Delphine Lapaillerie

Analysis of the Flavobacterium psychrophilum outer-membrane subproteome and identification of new antigenic targets for vaccine by immunomics

Flavobacterium psychrophilum is an important infectious Gram-negative bacterium causing cold-water disease (CWD) and rainbow trout fry syndrome. Outer-membrane proteins (OMPs) are key molecules with regard to the interface between the cell and its environment. Therefore, we sought to define the outer-membrane (OM) subproteome of F. psychrophilum in order to gain insight into the biology and pathogenesis of this bacterium and to identify the dominant antigens targeted by the rainbow trout (Oncorhynchus mykiss) immune system during infection. First, OMs were prepared from a cell-envelope suspension by differential Sarkosyl (sodium lauryl sarcosinate) solubility. We then isolated the OMPs and identified 36 proteins from 34 spots resolved by two-dimensional electrophoresis and LC-MS/MS. An immunoproteomic approach using antibodies from CWD-convalescent rainbow trout was then used to identify 25 immunoreactive F. psychrophilum antigens that may be relevant in pathogenesis and diagnosis. These included the previously characterized surface-exposed OMPs OmpA, OmpH/P18 and FspA, as well as newly described antigenic proteins. This study provides a number of novel candidate proteins for developing vaccine(s) against flavobacteriosis infection in aquaculture.

Proteomic analysis of an imatinib-resistant K562 cell line highlights opposing roles of heat shock cognate 70 and heat shock 70 proteins in resistance.

Understanding the molecular basis of resistance to imatinib, a tyrosine kinase inhibitor used as front‐line therapy in chronic myeloid leukemia, remains a challenge for successful treatment. In an attempt to identify new mechanisms of resistance, we performed a comparative proteomic analysis of an imatinib‐resistant cell line generated from the erythroblastic cell line K562 (K562‐r) for which no known mechanism of resistance has been detected. Bidimensional gel electrophoresis was carried out to compare the protein expression pattern of imatinib‐sensitive and of imatinib‐resistant K562 cells. Among the 400 matched spots on five pairs of gels, only 14 spots had a significantly increased or decreased expression leading to the identification of 24 proteins identified as scaffold proteins, metabolic enzymes, DNA translation and maturation, and chaperon proteins. Among the chaperon family, only Hsp70 and Hsc70 are overexpressed in K562‐r, results confirmed by Western blotting. We recently reported the participation of Hsp70 overexpression in imatinib resistance whereas a role for Hsc70 has yet to be determined. Hsc70 is not involved in imatinib resistance as the inhibition of its expression by siRNA does not restore sensitivity to imatinib. In contrast, the induced decreased expression of Hsc70 was accompanied by a greater overexpression of Hsp70. This proteomic study therefore suggests opposing roles of Hsp70 and Hsc70 in imatinib resistance.

Hunting down fungal secretomes using liquid-phase IEF prior to high resolution 2-DE.

The secreted proteins (secretome) of fungi play a key role in interactions of pathogenic and symbiotic fungi with plants. Using the plant pathogenic fungus Leptosphaeria maculans and symbiont Laccaria bicolor grown in culture, we have established a proteomic protocol for extraction, concentration and resolution of the fungal secretome. As no proteomic data were available on mycelium tissues from both L. maculans and L. bicolor, mycelial proteins were studied; they also helped verifying the purity of secretome samples. The quality of protein extracts was initially assessed by both 1‐DE and 2‐DE using first a broad pH range for IEF, and then narrower acidic and basic pH ranges, prior to 2‐DE. Compared with the previously published protocols for which only dozens of 2‐D spots were recovered from fungal secretome samples, up to approximately 2000 2‐D spots were resolved by our method. MS identification of proteins along several pH gradients confirmed this high resolution, as well as the presence of major secretome markers such as endopolygalacturonases, β‐glucanosyltransferases, pectate lyases and endoglucanases. Shotgun proteomic experiments evidenced the enrichment of secreted protein within the liquid medium. This is the first description of the proteome of L. maculans and L. bicolor, and the first application of liquid‐phase IEF to any fungal extracts.

Functional characterization of a chimeric soluble Fas ligand polymer with in vivo anti-tumor activity.

Binding of ligand FasL to its receptor Fas triggers apoptosis via the caspase cascade. FasL itself is homotrimeric, and a productive apoptotic signal requires that FasL be oligomerized beyond the homotrimeric state. We generated a series of FasL chimeras by fusing FasL to domains of the Leukemia Inhibitory Factor receptor gp190 which confer homotypic oligomerization, and analyzed the capacity of these soluble chimeras to trigger cell death. We observed that the most efficient FasL chimera, called pFasL, was also the most polymeric, as it reached the size of a dodecamer. Using a cellular model, we investigated the structure-function relationships of the FasL/Fas interactions for our chimeras, and we demonstrated that the Fas-mediated apoptotic signal did not solely rely on ligand-mediated receptor aggregation, but also required a conformational adaptation of the Fas receptor. When injected into mice, pFasL did not trigger liver injury at a dose which displayed anti-tumor activity in a model of human tumor transplanted to immunodeficient animals, suggesting a potential therapeutic use. Therefore, the optimization of the FasL conformation has to be considered for the development of efficient FasL-derived anti-cancer drugs targeting Fas.

Impact of foliar symptoms of "Esca proper" on proteins related to defense and oxidative stress of grape skins during ripening.

Esca is one of the major diseases affecting vineyards with direct impact on product yield; nevertheless, scientific studies concerning its impact on grape quality are scarce. As an attempt to better understand the mechanisms behind “Esca proper” development in grapes, this work focused on the identification of proteins whose expression is altered by the disease. 2‐DEs were performed on protein extracts from grape skins at different stages of maturity for two consecutive vintages. Grapes were collected in 2009 and in 2010 from plants that did not present signs of infection by Esca proper since the 2004 vintage and from plants that presented cast leaf symptoms at least once since 2004. For the first time, 13 proteins were shown to be influenced by Esca proper during the ripening process. Extensive bioinformatics analysis allowed the grouping of proteins involved in (i) stress tolerance and defense response, (ii) oxidative phosphorylation, (iii) oxidation–reduction processes in mitochondria, and (iv) oxidation–reduction processes in chloroplasts. Of these 13 proteins, cysteine synthase is the only one implicated in a metabolic pathway of oenological interest. This study shows how foliar symptoms of Esca proper may impact stress‐related pathways in grapes, which are characterized by modifications in the chain of oxidative phosphorylation and redox scavenging.

Modulation of chromatin structure by the FACT histone chaperone complex regulates HIV-1 integration

BackgroundInsertion of retroviral genome DNA occurs in the chromatin of the host cell. This step is modulated by chromatin structure as nucleosomes compaction was shown to prevent HIV-1 integration and chromatin remodeling has been reported to affect integration efficiency. LEDGF/p75-mediated targeting of the integration complex toward RNA polymerase II (polII) transcribed regions ensures optimal access to dynamic regions that are suitable for integration. Consequently, we have investigated the involvement of polII-associated factors in the regulation of HIV-1 integration.ResultsUsing a pull down approach coupled with mass spectrometry, we have selected the FACT (FAcilitates Chromatin Transcription) complex as a new potential cofactor of HIV-1 integration. FACT is a histone chaperone complex associated with the polII transcription machinery and recently shown to bind LEDGF/p75. We report here that a tripartite complex can be formed between HIV-1 integrase, LEDGF/p75 and FACT in vitro and in cells. Biochemical analyzes show that FACT-dependent nucleosome disassembly promotes HIV-1 integration into chromatinized templates, and generates highly favored nucleosomal structures in vitro. This effect was found to be amplified by LEDGF/p75. Promotion of this FACT-mediated chromatin remodeling in cells both increases chromatin accessibility and stimulates HIV-1 infectivity and integration.ConclusionsAltogether, our data indicate that FACT regulates HIV-1 integration by inducing local nucleosomes dissociation that modulates the functional association between the incoming intasome and the targeted nucleosome.

Hyper-proteolytic mutant of Beauveria bassiana, a new biological control agent against the tomato borer

The world tomato production is threatened by the invasive tomato borer Tuta absoluta. Difficulties in managing this pest were imposed mainly by the development of resistance in strains treated with conventional chemical insecticides. Resistance problems were even reported to insecticides of natural origin, leading to search for other control alternatives. P2 is a spontaneous mutant of the entomopathogenic fungus Beauveria bassiana. It was previously selected from a local strain (P1) and was characterized as hyper-producer of extracellular proteases. Here, the insecticidal potential of P1 and P2 strains was evaluated against T. absoluta larvae under laboratory conditions. Both strains were effective but P2 showed stronger effect than P1; median lethal concentration of P2 is tenfold lower than that of P1. Enzymatic assay analysis showed that extracellular enzymes are differently expressed by the two strains, especially proteases and chitinases which are known as cuticle degrading enzymes. The major expressed subtilisin-like protease (SBP) was upregulated at the transcriptional level in P2 strain. Proteomic analysis revealed four SBP isoforms which are highly over-expressed in this strain compared to P1. Post-translational regulation, most probably phosphorylation, was further suggested to control the SBP protease expression in B. bassiana P1 and P2 strains. The enzymatic profile in the two strains might explain their different insecticidal potential against the tomato borer. This is the first report showing such efficiency of Beauveria strains against this dangerous pest. Particularly, P2 strain showed high virulence reaching almost total larval mortality within 5xa0days post-application. It thus should be recommended as a new tool for the biocontrol of T. absoluta.

Modulation of the functional association between the HIV-1 intasome and the nucleosome by histone amino-terminal tails

BackgroundStable insertion of the retroviral DNA genome into host chromatin requires the functional association between the intasome (integrase·viral DNA complex) and the nucleosome. The data from the literature suggest that direct protein–protein contacts between integrase and histones may be involved in anchoring the intasome to the nucleosome. Since histone tails are candidates for interactions with the incoming intasomes we have investigated whether they could participate in modulating the nucleosomal integration process.ResultsWe show here that histone tails are required for an optimal association between HIV-1 integrase (IN) and the nucleosome for efficient integration. We also demonstrate direct interactions between IN and the amino-terminal tail of human histone H4 in vitro. Structure/function studies enabled us to identify amino acids in the carboxy-terminal domain of IN that are important for this interaction. Analysis of the nucleosome-binding properties of catalytically active mutated INs confirmed that their ability to engage the nucleosome for integration in vitro was affected. Pseudovirus particles bearing mutations that affect the IN/H4 association also showed impaired replication capacity due to altered integration and re-targeting of their insertion sites toward dynamic regions of the chromatin with lower nucleosome occupancy.ConclusionsCollectively, our data support a functional association between HIV-1 IN and histone tails that promotes anchoring of the intasome to nucleosomes and optimal integration into chromatin.