Project description:The goal of this study is to identify the different proteins secreted by Mycobacterium bovis in the culture media at different stages of bacterial growth. A field strain of M. bovis was allowed to grow in a culture media and culture supernatant was collected at three-time points representing approximately three different phases of growth. The supernatant was digested by trypsin and analyzed by LC-MS/MS analysis
Project description:Clostridioides difficile BI/NAP1/ribotype 027 is an epidemic hypervirulent strain found worldwide, including in Latin America. We examined the genomes and exoproteomes of two multilocus sequence type (MLST) clade 2 C. difficile strains considered hypervirulent: ICC-45 (ribotype SLO231/UK[CE]821), isolated in Brazil, and NAP1/027/ST01 (LIBA5756), isolated during a 2010 outbreak in Costa Rica. C. difficile isolates were cultured and extracellular proteins were analyzed using high-performance liquid chromatography-tandem mass spectrometry. Genomic analysis revealed that these isolates shared most of the gene composition. Only 83 and 290 NAP1/027 genes were considered singletons in ICC-45 and NAP1/027, respectively. Exoproteome analysis revealed 197 proteins, of which 192 were similar in both strains. Only five proteins were exclusive to the ICC-45 strain. These proteins were involved with catalytic and binding functions and indirectly interacted with proteins related to pathogenicity. Most proteins, including TcdA, TcdB, flagellin subunit, and cell surface protein, were overrepresented in the ICC-45 strain; 14 proteins, including mature S-layer protein, were present in higher proportions in LIBA5756. These data show close similarity between the genome and proteins in the supernatant of two strains with hypervirulent features isolated in Latin America and underscore the importance of epidemiological surveillance of the transmission and emergence of new strains.
Project description:Pectobacterium are Gram-negative rods of the family Pectobacteriaceae. They are the causative agent of soft rot diseases of crops and ornamental plants. However, their virulence mechanisms are not yet fully elucidated. Membrane vesicles (MVs) are universally released by bacteria and are be-lieved to play an important role in pathogenicity, and survival of bacteria in the environment. Our study investigates the role of MVs in the virulence of Pectobacterium. The results indicate that the morphology and yields of MVs depend on medium composition. In polygalacturonic acid (PGA) supplemented media, Pectobacterium produce MVs of a larger size (100-300 nm) apart of vesicles below 100 nm. Proteomic analyses revealed the presence of pectate degrading enzymes in MVs. The pectate plate test and enzymatic assay proved that those enzymes are active and able to de-grade pectates. What is more, pathogenicity test indicated that MVs derived from Pectobacterium were able to induce maceration of Zantedeschia sp. leaves. We also show that MVs of β-lactamase producing strains were able to suppress ampicillin activity and permit the growth of susceptible bacteria. Those findings indicate that MVs of Pectobacterium play an important role in host-pathogen interactions and niche competition with other bacteria. Our research also sheds some light on the mechanism of MVs production. We demonstrate that Pectobacterium strains, which overexpress the green fluorescence protein (GFP), produce more MVs than wild type strains. Moreover, proteomic analysis revealed that GFP was present in MVs. Therefore, we demonstrate that protein sequestration into MVs is not limited strictly to periplasmic proteins and is a common occurrence. Our research highlights the importance of MVs production as a mechanism of cargo delivery in Pectobacterium and an alternative secretion system.
Project description:Following exposure to vaccines, antigen-specific CD8+ T-cell responses develop as long-term memory pools. Novel vaccine strategies based on adenoviral vectors, e.g. those developed for HCV, are able to induce and sustain substantial CD8+ T-cell populations. How such populations evolve following vaccination remains to be defined at a transcriptional level. We addressed the transcriptional regulation of divergent CD8+ T-cell memory pools induced by an adenoviral vector encoding a model antigen (beta-galactosidase). We observe transcriptional profiles that mimic those following infection with persistent pathogens, murine and human cytomegalovirus (CMV). Key transcriptional hallmarks include up-regulation of homing receptors, and anti-apoptotic pathways, driven by conserved networks of transcription factors, including T-bet (TBX21). In humans, a novel adenovirus vaccine induced similar CMV-like phenotypes and underlying transcription factor regulation. These data clarify the core features of CD8+ T-cell memory following vaccination with adenovirus vectors and indicate a conserved pathway for memory development shared with persistent herpesviruses. Total RNA was extracted from sorted memory CD8 T cells induce by CMV and adenoviral vectors, and naïve CD8 cells
Project description:Nitrogen (N) is an essential macronutrient for agricultural production, and its excessive use causes not only economic but also environmental damage. However, improving nitrogen use efficiency (NUE) remains a complex task for achieving more productive crops under conditions of limited nitrogen availability. To the best of our knowledge, this is the first study aiming to integrate the morphophysiological responses and proteomic profiles of leaves and roots of popcorn (Zea mays everta). For this, two inbred lines contrasting for NUE, i.e., P2 (high NUE) and L80 (low NUE), were cultivated under high (100% of N supply) and low (10% of N supply) nitrogen conditions. Morphological and physiological traiits such as photochemical quenching (qP), non-photochemical quenching (NPQ), quantum yield of photosystem II (ΦPSII), and potential photosynthesis (Apot) were evaluated. P2 showed more pronounced vegetative growth under low N, as well as higher values of qP, NPQ, and ΦPSII. Comparative proteomic analysis of the leaves identified 215 differentially accumulated proteins (DAPs) in P2 and 168 DAPs in L80, while in roots, 127 DAPs were observed in P2 and 172 in L80. In leaves, DAPs involved in the response to oxidative stress, energy metabolism, and photosynthesis represented the main differences between P2 and L80. In roots, DAPs involved in nitrate transport, ammonium assimilation, and amino acid metabolism seem to have contributed to the improved NUE in P2. This study provides valuable insights into the molecular mechanisms underlying NUE and opens avenues for molecular breeding aimed at selecting superior genotypes for the development of a more sustainable agriculture.
Project description:Several neurodegenerative diseases including Alzheimer's Disease (AD) are characterized by ubiquitin-positive pathological protein aggregates. Here, an immunoaffinity approach is utilized to enrich ubiquitylated isopeptides after trypsin digestion from five AD and five age-matched control postmortem brain tissues. Label-free MS-based proteomic analysis identifies 4291 unique ubiquitylation sites mapping to 1682 unique proteins. Differential enrichment analysis shows that over 800 ubiquitylation sites are significantly altered between AD and control cases. Of these, ≈80% are increased in AD, including seven poly ubiquitin linkages, which is consistent with proteolytic stress and high burden of ubiquitylated pathological aggregates in AD. The microtubule associated protein Tau, the core component of neurofibrillary tangles, has the highest number of increased sites of ubiquitylation per any protein in AD. Tau poly ubiquitylation from AD brain homogenates is confirmed by reciprocal co-immunoprecipitation and by affinity capture using tandem ubiquitin binding entities. Co-modified peptides, with both ubiquitylation and phosphorylation sites, are also enriched in AD. Notably, many of the co-modified peptides mapped to Tau within KXGS motifs in the microtubule binding region suggesting that crosstalk between phosphorylation and ubiquitylation occurs on Tau in AD. Overall, these findings highlight the utility of MS to map ubiquitylated substrates in human brain and provides insight into mechanisms underlying pathological protein posttranslational modification in AD.
Project description:A gradient cell elongation with maximum in the lower side of pulvini and minimum in the upper side of pulvini pushes a maize stem upwards from a horizontal to a vertical position during gravitropsim. A differentiated gene expression between lower and upper pulvini was examined at 1 h, 6 and 24 h. Gene expression levels were compared in control (not bending), lower and upper (bending) pulvini. The samples were harvested at 1 h, 6 h and 24 h gravitropsim and total RNA were extracted using for microarray experiment.
Project description:Extracellular proteins are involved in a remarkable number of fundamental processes in cyanobacteria. Yet, there is limited knowledge regarding the identity and function of these proteins. Here, we introduce a solid-phase enhanced protein aggregation workflow that enables description of the cyanobacterial exoproteome with unprecedented depth. Application to cyanobacteria from three distinct habitats, Synechocystis sp. PCC 6803, Synechcoccus sp. PCC 11901 and Nostoc punctiforme PCC 73102, allowed the identification of up to 62% of all predicted secreted proteins. The approach was then extended to compare the Synechocystis sp. PCC 6803 wild-type secretome with that of a bloom-like aggregated state and a secretion-impaired mutant. Finally, we demonstrate that the workflow can be miniaturized and adapted to a 96-well format for high-throughput secretome analysis. Collectively, these findings challenge the general belief that cyanobacteria lack secretory proteins and point to a functional conservation of the secretome across species from different environments. Our approach can be applied to microbes from a wide range of habitats, with the potential to open new avenues of investigation in microbial exoproteomics.