Pascal Lenormand

AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX.

Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase‐independent mode of cell death mediated by apoptosis‐inducing factor (AIF). After poly(ADP‐ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C‐terminal proline‐rich binding domain (PBD, residues 543–559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA‐degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C‐terminal PBD abolishes AIF/H2AX interaction and AIF‐mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA‐deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase‐independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif.

Identification of Escherichia coli O-serogroups by restriction of the amplified O-antigen gene cluster (rfb-RFLP)

The precise serotyping of clinical Escherichia coli isolates is a crucial step for diagnostic and epidemiological purposes. Epidemiological knowledge associated with serotyping is so important that no alternative method may be considered if it does not correlate with serotyping. Unfortunately, E. coli are difficult to serotype. Genes specifically involved in O-antigen synthesis are clustered in E. coli, Shigella and Salmonella. Published oligonucleotide sequences complementary to JUMPstart and the gnd gene (the conserved flanking sequences upstream and downstream of O-antigen gene clusters, respectively) were used to amplify the O-antigen gene cluster of representative strains of 148 E. coli O-serogroups. A unique amplified fragment was observed for each serogroup (size ranging from 1.7 to 20 kbp). Clearly identifiable and reproducible O-patterns were obtained for the great majority of O-serogroups after MboII digestion of amplified products. The number of bands composing each pattern varied from five to 25. A database was built with the patterns obtained. A total of 147 O-patterns were obtained. Thirteen O-serogroups were subdivided into different O-patterns. However, each of 13 other O-patterns was shared by two or more O-serogroups. 0-serogroups of clinical isolates were deduced accurately from O-patterns in all cases, even for some rough or nonagglutinating isolates. The restriction method (rfb-RFLP) may prove to be better than serotyping since 100% of strains are typable, which is not the case with serotyping.

Phosphoproteomic analysis of Leishmania donovani pro- and amastigote stages.

Following transmission to the vertebrate host, the protozoan parasite Leishmania donovani differentiates into the pathogenic amastigote stage that is adapted for intracellular survival. This developmental transition is induced by environmental factors including elevated temperature and acidic pH and is likely transduced by signaling cascades involving protein kinases and their downstream phosphoprotein substrates. These signaling networks are highly adapted to the specific nutritional and physiological requirements of the organism and thus studying Leishmania phosphorylation may allow important insight into the parasite‐specific biology. We used a gel‐based approach to investigate qualitative and quantitative changes of the phosphoproteome of the major L. donovani life cycle stages. Phosphoproteins were purified by immobilized metal affinity chromatography (IMAC), separated by IEF and SDS‐PAGE using pH 4–7 IPG immobiline strips, revealed by fluorescent multiplex staining, and identified by MALDI‐MS and MS/MS. Our analysis allowed us to establish a first repertoire of the Leishmania phosphoproteome and to identify phosphoproteins implicated in stress‐ and heat shock response, RNA/protein turnover, metabolism, and signaling.

Taxonomic diversity of anaerobic glycerol dissimilation in the Enterobacteriaceae

A total of 1,123 strains representing 128 taxa in the Enterobacteriaceae (named species or subspecies and genomic species) were screened for the presence of glycerol dehydrogenases and 1,3-propanediol dehydrogenase. Only eight taxa, Citrobacter freundii sensu stricto, C. youngae, C. braakii, C. werkmanii, Citrobacter genomospecies 10 and 11, Enterobacter gergoviae and Klebsiella pneumoniae subsp. pneumoniae could grow fermentatively on glycerol and possessed both glycerol dehydrogenase type I (induced by glycerol and dihydroxyacetone) and 1,3-propanediol dehydrogenase which are typical enzymes of the anaerobic glycerol dissimilation pathway. Six other species, C. koseri, E. aerogenes, E. intermedium, K. oxytoca, K. planticola and K. terrigena could not grow fermentatively on glycerol and possessed a glycerol dehydrogenase type I but no 1,3-propanediol dehydrogenase. Other glycerol dehydrogenases types were found: type II (induced by glycerol and hydroxyacetone), type III (induced by glycerol only) and type IV (induced by hydroxyacetone only). They were widely distributed among the Enterobacteriaceae. Classification and identification may take advantage of tests exploring the dissimilation of glycerol.

60S ribosomal subunit assembly dynamics defined by semi-quantitative mass spectrometry of purified complexes

During the highly conserved process of eukaryotic ribosome formation, RNA follows a maturation path with well-defined, successive intermediates that dynamically associate with many pre-ribosomal proteins. A comprehensive description of the assembly process is still lacking. To obtain data on the timing and order of association of the different pre-ribosomal factors, a strategy consists in the use of pre-ribsomal particles isolated from mutants that block ribosome formation at different steps. Immunoblots, inherently limited to only a few factors, have been applied to evaluate the accumulation or decrease of pre-ribosomal intermediates under mutant conditions. For a global protein-level description of different 60S ribosomal subunit maturation intermediates in yeast, we have adapted a method of in vivo isotopic labelling and mass spectrometry to study pre-60S complexes isolated from strains in which rRNA processing was affected by individual depletion of five factors: Ebp2, Nog1, Nsa2, Nog2 or Pop3. We obtained quantitative data for 45 distinct pre-60S proteins and detected coordinated changes for over 30 pre-60S factors in the analysed mutants. These results led to the characterisation of the composition of early, intermediate and late pre-ribosomal complexes, specific for crucial maturation steps during 60S assembly in eukaryotes.

Hyperthermophilic archaea produce membrane vesicles that can transfer DNA

Thermococcales are hyperthermophilic archaea found in deep-sea hydrothermal vents. They have been recently reported to produce membrane vesicles (MVs) into their culture medium. Here, we have characterized the mode of production and determined the biochemical composition of MVs from two species of Thermococcales, Thermococcus gammatolerans and Thermococcus kodakaraensis. We observed that MVs are produced by a budding process from the cell membrane reminiscent of ectosome (microparticle) formation in eukaryotes. MVs and cell membranes from the same species have a similar protein and lipid composition, confirming that MVs are produced from cell membranes. The major protein present in cell membranes and MVs of both species is the oligopeptide binding protein OppA. This protein is also abundant in MVs from cells grown in minimal medium, suggesting that OppA could be involved in processes other than peptides scavenging. We have previously shown that MVs from Thermococcales harbour DNA and protect DNA against thermodegradation. Here, we show that T. kodakaraensis cells transformed with the shuttle plasmid pLC70 release MVs harbouring this plasmid. Notably, these MVs can be used to transfer pLC70 into plasmid-free cells, suggesting that MVs could be involved in DNA transfer between cells at high temperature.

Mycolactone Diffuses into the Peripheral Blood of Buruli Ulcer Patients - Implications for Diagnosis and Disease Monitoring

Background Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU), is unique among human pathogens in its capacity to produce a polyketide-derived macrolide called mycolactone, making this molecule an attractive candidate target for diagnosis and disease monitoring. Whether mycolactone diffuses from ulcerated lesions in clinically accessible samples and is modulated by antibiotic therapy remained to be established. Methodology/Principal Finding Peripheral blood and ulcer exudates were sampled from patients at various stages of antibiotic therapy in Ghana and Ivory Coast. Total lipids were extracted from serum, white cell pellets and ulcer exudates with organic solvents. The presence of mycolactone in these extracts was then analyzed by a recently published, field-friendly method using thin layer chromatography and fluorescence detection. This approach did not allow us to detect mycolactone accurately, because of a high background due to co-extracted human lipids. We thus used a previously established approach based on high performance liquid chromatography coupled to mass spectrometry. By this means, we could identify structurally intact mycolactone in ulcer exudates and serum of patients, and evaluate the impact of antibiotic treatment on the concentration of mycolactone. Conclusions/Significance Our study provides the proof of concept that assays based on mycolactone detection in serum and ulcer exudates can form the basis of BU diagnostic tests. However, the identification of mycolactone required a technology that is not compatible with field conditions and point-of-care assays for mycolactone detection remain to be worked out. Notably, we found mycolactone in ulcer exudates harvested at the end of antibiotic therapy, suggesting that the toxin is eliminated by BU patients at a slow rate. Our results also indicated that mycolactone titres in the serum may reflect a positive response to antibiotics, a possibility that it will be interesting to examine further through longitudinal studies.

Taxonomic Diversity of the D-Glucose Oxidation Pathway in the Enterobacteriaceae

Tests allowing the screening of large numbers of enterobacterial strains for the presence of the glucose oxidation pathway (glucose, gluconate, and 2-ketogluconate dehydrogenases) were devised or adapted. A total of 506 strains representing 111 taxa (named species or subspecies and unnamed genomic groups) were studied. The members of the genera Budvicia, Edwardsiella, Leminorella, Providencia, and Xenorhabdus and the species Citrobacter freundii, Erwinia carnegeana, Erwinia carotovora, Erwinia chrysanthemi, Erwinia nigrifluens, Erwinia salicis, Moellerella wisconsensis, Proteus penneri, Proteus vulgaris, Yersinia intermedia, Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia ruckeri were negative in all tests. Five species, Erwinia cypripedii, Ewingella americana, Rahnella aquatilis, Serratia marcescens (at 20°), and Tatumella ptyseos produced 2,5-diketogluconate from glucose without a requirement for pyrroloquinoline quinone (PQQ). When PQQ was provided (required for glucose oxidation), Serratia grimesii and Serratia liquefaciens produced 2,5-diketogluconate from glucose at 20°. Escherichia blattae had gluconate- and 2-ketogluconate dehydrogenases without glucose dehydrogenase. The members of the genera Hafnia, Obesumbacterium, and Pragia had only gluconate dehydrogenase. Other species had glucose dehydrogenase (with or without a requirement for PQQ) with or without gluconate dehydrogenase. Classification and identification may take advantage of tests exploring the glucose oxidation pathway.

Infection by chikungunya virus modulates the expression of several proteins in Aedes aegypti salivary glands

BackgroundArthropod-borne viral infections cause several emerging and resurging infectious diseases. Among the diseases caused by arboviruses, chikungunya is responsible for a high level of severe human disease worldwide. The salivary glands of mosquitoes are the last barrier before pathogen transmission.MethodsWe undertook a proteomic approach to characterize the key virus/vector interactions and host protein modifications that occur in the salivary glands that could be responsible for viral transmission by using quantitative two-dimensional electrophoresis.ResultsWe defined the protein modulations in the salivary glands of Aedes aegypti that were triggered 3 and 5 days after an oral infection (3 and 5 DPI) with chikungunya virus (CHIKV). Gel profile comparisons showed that CHIKV at 3 DPI modulated the level of 13 proteins, and at 5 DPI 20 proteins. The amount of 10 putatively secreted proteins was regulated at both time points. These proteins were implicated in blood-feeding or in immunity, but many have no known function. CHIKV also modulated the quantity of proteins involved in several metabolic pathways and in cell signalling.ConclusionOur study constitutes the first analysis of the protein response of Aedes aegypti salivary glands infected with CHIKV. We found that the differentially regulated proteins in response to viral infection include structural proteins and enzymes for several metabolic pathways. Some may favour virus survival, replication and transmission, suggesting a subversion of the insect cell metabolism by arboviruses. For example, proteins involved in blood-feeding such as the short D7, an adenosine deaminase and inosine-uridine preferring nucleoside hydrolase, may favour virus transmission by exerting an increased anti-inflammatory effect. This would allow the vector to bite without the bite being detected. Other proteins, like the anti-freeze protein, may support vector protection.

Comprehensive proteome analysis of Mycobacterium ulcerans and quantitative comparison of mycolactone biosynthesis

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a rapidly emerging human disease in which mycolactone, a cytotoxic and immunosuppressive macrocyclic polyketide, is responsible for massive skin destruction. The genome sequencing of M. ulcerans has recently been accomplished (http://genolist.pasteur.fr/BuruList/) enabling the first proteome study of this important human pathogen. Here, we present a comprehensive proteome analysis of different subcellular fractions and culture supernatant of in vitro grown M. ulcerans. By a combination of gel‐based and gel‐free techniques for protein and peptide separation with subsequent analysis by MS, we identified 1074 different proteins, corresponding to 25% of the protein‐coding DNA sequence. Interestingly, new information was obtained about central metabolism and lipid biosynthesis, and as many as 192 conserved hypothetical proteins were found. Comparative analysis of the wild‐type strain and an isogenic mycolactone‐deficient mutant, by 2‐DE and iTRAQ labeling of the cytoplasmic fraction, revealed differences in the expression profiles of proteins involved in lipid metabolism and information pathways, as well as stress responses.