Frédéric Hérault

A new cytolethal distending toxin (CDT) from Escherichia coli producing CNF2 blocks HeLa cell division in G2/M phase

Escherichia coli strain 1404, isolated from a septicaemic calf, carries a transferable plasmid called pVir which codes for the cytotoxic necrotizing factor type 2 (CNF2). A 4 h interaction between strain 1404 and HeLa cells induced the formation of giant mononucleated cells blocked in G2/M phase. Mating experiments between strain 1404 and a non‐pathogenic recipient strain demonstrated that the factor(s) encoded by pVir mediated the cell‐cycle arrest. A 3.3 kb DNA fragment isolated from a DNA bank of pVir was shown to code for the factor(s) causing the cell‐cycle arrest. Nucleotide sequence analysis revealed the presence of three genes encoding proteins sharing significant amino acid homology with the cytolethal distending toxins (CDTs) previously isolated from E. coli, Campylobacter jejuni and Shigella dysenteriae. Southern hybridization experiments demonstrated that the pVir of other CNF2‐producing E. coli strains contained sequences related to cdt. Although the amino acid sequences amongst CDT diverged significantly, the two other CDTs previously isolated from E. coli were also able to block the HeLa cell cycle. In conclusion, this study demonstrates the mode of action of CDT and will help us to elucidate the role of this emerging toxin family in microbial pathogenesis.

Transcriptome profiling of the feeding-to-fasting transition in chicken liver

BackgroundStarvation triggers a complex array of adaptative metabolic responses including energy-metabolic responses, a process which must imply tissue specific alterations in gene expression and in which the liver plays a central role. The present study aimed to describe the evolution of global gene expression profiles in liver of 4-week-old male chickens during a 48 h fasting period using a chicken 20 K oligoarray.ResultsA large number of genes were modulated by fasting (3532 genes with a pvalue corrected by Benjamini-Hochberg < 0.01); 2062 showed an amplitude of variation higher than +/- 40% among those, 1162 presented an human ortholog, allowing to collect functional information. Notably more genes were down-regulated than up-regulated, whatever the duration of fasting (16 h or 48 h). The number of genes differentially expressed after 48 h of fasting was 3.5-fold higher than after 16 h of fasting. Four clusters of co-expressed genes were identified by a hierarchical cluster analysis. Gene Ontology, KEGG and Ingenuity databases were then used to identify the metabolic processes associated to each cluster. After 16 h of fasting, genes involved in ketogenesis, gluconeogenesis and mitochondrial or peroxisomal fatty acid beta-oxidation, were up-regulated (cluster-1) whereas genes involved in fatty acid and cholesterol synthesis were down-regulated (cluster-2). For all genes tested, the microarray data was confirmed by quantitative RT-PCR. Most genes were altered by fasting as already reported in mammals. A notable exception was the HMG-CoA synthase 1 gene, which was up-regulated following 16 and 48 h of fasting while the other genes involved in cholesterol metabolism were down-regulated as reported in mammalian studies. We further focused on genes not represented on the microarray and candidates for the regulation of the target genes belonging to cluster-1 and -2 and involved in lipid metabolism. Data are provided concerning PPARa, SREBP1, SREBP2, NR1H3 transcription factors and two desaturases (FADS1, FADS2).ConclusionThis study evidences numerous genes altered by starvation in chickens and suggests a global repression of cellular activity in response to this stressor. The central role of lipid and acetyl-CoA metabolisms and its regulation at transcriptional level are confirmed in chicken liver in response to short-term fasting. Interesting expression modulations were observed for NR1H3, FADS1 and FADS2 genes. Further studies are needed to precise their role in the complex regulatory network controlling lipid metabolism.

Comparison of Muscle Transcriptome between Pigs with Divergent Meat Quality Phenotypes Identifies Genes Related to Muscle Metabolism and Structure

Background Meat quality depends on physiological processes taking place in muscle tissue, which could involve a large pattern of genes associated with both muscle structural and metabolic features. Understanding the biological phenomena underlying muscle phenotype at slaughter is necessary to uncover meat quality development. Therefore, a muscle transcriptome analysis was undertaken to compare gene expression profiles between two highly contrasted pig breeds, Large White (LW) and Basque (B), reared in two different housing systems themselves influencing meat quality. LW is the most predominant breed used in pig industry, which exhibits standard meat quality attributes. B is an indigenous breed with low lean meat and high fat contents, high meat quality characteristics, and is genetically distant from other European pig breeds. Methodology/Principal Findings Transcriptome analysis undertaken using a custom 15 K microarray, highlighted 1233 genes differentially expressed between breeds (multiple-test adjusted P-value<0.05), out of which 635 were highly expressed in the B and 598 highly expressed in the LW pigs. No difference in gene expression was found between housing systems. Besides, expression level of 12 differentially expressed genes quantified by real-time RT-PCR validated microarray data. Functional annotation clustering emphasized four main clusters associated to transcriptome breed differences: metabolic processes, skeletal muscle structure and organization, extracellular matrix, lysosome, and proteolysis, thereby highlighting many genes involved in muscle physiology and meat quality development. Conclusions/Significance Altogether, these results will contribute to a better understanding of muscle physiology and of the biological and molecular processes underlying meat quality. Besides, this study is a first step towards the identification of molecular markers of pork quality and the subsequent development of control tools.

Liver gene expression in relation to hepatic steatosis and lipid secretion in two duck species.

The susceptibility to development of hepatic steatosis is known to differ between Muscovy and Pekin ducks. Although some experiments were conducted to decipher these differences, few data have been produced to analyse the role of specific genes in this process. For this purpose, expression levels of genes involved in lipid (ATP citrate lyase, malic enzyme 1, fatty acid synthase, stearoyl-CoA desaturase 1, diacylglycerol O-acyl transferase 2, microsomal triglyceride transfer protein, apolipoprotein A1, apolipoprotein B, sterol regulatory element binding factor 1, hepatocyte nuclear factor 4, choline/ethanolamine phosphotransferase 1, carnitine palmitoyl transferase 1A, peroxisome proliferator-activated receptor alpha and sterol O-acyltransferase) and carbohydrate (activating transcription factor 4 or cAMP-response element binding protein, mitochondrial malate dehydrogenase 2 and carbohydrate responsive element binding protein) metabolism and in other functions were analysed in the liver of Pekin and Muscovy ducks fed ad libitum or overfed. A specific positive effect of feeding was observed on the expression of genes involved mainly in fatty acids and TG synthesis and glycolysis, and negative effect on genes involved in beta-oxidation. Interestingly, a strong species effect was also observed on stearoyl-CoA desaturase 1 and to a lesser extent on diacylglycerol O-acyl transferase 2 expression, leading to large differences in expression levels between Pekin and Muscovy overfed ducks, which could explain the difference in lipid metabolism and steatosis ability observed between the two duck species. These results should shed light on gene expression that might underlie susceptibility to hepatic steatosis in humans.

Joint analysis of quantitative trait loci and major-effect causative mutations affecting meat quality and carcass composition traits in pigs

BackgroundDetection of quantitative trait loci (QTLs) affecting meat quality traits in pigs is crucial for the design of efficient marker-assisted selection programs and to initiate efforts toward the identification of underlying polymorphisms. The RYR1 and PRKAG3 causative mutations, originally identified from major effects on meat characteristics, can be used both as controls for an overall QTL detection strategy for diversely affected traits and as a scale for detected QTL effects. We report on a microsatellite-based QTL detection scan including all autosomes for pig meat quality and carcass composition traits in an F2 population of 1,000 females and barrows resulting from an intercross between a Pietrain and a Large White-Hampshire-Duroc synthetic sire line. Our QTL detection design allowed side-by-side comparison of the RYR1 and PRKAG3 mutation effects seen as QTLs when segregating at low frequencies (0.03-0.08), with independent QTL effects detected from most of the same population, excluding any carrier of these mutations.ResultsLarge QTL effects were detected in the absence of the RYR1 and PRKGA3 mutations, accounting for 12.7% of phenotypic variation in loin colour redness CIE-a* on SSC6 and 15% of phenotypic variation in glycolytic potential on SSC1. We detected 8 significant QTLs with effects on meat quality traits and 20 significant QTLs for carcass composition and growth traits under these conditions. In control analyses including mutation carriers, RYR1 and PRKAG3 mutations were detected as QTLs, from highly significant to suggestive, and explained 53% to 5% of the phenotypic variance according to the trait.ConclusionsOur results suggest that part of muscle development and backfat thickness effects commonly attributed to the RYR1 mutation may be a consequence of linkage with independent QTLs affecting those traits. The proportion of variation explained by the most significant QTLs detected in this work is close to the influence of major-effect mutations on the least affected traits, but is one order of magnitude lower than effect on variance of traits primarily affected by these causative mutations. This suggests that uncovering physiological traits directly affected by genetic polymorphisms would be an appropriate approach for further characterization of QTLs.

Genetic variability of transcript abundance in pig skeletal muscle at slaughter: Relationships with meat quality traits

A family structured population of 325 pigs (females and barrows) was produced as an intercross between 2 commercial sire lines and was subjected to a systematic transcriptome analysis of LM samples obtained shortly after slaughter. Additionally, measurements of meat quality traits of fresh and cooked loin were gathered from the same animals. The transcriptome analysis was achieved by microarray hybridization, using a custom repertoire of 15,000 6mer DNA probes targeting transcripts expressed in growing pig skeletal muscle. These data allowed us to estimate the heritability of expression abundance for each of the quantified RNA species. The abundance of 9,765 RNA was estimated as heritable with a false discovery rate of 5%, from which 1,174 were deemed as highly heritable (h(2) > 0.50). We also observed a large number of transcripts whose LM expression abundance is genetically correlated with 4 meat quality traits: the loin pH measured at 45 min postmortem (pH45), 253 transcripts; the loin cooking loss (CL), 134 transcripts; the cooked loin shear force (SFc), 184 transcripts; and the loin color redness (a*) value, 190 transcripts. Heritable and meat quality genetically correlated transcripts showed an over-representation of biological processes involved in the induction of apoptosis (genetically correlated with CL), complement activation (genetically correlated with SFc), glucose metabolism (genetically correlated with a*), and cation channel activity (genetically correlated with pH45). Overall, the biological functions highlighted in the highly heritable transcripts and the lack of transcript that would be genetically correlated with LM glycolytic potential suggest that the genetic variability of the LM postmortem transcriptome is focused on muscle tissue response to postmortem ischemia and reflects more distantly the antemortem muscle physiology. Because of the contrasting distributions of the genetic correlations between LM RNA concentrations and the different meat quality traits studied, indirect selection strategies of meat quality traits based on measurements of selected LM RNA species could be only proposed for a subset of the analyzed meat characteristics (pH45, SFc, a*, CL). A substantial improvement in the efficiency of selection for these meat quality traits could result from measuring muscle RNA concentrations on selection candidates, if the same genetic parameters can be verified using in vivo-sampled muscles.

The Longissimus and Semimembranosus muscles display marked differences in their gene expression profiles in pig.

Background Meat quality depends on skeletal muscle structure and metabolic properties. While most studies carried on pigs focus on the Longissimus muscle (LM) for fresh meat consumption, Semimembranosus (SM) is also of interest because of its importance for cooked ham production. Even if both muscles are classified as glycolytic muscles, they exhibit dissimilar myofiber composition and metabolic characteristics. The comparison of LM and SM transcriptome profiles undertaken in this study may thus clarify the biological events underlying their phenotypic differences which might influence several meat quality traits. Methodology/Principal Findings Muscular transcriptome analyses were performed using a custom pig muscle microarray: the 15 K Genmascqchip. A total of 3823 genes were differentially expressed between the two muscles (Benjamini-Hochberg adjusted P value ≤0.05), out of which 1690 and 2133 were overrepresented in LM and SM respectively. The microarray data were validated using the expression level of seven differentially expressed genes quantified by real-time RT-PCR. A set of 1047 differentially expressed genes with a muscle fold change ratio above 1.5 was used for functional characterization. Functional annotation emphasized five main clusters associated to transcriptome muscle differences. These five clusters were related to energy metabolism, cell cycle, gene expression, anatomical structure development and signal transduction/immune response. Conclusions/Significance This study revealed strong transcriptome differences between LM and SM. These results suggest that skeletal muscle discrepancies might arise essentially from different post-natal myogenic activities.

Quantitative real-time PCR primer design, cDNA amplification and sequence analysis from 22 genes mainly associated with lipid metabolism in Pekin (Anas platyrhynchos) and Muscovy (Cairina moschata) ducks.

Few genomic tools are available in ducks. To produce some new resources, we have designed Pekin (Anas platyrhynchos) and Muscovy (Cairina moschata) duck-specific primers for 22 genes involved mainly in lipid metabolism, and to a lesser extent in carbohydrate metabolism and other functions. Primers were designed according to duck sequences when available and otherwise from the corresponding conserved regions in chicken and human sequences. These primers allowed quantitative RT-PCR amplification of RNA from Pekin and Muscovy ducks. Amplified cDNA products from both species were sequenced and were found to be very similar to chicken sequences (about 94%). This work provides additional genomic resources and polymorphism information for some genes in duck species and represents a first step towards gene expression analyses in Pekin and Muscovy ducks.

Combined GWAS and LDLA approaches to improve genome-wide quantitative trait loci detection affecting carcass and meat quality traits in pig

Many QTL affecting meat quality and carcass traits have been reported. However, in most of the cases these QTL have been detected in non-commercial populations. Therefore, a family structured population of 457 F2 pigs issued from an inter-cross between 2 commercial sire lines was used to detect QTL affecting meat quality and carcass traits. All animals were genotyped using the Illumina PorcineSNP60 BeadChip platform. Genome-wide association studies were used in combination with linkage disequilibrium-linkage analysis to identify QTL. A total of 32 QTL were detected. Nine of these QTL exceeded the genome-wide 5% significance threshold. We detected 18 QTL affecting carcass composition traits and 16 QTL affecting meat quality traits. Using post-QTL bioinformatics analysis we highlighted 26 functional candidate genes related to fatness, muscle development, meat color and meat pH. Finally, our results shed light on the advantage of using different QTL detection methodologies to get a global overview of the QTL present in the studied population.