Delineation of the pan-proteome of fish-pathogenic Streptococcus agalactiae strains using a label-free shotgun approach.

BACKGROUND: Streptococcus agalactiae (GBS) is a major pathogen of Nile tilapia, a global commodity of the aquaculture sector. The aims of this study were to evaluate protein expression in the main genotypes of GBS isolated from diseased fishes in Brazil using a label-free shotgun nano-liquid chromatography-ultra definition mass spectrometry (nanoLC-UDMSE) approach and to compare the differential abundance of proteins identified in strains isolated from GBS-infected fishes and humans. RESULTS: A total of 1070 protein clusters were identified by nanoLC-UDMSE in 5 fish-adapted GBS strains belonging to sequence types ST-260 and ST-927 and the non-typeable (NT) lineage and 1 human GBS strain (ST-23). A total of 1065 protein clusters corresponded to the pan-proteome of fish-adapted GBS strains; 989 of these were identified in all fish-adapted GBS strains (core proteome), and 62 were shared by at least two strains (accessory proteome). Proteins involved in the stress response and in the regulation of gene expression, metabolism and virulence were detected, reflecting the adaptive ability of fish-adapted GBS strains in response to stressor factors that affect bacterial survival in the aquatic environment and bacterial survival and multiplication inside the host cell. Measurement of protein abundance among different hosts showed that 5 and 26 proteins were exclusively found in the human- and fish-adapted GBS strains, respectively; the proteins exclusively identified in fish isolates were mainly related to virulence factors. Furthermore, 215 and 269 proteins were up- and down-regulated, respectively, in the fish-adapted GBS strains in comparison to the human isolate. CONCLUSIONS: Our study showed that the core proteome of fish-adapted GBS strains is conserved and demonstrated high similarity of the proteins expressed by fish-adapted strains to the proteome of the human GBS strain. This high degree of proteome conservation of different STs suggests that, a monovalent vaccine may be effective against these variants.

Redesigning N-glycosylation sites in a GH3 ß-xylosidase improves the enzymatic efficiency.

BACKGROUND: ß-Xylosidases are glycoside hydrolases (GHs) that cleave xylooligosaccharides and/or xylobiose into shorter oligosaccharides and xylose. Aspergillus nidulans is an established genetic model and good source of carbohydrate-active enzymes (CAZymes). Most fungal enzymes are N-glycosylated, which influences their secretion, stability, activity, signalization, and protease protection. A greater understanding of the N-glycosylation process would contribute to better address the current bottlenecks in obtaining high secretion yields of fungal proteins for industrial applications. RESULTS: In this study, BxlB-a highly secreted GH3 ß-xylosidase from A. nidulans, presenting high activity and several N-glycosylation sites-was selected for N-glycosylation engineering. Several glycomutants were designed to investigate the influence of N-glycans on BxlB secretion and function. The non-glycosylated mutant (BxlBnon-glyc) showed similar levels of enzyme secretion and activity compared to the wild-type (BxlBwt), while a partially glycosylated mutant (BxlBN1;5;7) exhibited increased activity. Additionally, there was no enzyme secretion in the mutant in which the N-glycosylation context was changed by the introduction of four new N-glycosylation sites (BxlBCC), despite the high transcript levels. BxlBwt, BxlBnon-glyc, and BxlBN1;5;7 formed similar secondary structures, though the mutants had lower melting temperatures compared to the wild type. Six additional glycomutants were designed based on BxlBN1;5;7, to better understand its increased activity. Among them, the two glycomutants which maintained only two N-glycosylation sites each (BxlBN1;5 and BxlBN5;7) showed improved catalytic efficiency, whereas the other four mutants' catalytic efficiencies were reduced. The N-glycosylation site N5 is important for improved BxlB catalytic efficiency, but needs to be complemented by N1 and/or N7. Molecular dynamics simulations of BxlBnon-glyc and BxlBN1;5 reveals that the mobility pattern of structural elements in the vicinity of the catalytic pocket changes upon N1 and N5 N-glycosylation sites, enhancing substrate binding properties which may underlie the observed differences in catalytic efficiency between BxlBnon-glyc and BxlBN1;5. CONCLUSIONS: This study demonstrates the influence of N-glycosylation on A. nidulans BxlB production and function, reinforcing that protein glycoengineering is a promising tool for enhancing thermal stability, secretion, and enzymatic activity. Our report may also support biotechnological applications for N-glycosylation modification of other CAZymes.

Evaluation of sample preparation protocols for proteomic analysis of sunflower leaves.

Six protocols for extraction of proteins from sunflower (Helianthus annuus L.) leaves were evaluated for their abilities in both removing interferents and attaining the best resolution in two-dimensional gel electrophoresis. "Classical" phenol extraction followed by precipitation with ammonium acetate in methanol displayed the most efficient protocol, which allowed the detection of 244 protein spots with ca. 485mug of protein in gel electrophoresis. Tandem mass spectrometry was performed to identify proteins in 61 spots, and cross species identification was used for this task. Proteins from twenty two spots were identified, and 12 of these proteins are up to now not included into the ExPASy sunflower protein databank.