Next-Generation Proteomics Reveals a Greater Antioxidative Response to Drought in Coffea arabica Than in Coffea canephora

Drought is a major threat to coffee, compromising the quality and quantity of its production. We have analyzed the core proteome of 18 Coffea canephora cv. Conilon Clone 153 and C. arabica cv. Icatu plants and assessed their responses to moderate (MWD) and severe (SWD) water deficits. Label-free quantitative shotgun proteomics identified 3000 proteins in both genotypes, but less than 0.8% contributed to ca. 20% of proteome biomass. Proteomic changes were dependent on the severity of drought, being stronger under SWD and with an enrolment of different proteins, functions, and pathways than under MWD. The two genotypes displayed stress-responsive proteins under SWD, but only C. arabica showed a higher abundance of proteins involved in antioxidant detoxification activities. Overall, the impact of MWD was minor in the two genotypes, contrary to previous studies. In contrast, an extensive proteomic response was found under SWD, with C. arabica having a greater potential for acclimation/resilience than C. canephora. This is likely supported by a wider antioxidative response and an ability to repair photosynthetic structures, being crucial to develop new elite genotypes that assure coffee supply under water scarcity levels.

The Bacillus phage SPβ and its relatives: A temperate phage model system reveals new strains, species, prophage integration loci, conserved proteins and lysogeny management components

The Bacillus phage SPβ has been known for about 50 years, but only a few strains are avalible. We isolated four new wild type strains of the SPbeta species. Phage vB_BsuS-Goe14 introduces its prophage into the spoVK locus, previously not observed to be used by SPβ-like phages. We could also reveal the SPβ-like phage genome replication strategy, the genome packaging mode, and the phage genome opening point. We extracted 55 SPβ-like prophages from public Bacillus genomes, thereby discovering three more integration loci and one additional type of integrase. The identified prophages resembled four new species clusters and three species orphans in the genus Spbetavirus. The determined core proteome of all SPβ-like prophages consists of 38 proteins. The integration cassette proved to be not conserved even though present in all strains. It consists of distinct integrases. Analysis of SPβ transcriptomes revealed three conserved genes, yopQ, yopR, and yokI, to be transcribed from a dormant prophage. While yopQ and yokI could be deleted from the prophage without activating the prophage, damaging of yopR led to a clear-plaque phenotype. Under the applied laboratory conditions, the yokI mutant showed an elevated virion release implying the YokI protein being a component of the arbitrium system.

Proteomics of spatially identified tissues in whole organs

Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of biological specimens imaged in 3D as a whole are lacking. Here, we present DISCO-MS, a technology combining whole-organ imaging, deep learning-based image analysis, and ultra-high sensitivity mass spectrometry. DISCO-MS yielded qualitative and quantitative proteomics data indistinguishable from uncleared samples in both rodent and human tissues. Using DISCO-MS, we investigated microglia activation locally along axonal tracts after brain injury and revealed known and novel biomarkers. Furthermore, we identified initial individual amyloid-beta plaques in the brains of a young familial Alzheimer's disease mouse model, characterized the core proteome of these aggregates, and highlighted their compositional heterogeneity. Thus, DISCO-MS enables quantitative, unbiased proteome analysis of target tissues following unbiased imaging of entire organs, providing new diagnostic and therapeutic opportunities for complex diseases, including neurodegeneration.

A possible role of gas-phase electrophoretic mobility molecular analysis (nES GEMMA) in extracellular vesicle research

The emerging role of extracellular vesicles (EVs) as biomarkers and their envisioned therapeutic use require advanced techniques for their detailed characterization. In this context, we investigated gas-phase electrophoresis on a nano electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA, aka nES differential mobility analyzer, nES DMA) as an alternative to standard analytical techniques. In gas-phase electrophoresis, single-charged, surface-dry, native, polydisperse, and aerosolized analytes, e.g., proteins or bio-nanoparticles, are separated according to their electrophoretic mobility diameter, i.e., globular size. Subsequently, monodisperse particles are counted after a nucleation step in a supersaturated atmosphere as they pass a focused laser beam. Hence, particle number concentrations are obtained in accordance with recommendations of the European Commission for nanoparticle characterization (2011/696/EU from October 18th, 2011). Smaller sample constituents (e.g., co-purified proteins) can be detected next to larger ones (e.g., vesicles). Focusing on platelet-derived EVs, we compared different vesicle isolation techniques. In all cases, nanoparticle tracking analysis (NTA) confirmed the presence of vesicles. However, nES GEMMA often revealed a significant co-purification of proteins from the sample matrix, precluding gas-phase electrophoresis of less-diluted samples containing higher vesicle concentrations. Therefore, mainly peaks in the protein size range were detected. Mass spectrometry revealed that these main contaminants belonged to the group of globulins and coagulation-related components. An additional size exclusion chromatography (SEC) step enabled the depletion of co-purified, proteinaceous matrix components, while a label-free quantitative proteomics approach revealed no significant differences in the detected EV core proteome. Hence, the future in-depth analysis of EVs via gas-phase electrophoresis appears feasible. Graphical abstract

A Comprehensive Computer Aided Vaccine Design Approach to Propose a Multi-Epitopes Subunit Vaccine against Genus Klebsiella Using Pan-Genomics, Reverse Vaccinology, and Biophysical Techniques

Klebsiella is a genus of nosocomial bacterial pathogens and is placed in the most critical list of World Health Organization (WHO) for development of novel therapeutics. The pathogens of the genus are associated with high mortality and morbidity. Owing to their strong resistance profile against different classes of antibiotics and nonavailability of a licensed vaccine, urgent efforts are required to develop a novel vaccine candidate that can tackle all pathogenic species of the Klebsiella genus. The present study aims to design a broad-spectrum vaccine against all species of the Klebsiella genus with objectives to identify the core proteome of pathogen species, prioritize potential core vaccine proteins, analyze immunoinformatics of the vaccine proteins, construct a multi-epitopes vaccine, and provide its biophysical analysis. Herein, we investigated all reference species of the genus to reveal their core proteome. The core proteins were then subjected to multiple reverse vaccinology checks that are mandatory for the prioritization of potential vaccine candidates. Two proteins (TonB-dependent siderophore receptor and siderophore enterobactin receptor FepA) were found to fulfill all vaccine parameters. Both these proteins harbor several potent B-cell-derived T-cell epitopes that are antigenic, nonallergic, nontoxic, virulent, water soluble, IFN-γ producer, and efficient binder of DRB*0101 allele. The selected epitopes were modeled into a multi-epitope peptide comprising linkers and Cholera Toxin B adjuvant. For docking with innate immune and MHC receptors and afterward molecular dynamics simulations and binding free energy analysis, the vaccine structure was modeled for tertiary structure and refined for structural errors. To assess the binding affinity and presentation of the designed vaccine construct, binding mode and interactions analysis were performed using molecular docking and molecular dynamics simulation techniques. These biophysical approaches illustrated the vaccine as a good binder to the immune receptors and revealed robust interactions energies. The vaccine sequence was further translated to nucleotide sequence and cloned into an appropriate vector for expressing it at high rate in Escherichia coli K12 strain. In addition, the vaccine was illustrated to generate a good level of primary, secondary, and tertiary immune responses, proving good immunogenicity of the vaccine. Based on the reported results, the vaccine can be a good candidate to be evaluated for effectiveness in wet laboratory validation studies.

Biotechnological Importance of Torulaspora delbrueckii: From the Obscurity to the Spotlight

Torulaspora delbrueckii has attracted interest in recent years, especially due to its biotechnological potential, arising from its flavor- and aroma-enhancing properties when used in wine, beer or bread dough fermentation, as well as from its remarkable resistance to osmotic and freezing stresses. In the present review, genomic, biochemical, and phenotypic features of T. delbrueckii are described, comparing them with other species, particularly with the biotechnologically well-established yeast, Saccharomyces cerevisiae. We conclude about the aspects that make this yeast a promising biotechnological model to be exploited in a wide range of industries, particularly in wine and bakery. A phylogenetic analysis was also performed, using the core proteome of T. delbrueckii, to compare the number of homologous proteins relative to the most closely related species, understanding the phylogenetic placement of this species with robust support. Lastly, the genetic tools available for T. delbrueckii improvement are discussed, focusing on adaptive laboratorial evolution and its potential.

Taxonomy of Rhizobiaceae revisited: proposal of a new framework for genus delimitation

The alphaproteobacterial family Rhizobiaceae is highly diverse, with 168 species with validly published names classified into 17 genera with validly published names. Most named genera in this family are delineated based on genomic relatedness and phylogenetic relationships, but some historically named genera show inconsistent distribution and phylogenetic breadth. Most problematic is Rhizobium, which is notorious for being highly paraphyletic, as most newly described species in the family being assigned to this genus without consideration for their proximity to existing genera, or the need to create novel genera. In addition, many Rhizobiaceae genera lack synapomorphic traits that would give them biological and ecological significance. We propose a common framework for genus delimitation within the family Rhizobiaceae. We propose that genera in this family should be defined as monophyletic groups in a core-genome gene phylogeny, that are separated from related species using a pairwise core-proteome average amino acid identity (cpAAI) threshold of approximately 86%. We further propose that the presence of additional genomic or phenotypic evidence can justify the division of species into separate genera even if they all share greater than 86% cpAAI. Applying this framework, we propose to reclassify Rhizobium rhizosphaerae and Rhizobium oryzae into the new genus Xaviernesmea gen. nov. Data is also provided to support the recently proposed genus "Peteryoungia", and the reclassifications of Rhizobium yantingense as Endobacterium yantingense comb. nov., Rhizobium petrolearium as Neorhizobium petrolearium comb. nov., Rhizobium arenae as Pararhizobium arenae comb. nov., Rhizobium tarimense as Pseudorhizobium tarimense comb. nov., and Rhizobium azooxidefex as Mycoplana azooxidifex comb. nov. Lastly, we present arguments that the unification of the genera Ensifer and Sinorhizobium in Opinion 84 of the Judicial Commission is no longer justified by current genomic and phenotypic data. We thus argue that the genus Sinorhizobium is not illegitimate and now encompasses 17 species.

Screening of potential vaccine candidates against pathogenic Brucella spp. using compositive reverse vaccinology

Brucella spp. are Gram-negative, facultative intracellular bacteria that cause brucellosis in humans and various animals. The threat of brucellosis has increased, yet currently available live attenuated vaccines still have drawbacks. Therefore, subunit vaccines, produced using protein antigens and having the advantage of being safe, cost-effective and efficacious, are urgently needed. In this study, we used core proteome analysis and a compositive RV methodology to screen potential broad-spectrum antigens against 213 pathogenic strains of Brucella spp. with worldwide geographic distribution. Candidate proteins were scored according to six biological features: subcellular localization, antigen similarity, antigenicity, mature epitope density, virulence, and adhesion probability. In the RV analysis, a total 32 candidate antigens were picked out. Of these, three proteins were selected for assessment of immunogenicity and preliminary protection in a mouse model: outer membrane protein Omp19 (used as a positive control), type IV secretion system (T4SS) protein VirB8, and type I secretion system (T1SS) protein HlyD. These three antigens with a high degree of conservation could induce specific humoral and cellular immune responses. Omp19, VirB8 and HlyD could substantially reduce the organ bacterial load of B. abortus S19 in mice and provide varying degrees of protection. In this study, we demonstrated the effectiveness of this unique strategy for the screening of potential broad-spectrum antigens against Brucella. Further evaluation is needed to identify the levels of protection conferred by the vaccine antigens against wild-type pathogenic Brucella species challenge.

Proteomics-Based Data Integration of Wheat Cultivars Facing Fusarium graminearum Strains Revealed a Core-Responsive Pattern Controlling Fusarium Head Blight

Fusarium head blight (FHB), mainly occurring upon Fusarium graminearum infection in a wide variety of small-grain cereals, is supposed to be controlled by a range of processes diverted by the fungal pathogen, the so-called susceptibility factors. As a mean to provide relevant information about the molecular events involved in FHB susceptibility in bread wheat, we studied an extensive proteome of more than 7,900 identified wheat proteins in three cultivars of contrasting susceptibilities during their interaction with three F. graminearum strains of different aggressiveness. No cultivar-specific proteins discriminated the three wheat genotypes, demonstrating the establishment of a core proteome regardless of unequivocal FHB susceptibility differences. Quantitative protein analysis revealed that most of the FHB-induced molecular adjustments were shared by wheat cultivars and occurred independently of the F. graminearum strain aggressiveness. Although subtle abundance changes evidenced genotype-dependent responses to FHB, cultivar distinction was found to be mainly due to basal abundance differences, especially regarding the chloroplast functions. Integrating these data with previous proteome mapping of the three F. graminearum strains facing the three same wheat cultivars, we demonstrated strong correlations between the wheat protein abundance changes and the adjustments of fungal proteins supposed to interfere with host molecular functions. Together, these results provide a resourceful dataset that expands our understanding of the specific molecular events taking place during the wheat–F. graminearum interaction.