1. Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signalingAbida Zahirović, Špela Zupančič, Andraž Verdir, Sebastjan Nemec, Slavko Kralj, Luka Snoj, Aleš Berlec, 2025, pregledni znanstveni članek Povzetek: Introduction: With growing evidence of clinical efficacy of probiotics in various diseases, safety concerns have arisen regarding the therapeutic use of live probiotic bacteria, especially in critically ill, immunocompromised, and pediatric populations. Serious probiotic-related adverse effects have been reported in these patients, including bloodstream infection and sepsis. This has led to an increased interest in developing postbiotics (non-viable bacterial products) that may exert beneficial effects on the host without the risks associated with administration of live microorganisms. The aim of this study was to explore postbiotic potential of recombinant Lactococcus lactis bacteria that have been engineered to display interleukin 6 (IL6)-targeting affibody (ZIL6) on their surface and are intended for treatment of inflammatory intestinal diseases. Methods: Five different killing treatments were applied to kill bacteria (heat, ethanol, sonication, UV, and gamma irradiation) and their effect on bacterial viability, morphology and functionality was examined in vitro using a combination of different techniques, including microscopy, flow cytometry, immunoassays and cell-based reporter assay. Results: The results showed that ZIL6 affibody displayed on L. lactis via non-covalent anchoring withstood the treatments applied to kill bacteria and remained functional after the loss of microbial viability. The degree of functionality was dependent on the type of treatment. Heat-killed cells retained 50% of the activity of live strain, while most of the activity was preserved after exposure of bacteria to ethanol, sonication, UV and gamma irradiation. The applied treatments varied in killing efficacy, whereby ethanol and heat rendered bacteria non-viable, UV and gamma irradiation yielded non-replicative cells, whereas sonication was ineffective in killing L. lactis. Among non-viable cells, ethanol-killed bacteria exhibited the greatest activity and showed high maximum binding capacity of 200 ng IL6 per mg dry cell weight, possessed strong nanomolar affinity for IL6, and inhibited up to 78% of IL6-induced STAT3 signaling. Conclusion: The study demonstrates that functional non-viable bacterial cells can be derived from the recombinant L. lactis with therapeutic proteins displayed on their surface and provides a good foundations for further studies of their postbiotic potential in adjunctive therapy of inflammatory intestinal diseases.
Ključne besede: affibody, probiotics, postbiotics, Lactococcus lactis
Objavljeno v DiRROS: 03.11.2025; Ogledov: 186; Prenosov: 95
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2. Modified vaginal lactobacilli expressing fluorescent and luminescent proteins for more effective monitoring of their release from nanofibers, safety and cell adhesionSpase Stojanov, Tina Vida Plavec, Špela Zupančič, Aleš Berlec, 2024, izvirni znanstveni članek Povzetek: Electrospun nanofibers offer a highly promising platform for the delivery of vaginal lactobacilli, providing an innovative approach to preventing and treating vaginal infections. To advance the application of nanofibers for the delivery of lactobacilli, tools for studying their safety and efficacy in vitro need to be established. In this study, fluorescent (mCherry and GFP) and luminescent (NanoLuc luciferase) proteins were expressed in three vaginal lactobacilli (Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus jensenii) and a control Lactiplantibacillus plantarum with the aim to use this technology for close tracking of lactobacilli release from nanofibers and their adhesion on epithelial cells. The recombinant proteins influenced the growth of the bacteria, but not their ability to produce hydrogen peroxide. Survival of lactobacilli in nanofibers immediately after electrospinning varied among species. Bacteria retained fluorescence upon incorporation into PEO nanofibers, which was vital for evaluation of their rapid release. In addition, fluorescent labelling facilitated efficient tracking of bacterial adhesion to Caco-2 epithelial cells, while luminescence provided important quantitative insights into bacterial attachment, which varied from 0.5 to 50% depending on the species. The four lactobacilli in dispersion or in nanofibers were not detrimental for the viability of Caco-2 cells, and did not demonstrate hemolytic activity highlighting the safety profiles of both bacteria and PEO nanofibers. To summarize, this study contributes to the development of a promising delivery system, tailored for local administration of safe vaginal lactobacilli.
Objavljeno v DiRROS: 17.01.2025; Ogledov: 590; Prenosov: 210
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3. Incorporation of recombinant proteins into extracellular vesicles by Lactococcus cremorisTina Vida Plavec, Kristina Žagar, Giulia Della Pelle, Špela Zupančič, Robert Vidmar, Aleš Berlec, 2025, izvirni znanstveni članek Povzetek: Extracellular vesicles (EVs) are nanosized lipid bilayer particles released by various cellular organisms that carry an array of bioactive molecules. EVs have diagnostic potential, as they play a role in intercellular interspecies communication, and could be applied in drug delivery. In contrast to mammalian cell-derived EVs, the study of EVs from bacteria, particularly Gram-positive bacteria, received less research attention. This study aimed to investigate the production of EVs by lactic acid bacterium Lactococcus cremoris NZ9000 and to examine the impact of recombinant protein expression on their formation and protein content. Four different recombinant proteins were expressed in L. cremoris NZ9000, in different forms of expression and combinations, and the produced EVs were isolated using the standard ultracentrifugation method. The presence of vesicular structures (50–200 nm) in the samples was confirmed by transmission electron microscopy and by flow cytometry using membrane-specific stain. Mass spectrometry analyses confirmed the presence of recombinant proteins in the EVs fraction, with amounts ranging from 13.17 to 100%, highlighting their significant incorporation into the vesicles, together with intrinsic L. cremoris NZ9000 proteins that were either more abundant in the cytoplasm (ribosomal proteins, metabolic enzymes) or present in the membrane. The presence of the most abundant lactococcal proteins in EVs fraction suggests that protein cargo-loading of EVs in L. cremoris NZ9000 is not regulated. However, our data suggests that L. cremoris NZ9000 genetically engineered to express recombinant proteins can produce EVs containing these proteins in scalable manner. As L. cremoris NZ9000 is considered safe bacterium, EVs from L. cremoris NZ9000 could have several advantages over EVs from other bacteria, implying possible biotechnological applications, e.g. in therapeutic protein delivery.
Ključne besede: Lactococcus cremoris, extracellular vesicles, recombinant proteins, delivery vehicle
Objavljeno v DiRROS: 16.01.2025; Ogledov: 823; Prenosov: 386
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