Nuclear envelope breakdown in starfish oocytes proceeds by partial NPC disassembly followed by a rapidly spreading fenestration of nuclear membranes

Breakdown of the nuclear envelope (NE) was analyzed in live starfish oocytes using a size series of fluorescently labeled dextrans, membrane dyes, and GFP-tagged proteins of the nuclear pore complex (NPC) and the nuclear lamina. Permeabilization of the nucleus occurred in two sequential phases. In phase I the NE became increasingly permeable for molecules up to ∼40 nm in diameter, concurrent with a loss of peripheral nuclear pore components over a time course of 10 min. The NE remained intact on the ultrastructural level during this time. In phase II the NE was completely permeabilized within 35 s. This rapid permeabilization spread as a wave from one epicenter on the animal half across the nuclear surface and allowed free diffusion of particles up to ∼100 nm in diameter into the nucleus. While the lamina and nuclear membranes appeared intact at the light microscopic level, a fenestration of the NE was clearly visible by electron microscopy in phase II. We conclude that NE breakdown in starfish oocytes is triggered by slow sequential disassembly of the NPCs followed by a rapidly spreading fenestration of the NE caused by the removal of nuclear pores from nuclear membranes still attached to the lamina

Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretory strains

ABSTRACT: BACKGROUND: Due to its capacity to produce large amounts of cellulases, Trichoderma reesei is increasingly been researched in various fields of white biotechnology, especially in biofuel production from lignocellulosic biomass. The commercial enzyme mixtures produced at industrial scales are not well characterized, and their proteinaceous components are poorly identified and quantified. The development of proteomic methods has made it possible to comprehensively overview the enzymes involved in lignocellulosic biomass degradation which are secreted under various environmental conditions. RESULTS: The protein composition of the secretome produced by industrial T. reesei (strain CL847) grown on a medium promoting the production of both cellulases and hemicellulases was explored using two-dimensional electrophoresis and MALDI-TOF or LC-MS/MS protein identification. A total of 22 protein species were identified. As expected, most of them are potentially involved in biomass degradation. The 2D map obtained was then used to compare the secretomes produced by CL847 and another efficient cellulolytic T. reesei strain, Rut-C30, the reference cellulase-overproducing strain using lactose as carbon source and inducer of cellulases. CONCLUSION: This study provides the most complete mapping of the proteins secreted by T. reesei to date. We report on the first use of proteomics to compare secretome composition between two cellulase-overproducing strains Rut-C30 and CL847 grown under similar conditions. Comparison of protein patterns in both strains highlighted many unexpected differences between cellulase cocktails. The results demonstrate that 2D electrophoresis is a promising tool for studying cellulase production profiles, whether for industrial characterization of an entire secretome or for a more fundamental study on cellulase expression at genome-wide scale

Metagenomic Approaches for Investigating the Role of the Microbiome in Gut Health and Inflammatory Diseases

The human gut microbiota makes fundamental contributions to host metabolism and immune system. Therefore, perturbations in its composition, a process known as dysbiosis, have an important role in the development of several chronicle diseases, mainly intestinal inflammatory disorders. Culture-independent molecular methods are allowing scientific community to uncover substantive findings, thus giving a more detailed description of the human intestinal microbiota. This chapter presents a review on current metagenomic approaches, based on next-generation sequencing technologies, for investigating bacterial taxonomic classification and predictive function associated with the human gut in health and disease. In this context, we describe recent studies that have been trying to elucidate important alterations in microbiome composition across individuals according to delivery mode, aging, diet and medication that might be linked to susceptibility to immune-mediated diseases. A description of the main bacterial taxa and genes acting in dysbiosis during inflammation, focusing on chronic inflammatory bowel diseases and colorectal cancer, is also explored in this chapter

Development of serological proteome analysis of mastitis by Staphylococcus aureus in ewes.

International audienceStaphylococcus aureus is a major agent of mastitis in ruminants worldwide. So far, efficient measures for its prophylaxis (including vaccination) have proven to be unsuccessful and there is a need for a better understanding of the host response to udder infection by S. aureus. Serological proteome analysis (SERPA) is a promising technique that can be used to identify S. aureus immuno-dominant determinants providing that bacterial culture conditions used to grow S. aureus strains for protein sample preparation mimic the context of mastitis. A S. aureus strain was used in experimental mastitis to generate sheep serum used to determine the best growth conditions for SERPA. Sera collected in the field from different ewes suffering from mastitis by S. aureus were used to confirm experimental observations. Three different culture media (BHI, whey and iron-depleted RPMI) were tested. The influence of aeration and growth phase on protein production was also evaluated by immuno-detection of protein samples prepared from cultures grown in different conditions and obtained from different culture fractions (supernatant, cell wall, and total lysates). Our results showed that culturing in iron-depleted RPMI with (secreted proteins, prepared from stationary phase) or without aeration (cell wall proteins, prepared from early stationary phase, and total proteins, prepared from exponential phase) is the condition that best mimics growth in vivo during mastitis and this in vitro growth condition is to be used henceforth in experiments involving SERPA

Mutation of the surface layer protein SlpB has pleiotropic effects in the probiotic propionibacterium freudenreichii CIRM-BIA 129

Propionibacterium freudenreichii is a beneficial Gram-positive bacterium, traditionally used as a cheese-ripening starter, and currently considered as an emerging probiotic. As an example, the P. freudenreichii CIRM-BIA 129 strain recently revealed promising immunomodulatory properties. Its consumption accordingly exerts healing effects in different animal models of colitis, suggesting a potent role in the context of inflammatory bowel diseases. This anti-inflammatory effect depends on surface layer proteins (SLPs). SLPs may be involved in key functions in probiotics, such as persistence within the gut, adhesion to host cells and mucus, or immunomodulation. Several SLPs coexist in P. freudenreichii CIRM-BIA 129 and mediate immunomodulation and adhesion. A mutant P. freudenreichii CIRM-BIA 129ΔslpB (CB129ΔslpB) strain was shown to exhibit decreased adhesion to intestinal epithelial cells. In the present study, we thoroughly analyzed the impact of this mutation on cellular properties. Firstly, we investigated alterations of surface properties in CB129ΔslpB. Surface extractable proteins, surface charges (ζ-potential) and surface hydrophobicity were affected by the mutation. Whole-cell proteomics, using high definition mass spectrometry, identified 1,288 quantifiable proteins in the wild-type strain, i.e., 53% of the theoretical proteome predicted according to P. freudenreichii CIRM-BIA 129 genome sequence. In the mutant strain, we detected 1,252 proteins, including 1,227 proteins in common with the wild-type strain. Comparative quantitative analysis revealed 97 proteins with significant differences between wild-type and mutant strains. These proteins are involved in various cellular process like signaling, metabolism, and DNA repair and replication. Finally, in silico analysis predicted that slpB gene is not part of an operon, thus not affecting the downstream genes after gene knockout. This study, in accordance with the various roles attributed in the literature to SLPs, revealed a pleiotropic effect of a single slpB mutation, in the probiotic P. freudenreichii. This suggests that SlpB may be at a central node of cellular processes and confirms that both nature and amount of SLPs, which are highly variable within the P. freudenreichii species, determine the probiotic abilities of strains.Fil: do Carmo, Fillipe L. R.. Institut National de la Recherche Agronomique; Francia. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Marques Da Silva, Wanderson. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Tavares, Guilherme C.. Universidade Federal de Minas Gerais; BrasilFil: Ibraim, Izabela C.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Cordeiro, Barbara F.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Oliveira, Emiliano R.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Rabah, Houem. Institut National de la Recherche Agronomique; FranciaFil: Cauty, Chantal. Institut National de la Recherche Agronomique; FranciaFil: da Silva, Sara H.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Canário Viana, Marcus V.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Caetano, Ana C. B.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: dos Santos, Roselane G.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: de Oliveira Carvalho, Rodrigo D.. Instituto de Ciencias Da Saúde; BrasilFil: Jardin, Julien. Institut National de la Recherche Agronomique; FranciaFil: Pereira, Felipe L.. Universidade Federal de Minas Gerais; BrasilFil: Folador, Edson L.. Universidade Estadual da Paraiba; BrasilFil: Le Loir, Yves. Institut National de la Recherche Agronomique; FranciaFil: Figueiredo, Henrique C. P.. Universidade Federal de Minas Gerais; BrasilFil: Jan, Gwénaël. Institut National de la Recherche Agronomique; FranciaFil: Azevedo, Vasco. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; Brasi

A Temporal -omic Study of Propionibacterium freudenreichii CIRM-BIA1T Adaptation Strategies in Conditions Mimicking Cheese Ripening in the Cold

Propionibacterium freudenreichii is used as a ripening culture in Swiss cheese manufacture. It grows when cheeses are ripened in a warm room (about 24°C). Cheeses with an acceptable eye formation level are transferred to a cold room (about 4°C), inducing a marked slowdown of propionic fermentation, but P. freudenreichii remains active in the cold. To investigate the P. freudenreichii strategies of adaptation and survival in the cold, we performed the first global gene expression profile for this species. The time-course transcriptomic response of P. freudenreichii CIRM-BIA1T strain was analyzed at five times of incubation, during growth at 30°C then for 9 days at 4°C, under conditions preventing nutrient starvation. Gene expression was also confirmed by RT-qPCR for 28 genes. In addition, proteomic experiments were carried out and the main metabolites were quantified. Microarray analysis revealed that 565 genes (25% of the protein-coding sequences of P. freudenreichii genome) were differentially expressed during transition from 30°C to 4°C (P<0.05 and |fold change|>1). At 4°C, a general slowing down was observed for genes implicated in the cell machinery. On the contrary, P. freudenreichii CIRM-BIA1T strain over-expressed genes involved in lactate, alanine and serine conversion to pyruvate, in gluconeogenesis, and in glycogen synthesis. Interestingly, the expression of different genes involved in the formation of important cheese flavor compounds, remained unchanged at 4°C. This could explain the contribution of P. freudenreichii to cheese ripening even in the cold. In conclusion, P. freudenreichii remains metabolically active at 4°C and induces pathways to maintain its long-term survival

Development of a anti inflammatory cheese

International audienc

Peculiar properties of propionibacteria lead to unexpected probiotic properties

Cheeses and other fermented dairy products constitute a tremendous source of live and active bacteria in our diet. Depending on the product, on the strain and on the health status of individuals, this major bacterial intake can have beneficial effects, or not! Recognized molecular, cellular and animal models have been developed to detect and study probiotic bacterial strains. These tools may also reveal unexpected probiotic potential in bacterial strains within the tremendous biodiversity present in traditional fermented products. In our team, we focused on dairy propionibacteria, taking advantage of a unique biological resource center. Propionibacterium freudenreichii is a beneficial bacterium used in the food industry as a vitamin producer, as a bio-preservative and as a cheese ripening starter. We hypothesized that strains considered for techno-functional reasons (aroma, texture), may also play a beneficial role as probiotics.Food-grade dairy propionibacteria are nutraceutical producers. They release short chain fatty acids (SCFAs), folic acid and cobalamin, which play a role in intestinal physiology. SCFAs induce apoptosis via the mitochondrial death pathway. We investigate the potential of dairy propionibacteria-fermented products to synergize with drugs used in gastric and colorectal cancer.Some strains are known to adhere to intestinal epithelial cells and mucus and to modulate important functions of the gut mucosa, including cell proliferation and immune response. Selected strains show promising immunomodulatory properties with an anti-inflammatory protective evidenced effect in vitro and in vivo. We thus developed experimental monoxenic fermented dairy products, exclusively fermented by an anti-inflammatory selected strain of P. freudenreichii. Key surface proteins, involved in its immunomodulatory effects, are expressed within these products. Their consumption exerted an anti-inflammatory effect in vivo.These results open new perspectives in the field of prevention and/or treatment of intestinal cancer (CRC) and inflammatory diseases (IBD). The synergy with pro-apoptotic chemotherapy molecules suggests that such a fermented product, or the Propionibacterium alone, may be proposed as a food supplement to enhance the effects of anticancer treatments. The preventive effect in the context of induced colitis suggests that it could potentialize IBD treatment.This work reveals an unexpected probiotic potential in industrial dairy propionibacteria starters. It opens exciting perspectives in a context of growing occurrence of IBD, multifactor diseases related to diet, and provides a new functional fermented product for preclinical and clinical studies aimed at prevention or treatment of IBD