAHD1, we also show that changes in BAHD1 levels alter expression of steroid/lipid metabolism genes. Biochemical analysis of the BAHD1-associated multiprotein complex identifies MIER proteins as novel partners of BAHD1 and suggests that BAHD1-MIER interaction forms a hub for histone deacetylases and methyltransferases, chromatin readers and transcription factors. We further show that overexpression of BAHD1 leads to an increase of MIER1 enrichment on the inactive X chromosome (Xi). In addition, BAHD1 and MIER1/3 repress expression of the steroid hormone receptor genes ESR1 and PGR, both playing important roles in placental development and energy metabolism. Moreover, modulation of BAHD1 expression in HEK293 cells triggers epigenetic changes at the ESR1 locus. Together, these results identify BAHD1 as a core component of a chromatin-repressive complex regulating placental morphogenesis and body fat storage and suggest that its dysfunction may contribute to several human diseases

From array-based hybridization of Helicobacter pylori isolates to the complete genome sequence of an isolate associated with MALT lymphoma

<p>Abstract</p> <p>Background</p> <p><it>elicobacter pylori </it>infection is associated with several gastro-duodenal inflammatory diseases of various levels of severity. To determine whether certain combinations of genetic markers can be used to predict the clinical source of the infection, we analyzed well documented and geographically homogenous clinical isolates using a comparative genomics approach.</p> <p>Results</p> <p>A set of 254 <it>H. pylori </it>genes was used to perform array-based comparative genomic hybridization among 120 French <it>H. pylori </it>strains associated with chronic gastritis (n = 33), duodenal ulcers (n = 27), intestinal metaplasia (n = 17) or gastric extra-nodal marginal zone B-cell MALT lymphoma (n = 43). Hierarchical cluster analyses of the DNA hybridization values allowed us to identify a homogeneous subpopulation of strains that clustered exclusively with <it>cag</it>PAI minus MALT lymphoma isolates. The genome sequence of B38, a representative of this MALT lymphoma strain-cluster, was completed, fully annotated, and compared with the six previously released <it>H. pylori </it>genomes (i.e. J99, 26695, HPAG1, P12, G27 and Shi470). B38 has the smallest <it>H. pylori </it>genome described thus far (1,576,758 base pairs containing 1,528 CDSs); it contains the <it>vacA</it>s2m2 allele and lacks the genes encoding the major virulence factors (absence of <it>cag</it>PAI, <it>bab</it>B, <it>bab</it>C, <it>sab</it>B, and <it>hom</it>B). Comparative genomics led to the identification of very few sequences that are unique to the B38 strain (9 intact CDSs and 7 pseudogenes). Pair-wise genomic synteny comparisons between B38 and the 6 <it>H. pylori </it>sequenced genomes revealed an almost complete co-linearity, never seen before between the genomes of strain Shi470 (a Peruvian isolate) and B38.</p> <p>Conclusion</p> <p>These isolates are deprived of the main <it>H. pylori </it>virulence factors characterized previously, but are nonetheless associated with gastric neoplasia.</p