Changes in the relative abundance of two Saccharomyces species from oak forests to wine fermentations

Saccharomyces cerevisiae and its sibling species S. paradoxus are known to inhabit temperate arboreal habitats across the globe. Despite their sympatric distribution in the wild, S. cerevisiae is predominantly associated with human fermentations. The apparent ecological differentiation of these species is particularly striking in Europe where S. paradoxus is abundant in forests and S. cerevisiae is abundant in vineyards. However, ecological differences may be confounded with geographic differences in species abundance. To compare the distribution and abundance of these two species we isolated Saccharomyces strains from over 1,200 samples taken from vineyard and forest habitats in Slovenia. We isolated numerous strains of S. cerevisiae and S. paradoxus as well as small number of S. kudriavzevii strains from both vineyard and forest environments. We find S. cerevisiae less abundant than S. paradoxus on oak trees both within and outside the vineyard, but more abundant on grapevines and associated substrates. Analysis of the uncultured microbiome shows that both S. cerevisiae and S. paradoxus are rare species in soil and bark samples, but can be much more common in grape must. In contrast to S. paradoxus, European strains of S. cerevisiae have acquired multiple traits thought to be important for life in the vineyard and dominance of wine fermentations. We conclude that S. cerevisiae and S. paradoxus currently share both vineyard and non-vineyard habitats in Slovenia and we discuss factors relevant to their global distribution and relative abundance

Potential of bovine kappa - casein as biomarker for detection of adulteration of goat’s milk with cow’s milk

Korištenjem dvodimenzionalne elektroforeze dokazano je da kravlji kapa-kazein može biti odgovarajući biomarker za utvrđivanje patvorenja kozjeg mlijeka kravljim mlijekom, ne samo u sirovom mlijeku, već i za mlijeko koje se termički obrađuje pasterizacijom ili ultra-visokom temperaturom. Prisutnost kravljeg mlijeka u kozjem mlijeku moguće je detektirati na razini od 2 %. Nadalje, položaj proteinskih mrlja kapa-kazeina na 2-D gelovima ostao je nepromijenjen uspoređujući uzorke mlijeka različitog geografskog podrijetla, Belgije i Slovenije. Ovi rezultati pokazuju da ni toplinska obrada niti različito geografsko podrijetlo ne utječu na položaj kravljih proteinskih mrlja kapa-kazeina na 2-D gelovima.Using two-dimensional (2-D) electrophoresis it was shown that bovine kappa-casein could be an appropriate biomarker of adulteration of goat’s milk with cow’s milk not only in raw milk, but also for milk thermally processed by pasteurization or treated with ultra-high-temperature. The presence of cow’s milk in goat’s milk was detected at level of 2 %. Furthermore, position of bovine kappa-casein spots on 2-D gels remained unchanged even with samples from two different geographical origins, Belgium and Slovenia. These results show that neither thermal processing nor different geographical area seem to affect the position of bovine kappa-casein spots on 2-D gels

Chitin-Binding Protein of Verticillium nonalfalfae Disguises Fungus from Plant Chitinases and Suppresses Chitin-Triggered Host Immunity

During fungal infections, plant cells secrete chitinases, which digest chitin in the fungal cell walls. The recognition of released chitin oligomers via lysin motif (LysM)-containing immune host receptors results in the activation of defense signaling pathways. We report here that Verticillium nonalfalfae, a hemibiotrophic xylem-invading fungus, prevents these digestion and recognition processes by secreting a carbohydrate-binding motif 18 (CBM18)-chitin-binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by the Vna8.213 gene, which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however, Vna8.213 knockout mutants exhibited wilting symptoms similar to the wild-type fungus, suggesting that Vna8.213 activity is functionally redundant during fungal infection of hop. In a binding assay, recombinant VnaChtBP bound chitin and chitin oligomers in vitro with submicromolar affinity and protected fungal hyphae from degradation by plant chitinases. Moreover, the chitin-triggered production of reactive oxygen species from hop suspension cells was abolished in the presence of VnaChtBP, indicating that VnaChtBP also acts as a suppressor of chitin-triggered immunity. Using a yeast-two-hybrid assay, circular dichroism, homology modeling, and molecular docking, we demonstrated that VnaChtBP forms dimers in the absence of ligands and that this interaction is stabilized by the binding of chitin hexamers with a similar preference in the two binding sites. Our data suggest that, in addition to chitin-binding LysM (CBM50) and Avr4 (CBM14) fungal effectors, structurally unrelated CBM18 effectors have convergently evolved to prevent hydrolysis of the fungal cell wall against plant chitinases and to interfere with chitin-triggered host immunity.</p

Combination of phage and Gram-positive bacterial display of human antibody repertoires enables isolation of functional high affinity binders

Surface display couples genotype with a surface exposed phenotype and thereby allows screening of gene-encoded protein libraries for desired characteristics. Of the various display systems available, phage display is by far the most popular, mainly thanks to its ability to harbour large size libraries. Here, we describe the first use of a Gram-positive bacterial host for display of a library of human antibody genes which, when combined with phage display, provides ease of use for screening, sorting and ranking by flow cytometry. We demonstrate the utility of this method by identifying low nanomolar affinity scFv fragments towards human epidermal growth factor receptor 2 (HER2). The ranking and performance of the scFv isolated by flow sorting in surface-immobilised form was retained when expressed as soluble scFv and analysed by biolayer interferometry, as well as after expression as full-length antibodies in mammalian cells. We also demonstrate the possibility of using Gram-positive bacterial display to directly improve the affinity of the identified binders via an affinity maturation step using random mutagenesis and flow sorting. This combined approach has the potential for a more complete scan of the antibody repertoire and for affinity maturation of human antibody formats

Functional analysis of Verticillium nonalfalfae candidate effector proteins

Pri hmelju (Humulus lupulus L.) je poznana letalna oblika verticilijske uvelosti, ki se odraža z bolezenskimi znaki uvelosti, klorozami, nekrozami in končnim propadom rastline. Bolezen povzroča fitopatogena gliva Verticillium nonalfalfae (Vna), ki za uspešno kolonizacijo rastline izloča efektorske proteine. Ti jo ščitijo pred obrambnim odzivom rastline, modulirajo imunski odziv ali vplivajo na fiziologijo rastline. Razumevanje funkcije kandidatnih sekretornih efektorskih proteinov (CSEPs) in poznavanje molekul gostitelja, s katerimi določen CSEP interagira, je ključno pri načrtovanju novih strategij zatiranja verticiljske uvelosti in lahko pospeši žlahtnjenje kultivarjev hmelja odpornih na Vna. V okviru te doktorske disertacije smo okarakterizirali kandidatna efektorska proteina VnaChtBP in VnaSSP4.2, ki smo ju izbrali na osnovi predhodnih študij. S konfokalno mikroskopijo smo pri modelni rastlini Nicotiana benthamiana ugotovili nukleo-citosolno subcelično lokalizacijo obeh izbranih CSEPs. Z infiltracijo rekombinantnih proteinov VnaChtBP in VnaSSP4.2 v različne rastline nismo uspeli potrditi hipoteze o njihovi vpletenosti v PTI ali ETI odziv. Ugotovili smo, da se efektor VnaChtBP specifično veže na hitin in ščiti glivo pred razgradnjo z rastlinskimi hitinazami. Pokazali smo, da se rekombinantni protein VnaSSP4.2 veže na molekule PIP, kardiolipin in sulfatid, ki so opisani kot modulatorji imunskega odziva.Outbreaks of a lethal type of Verticillium wilt of hops (Humulus lupulus L.) are reported yearly. This disease is caused by a phytopathogenic fungus Verticillium nonalfalfae (Vna) and presents in wilting, chlorosis, necrosis, and plant death. In order to successfully colonize its host, Vna secrets effector proteins, which protect the fungus and modulate the plant immune system or plant physiology. Understanding the function of candidate secreted effector proteins (CSEPs) and knowledge of host interacting molecules is a crucial contribution to development of effective strategies for pest control and can accelerate breeding of Vna resistant cultivars. In the presented doctoral thesis, we characterized candidate effector proteins VnaChtBP and VnaSSP4.2, which were selected based on previous studies. Using confocal microscopy, a nucleo-cytosolic subcellular localization was determined for both effectors expressed in model plant Nicotiana benthamiana. Infiltration of recombinant proteins to various plants did not confirm the hypothesis of VnaChtBP or VnaSSP4.2 involvement in PTI or ETI responses. We were able to show that the recombinant protein VnaSSP4.2 binds to PIP, cadiolipin and sulfatide molecules, all known modulators of plant immunity. We determined that the effector VnaChtBP binds specifically to chitin and protects the fungus from degradation by plant chitinases

Mammalian cell surface display system for conformational epitope determination of SARS-CoV-2 neutralizing antibodies

Cell surface display of recombinant proteins for epitope mapping has become a powerful tool for biotechnology and biomedical applications. One critical aspect for identifying the full and correct epitope is to present the correctly folded and post-translationally modified protein antigen on the cell surface for true epitope-paratope interaction. Being the gold standard for production of complex proteins, Chinese hamster ovarian cells are here used for a mammalian cell display system. Additionally, to enable a fast workflow for alanine substitution of a large panel of clones, a web-based design tool and automated cloning process was established. A proof-of-concept is presented with the aligned epitope of the crystal structure for the CR3022 antibody with our mammalian cell display determined epitope. Furthermore, three additional antibody epitopes against SARS-CoV-2 RBD are presented. This study presents a detailed view of epitope-paratope interactions, which is of great importance for future clinical interventions against SARS-CoV-2. QC 20210607</p

Mammalian cell surface display system for conformational epitope determination of SARS-CoV-2 neutralizing antibodies

Cell surface display of recombinant proteins for epitope mapping has become a powerful tool for biotechnology and biomedical applications. One critical aspect for identifying the full and correct epitope is to present the correctly folded and post-translationally modified protein antigen on the cell surface for true epitope-paratope interaction. Being the gold standard for production of complex proteins, Chinese hamster ovarian cells are here used for a mammalian cell display system. Additionally, to enable a fast workflow for alanine substitution of a large panel of clones, a web-based design tool and automated cloning process was established. A proof-of-concept is presented with the aligned epitope of the crystal structure for the CR3022 antibody with our mammalian cell display determined epitope. Furthermore, three additional antibody epitopes against SARS-CoV-2 RBD are presented. This study presents a detailed view of epitope-paratope interactions, which is of great importance for future clinical interventions against SARS-CoV-2. QC 20210607</p

Clinical and histopathological features of gelsolin amyloidosis associated with a novel GSN variant p.Glu580Lys

Gelsolin amyloidosis typically presents with corneal lattice dystrophy and is most frequently associated with pathogenic GSN variant p.Asp214Asn. Here we report clinical and histopathological features of gelsolin amyloidosis associated with a novel GSN variant p.Glu580Lys. We studied DNA samples of seven members of a two-generation family. Exome sequencing was performed in the proband, and targeted Sanger sequencing in the others. The heterozygous GSN variant p.Glu580Lys was identified in six patients. The patients exhibited corneal dystrophy (5/6), loose skin (5/6) and/or heart arrhythmia (3/6) and one presented with bilateral optic neuropathy. The impact of the mutation on the protein structure was evaluated in silico. The substitution is located in the fifth domain of gelsolin protein, homologous to the second domain harboring the most common pathogenic variant p.Asp214Asn. Structural investigation revealed that the mutation might affect protein folding. Histopathological analysis showed amyloid deposits in the skin. The p.Glu580Lys is associated with corneal dystrophy, strengthening the association of the fifth domain of gelsolin protein with the typical amyloidosis phenotype. Furthermore, optic neuropathy may be related to the disease and is essential to identify before discussing corneal transplantation