Modulation of Campylobacter jejuni adhesion to biotic model surfaces by fungal lectins and protease inhibitors

Campylobacter jejuni, a Gram-negative bacterium, is one of the most common causes of foodborne illness worldwide. Its adhesion mechanism is mediated by several bacterial factors, including flagellum, protein adhesins, lipooligosaccharides, proteases, and host factors, such as surface glycans on epithelial cells and mucins. Fungal lectins, specialized carbohydrate-binding proteins, can bind to specific glycans on host and bacterial cells and thus influence pathogenesis. In this study, we investigated the effects of fungal lectins and protease inhibitors on the adhesion of C. jejuni to model biotic surfaces (mucin, fibronectin, and collagen) and Caco-2 cells as well as the invasion of Caco-2 cells. The lectins Marasmius oreades agglutinin (MOA) and Laccaria bicolor tectonin 2 (Tec2) showed remarkable efficacy in all experiments. In addition, different pre-incubations of lectins with C. jejuni or Caco-2 cells significantly inhibited the ability of C. jejuni to adhere to and invade Caco-2 cells, but to varying degrees. Pre-incubation of Caco-2 cells with selected lectins reduced the number of invasive C. jejuni cells the most, while simultaneous incubation showed the greatest reduction in adherent C. jejuni cells. These results suggest that fungal lectins are a promising tool for the prevention and treatment of C. jejuni infections. Furthermore, this study highlights the potential of fungi as a rich reservoir for novel anti-adhesive agents

Response of yeast Saccharomyces cerevisiae to formation of TDP-43 protein aggregates

Kvasovke Saccharomyces cerevisiae imajo pomembno vlogo kot modelni organizem za razumevanje razvoja različnih nevrodegenerativnih bolezni. Prav njihova enostavnost za kultivacijo in zadostna podobnost celicam sesalcev sta pripomogla k njihovi splošni uporabi pri različnih znanstvenih študijah. Nevrodegenerativne bolezni predstavljajo težavo in izziv modernega časa, saj so rezultat staranja populacije in podaljševanja življenjske dobe ljudi. Med redke nevrodegenerativne bolezni prištevamo amiotrofično lateralno sklerozo (ALS). Prizadene motorične nevrone v možganih in hrbtenjači ter povzroči njihovo degeneracijo s citoplazemskimi agregati proteina TDP-43. Cilj naloge je bil preučiti odziv kvasovk na prisotnost citotoksičnih agregatov proteina TDP-43-GFP. Kvasovke, ki smo jih gojili v zaporednih šaržnih kultivacijah, smo opazovali s pomočjo fluorescenčne mikroskopije v različnih fazah rasti. Hkrati smo merili fluorescenčno intenziteto za indikacijo izražanja vstavljenega humanega gena TDP-43-GFP, prirast biomase za vpliv agregatov na celično rast in razmnoževanje ter metabolno aktivnost celic kot odraz energijskih potreb za celični stresni odziv na prisotnost agregatov. Z eksperimenti smo razbrali stresni vpliv agregatov na celice in posledično njihov odziv. Ugotovili smo, da je kultura, ki je bila prvotno kultivirana v ekspresijskem ter nato v represijskem gojišču, ob ponovni kultivaciji v ekspresijskem gojišču po 8 urah kultivacije boljše pripravljena na agregacijski stres. To je razvidno kot povišana celična metabolna aktivnost ter zmanjšanje izražanja gena TDP-43 za polovico v primerjavi z ekspresijsko kulturo prve kultivacije. Naši rezultati bi lahko pomembno pripomogli k razumevanju razvoja ALS pri nastanku in odstranitvi agregatov proteina TDP-43.Yeast Saccharomyces cerevisiae play an important role as a model organism for understanding the development of various neurodegenerative diseases. Their ease of cultivation and sufficient resemblance to mammalian cells have contributed to their general use in various scientific studies. Neurodegenerative diseases are a problem and a challenge of modern times, as they are the result of an aging population and the extended expectancy of human life. Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disease. It affects motor neurons in the brain and spinal cord and causes their degeneration with cytoplasmic aggregates of the TDP-43 protein. The aim of the thesis was to study the yeast response to the presence of cytotoxic aggregates of protein TDP-43-GFP. Yeast were grown in successive batch cultivations and were observed using a fluorescence microscope at different stages of growth. At the same time, fluorescence intensity was measured to indicate the expression of the inserted human TDP-43-GFP gene, biomass yield for the effect of aggregates on cell growth and reproduction, and cellular metabolic activity as a reflection of energy needs for cellular stress response to the presence of aggregates. Experiments have revealed the stress effect of aggregates on cells and their response. Interestingly, the culture initially cultured in expression and then in the repression medium and yet again re-cultured in the expression medium was better prepared for aggregation stress after 8 hours of cultivation, which was reflected in lower gene expression intensity, higher biomass yield, and increased initial cellular metabolism. Our results could significantly help to understand the development of ALS in the formation and removal of TDP-43 protein aggregates

Life Cycle Assessment of Pilot-Scale Bio-Refining of Invasive Japanese Knotweed Alien Plant towards Bio-Based Bioactive Compounds

Japanese knotweed is an invasive alien plant species with characteristic rapid expansion in Europe and North America and resistance to extermination. It displaces autochthonous biodiversity and causes major damage to infrastructure, thus causing global ecological and economic damage. The Japanese knotweed plant is usually eradicated using various chemical, biological, or mechanical techniques, which at a large scale include heavy equipment, usually followed by incineration. Therefore, excavation is preferred to eradication techniques, and as a biomass waste recovery method due to the extraction of high-value biocompounds. This is supported by the fact that the Japanese knotweed possesses various bioactive compounds with beneficial effects on human health. Its rhizome bark extract produces strong and stable antioxidant activity over time, as well as apoptotic, antibacterial, and other beneficial activities. In this work, an environmental impact assessment, including greenhouse gas footprint, acidification, eutrophication, and ecotoxicity for extraction route of the Japanese knotweed rhizome bark, is performed. A comparative case study between the lab-based and proposed pilot-scale production of active added-value extract was evaluated. The results show the pilot-scale production exhibits lower environmental burdens, mainly due to greater electricity requirements for the lab-scale alternative

Antioxidant and antimicrobial biofoil based on chitosan and Japanese knotweed (Fallopia japonica, Houtt.)

A 70% ethanol(aq) extract of the rhizome bark of the invasive alien plant species Japanese knotweed (JKRB) with potent (in the range of vitamin C) and stable antioxidant activity was incorporated in 1% w/v into a chitosan biofoil, which was then characterized on a lab-scale. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay confirmed the antioxidant activity of the JKRB biofoil upon contact with the food simulants A, B, C, and D1 (measured half-maximal inhibitory concentrations—IC50) and supported the Folin–Ciocalteu assay result. The migration of the antioxidant marker, (−)-epicatechin, into all food simulants (A, B, C, D1, D2, and E) was quantified using liquid chromatography hyphenated to mass spectrometry (LC-MS). Calculations showed that 1 cm2 of JKRB biofoil provided antioxidant activity to ~0.5 L of liquid food upon 1 h of contact. The JKRB biofoil demonstrated antimicrobial activity against Gram-positive bacteria. The incorporation of JKRB into the chitosan biofoil resulted in improved tensile strength from 0.75 MPa to 1.81 MPa, while elongation decreased to 28%. JKRB biofoil’s lower moisture content compared to chitosan biofoil was attributed to the formation of hydrogen bonds between chitosan biofoil and JKRB compounds, further confirmed with attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The JKRB biofoil completely degraded in compost in 11 days. The future upscaled production of JKRB biofoil from biowastes for active packaging may support the fights against plastic waste, food waste, and the invasiveness of Japanese knotweed, while greatly contributing to the so-called ‘zero-waste’ strategy and the reduction in greenhouse gas emissions

The role of luxS in Campylobacter jejuni beyond intercellular signaling

The full role of the luxS gene in the biological processes, such as essential amino acid synthesis, nitrogen and pyruvate metabolism, and flagellar assembly, of Campylobacter jejuni has not been clearly described to date. Therefore, in this study, we used a comprehensive approach at the cellular and molecular levels, including transcriptomics and proteomics, to investigate the key role of the luxS gene and compared C. jejuni 11168ΔluxS (luxS mutant) and C. jejuni NCTC 11168 (wild type) strains. Transcriptomic analysis of the luxS mutant grown under optimal conditions revealed upregulation of luxS mutant metabolic pathways when normalized to wild type, including oxidative phosphorylation, carbon metabolism, citrate cycle, biosynthesis of secondary metabolites, and biosynthesis of various essential amino acids. Interestingly, induction of these metabolic pathways was also confirmed by proteomic analysis, indicating their important role in energy production and the growth of C. jejuni. In addition, genes important for the stress response of C. jejuni, including nutrient starvation and oxidative stress, were upregulated. This was also evident in the better survival of the luxS mutant under starvation conditions than the wild type. At the molecular level, we confirmed that metabolic pathways were upregulated under optimal conditions in the luxS mutant, including those important for the biosynthesis of several essential amino acids. This also modulated the utilization of various carbon and nitrogen sources, as determined by Biolog phenotype microarray analysis. In summary, transcriptomic and proteomic analysis revealed key biological differences in tricarboxylic acid (TCA) cycle, pyruvate, nitrogen, and thiamine metabolism as well as lipopolysaccharide biosynthesis in the luxS mutant

DataSheet_1_Modulation of Campylobacter jejuni adhesion to biotic model surfaces by fungal lectins and protease inhibitors.docx

Campylobacter jejuni, a Gram-negative bacterium, is one of the most common causes of foodborne illness worldwide. Its adhesion mechanism is mediated by several bacterial factors, including flagellum, protein adhesins, lipooligosaccharides, proteases, and host factors, such as surface glycans on epithelial cells and mucins. Fungal lectins, specialized carbohydrate-binding proteins, can bind to specific glycans on host and bacterial cells and thus influence pathogenesis. In this study, we investigated the effects of fungal lectins and protease inhibitors on the adhesion of C. jejuni to model biotic surfaces (mucin, fibronectin, and collagen) and Caco-2 cells as well as the invasion of Caco-2 cells. The lectins Marasmius oreades agglutinin (MOA) and Laccaria bicolor tectonin 2 (Tec2) showed remarkable efficacy in all experiments. In addition, different pre-incubations of lectins with C. jejuni or Caco-2 cells significantly inhibited the ability of C. jejuni to adhere to and invade Caco-2 cells, but to varying degrees. Pre-incubation of Caco-2 cells with selected lectins reduced the number of invasive C. jejuni cells the most, while simultaneous incubation showed the greatest reduction in adherent C. jejuni cells. These results suggest that fungal lectins are a promising tool for the prevention and treatment of C. jejuni infections. Furthermore, this study highlights the potential of fungi as a rich reservoir for novel anti-adhesive agents.</p