Aleš Berlec

Introduction: Lactic acid bacteria are commonly used to convert carbohydrates into valuable products like lactic acid, but they currently rely on carbon sources from food crops, raising sustainability and cost concerns. Lignocellulosic waste is a more sustainable alternative, yet these bacteria cannot naturally break down cellulose to grow on it. Efficient cellulose degradation requires synergetic action of multiple cellulolytic enzymes, each operating through distinct mechanisms. Methods: To design genetic constructs for co-expression of gene pairs encoding different heterologous cellulases (Cel5I, Cel5H or Cel9A) from separate expression cassettes in Lactococcus cremoris , the BglBrick approach was used. The activity of culture supernatants containing co-expressed cellulases was assessed on microcrystalline cellulose and phosphoric acid swollen cellulose (PASC), and compared with that of co-cultures and cultures expressing individual enzymes. Subsequently, growth of selected developed strains on PASC was evaluated. Results: The A1 strain that co-produced cellulase pair Cel5H-Cel9A achieved the highest activity on PASC and one of the highest activities measured on microcrystalline cellulose. Additionally, the A1 strain showed the highest degradation of PASC during the 16 days of anaerobic cultivation and one of the highest lactic acid production levels. Discussion: In this study, co-expression of gene pairs that encode different heterologous cellulases from separate promoters in L. cremoris was achieved for the first time. This approach allows each cellulase to be tuned independently, enabling optimization of their expression levels to maximize synergistic effects and enhance the cellulolytic performance of the engineered bacteria.

Campylobacter jejuni, the most common foodborne pathogen, relies on adhesion, invasion, and biofilm formation for successful virulence and persistence in the food-processing environments. In this study, we investigated the effects of the transient bacterium with probiotic properties Lactococcus cremoris on C. jejuni virulence via an adhesion and invasion assay using human colon adenocarcinoma Caco-2 cells, culturability via colony-forming units, and proteomic adaptation in co-culture via label-free quantitative mass spectrometry. Caco-2 cell adhesion and invasion assay revealed that L. cremoris decreases C. jejuni adhesion (by 50%) and invasion (by 92%), particularly when inoculated prior to the pathogen. In mature (48 h old) dual-species biofilms, C. jejuni culturability decreased by 88%, while L. cremoris culturability remained unaffected, compared to single-species biofilms. Proteomic analysis of biofilm cells showed metabolic reprogramming in C. jejuni, with increased levels of proteins related to the TCA cycle, stress response, amino acid and nucleotide metabolism, chemotaxis, and energy production. The most upregulated proteins included the tungsten transporter TupA, LuxS, Dps, and hydrogenase subunits HydAB, indicating adaptations to nutrient limitation and oxidative stress. Nevertheless, the decreased culturability of C. jejuni suggests it is overwhelmed under the competitive conditions of dual-species biofilms. Our findings demonstrate that L. cremoris exerts strong antagonistic effects on C. jejuni, restricting its virulence, and triggering significant metabolic shifts in dual-species biofilms. These results highlight the potential of L. cremoris as a preventative strategy against C. jejuni adhesion, colonisation, and transmission.

Background: Genetically modified Lactococcus cremoris strains producing heterologous cellulases emerge as promising candidates for cost-effective bioprocessing of plant biowaste into high-value organic compounds. A key challenge in enabling efficient cellulose degradation by L. cremoris remains the effective secretion of heterologous cellulases. A potential solution would be to use genetic engineering to enhance the permeabilization of bacterial cell membrane and cell wall to facilitate the release of cellulases. This could be achieved by the co-expression of cellulase genes with genes encoding the prophage lytic proteins such as holin and endolysin. Results: Co-expression of genes encoding the prophage protein holin and heterologous cellulases Cel5I, Cel9A and Cel5H in L. cremoris significantly improved secretion of Cel5I (up to 15-fold) and Cel9A (at least 3-fold), as well as surface display of Cel5I, Cel5H and Cel9A. Comparable levels of secretion and surface display of cellulases were observed regardless of whether the holin gene was co-expressed from a plasmid or inserted into the genome. In contrast, co-expression of gene encoding the prophage protein endolysin did not improve secretion of cellulase even when produced simultaneously with its partner protein holin. Conclusions: Co-production of holin with heterologous cellulases appears as a promising strategy for improving cellulolytic capability of L. cremoris by increasing their secretion. This might advance the development of consolidated bioprocesses involving lactic acid bacteria for valorisation of cellulosic material. Additionally, the same approach might likewise be employed to enhance the secretion and surface display of other heterologous recombinant proteins.

Biofilms, structured communities of microbial cells embedded in extracellular polymeric substances, are notorious for their resilience against conventional antimicrobial treatments. They contribute significantly to chronic infections and industrial biofouling, necessitating innovative strategies for their eradication. Magnetic iron oxide nanoparticles have emerged as a promising tool in combating biofilms due to their biocompatibility and unique physicochemical properties, which enable magnetic delivery of antibacterial agents, magnetic hyperthermia, magneto-mechanical actuation including mechanical biofilm disruption, and reversible dynamic magnetic assembly into hierarchical structures. This review describes developing stages of magnetic nanoscale weapons against biofilms ranging from individual iron oxide nanoparticles to complex hierarchical nanoparticle assemblies in the form of magnetic robots and their swarms. A vast array of possible antibiofilm and antibacterial functionalities originating from iron ions, individual iron oxide nanoparticles, spherical nanoparticle assemblies, magnetic robots, and swarms of robots are presented. Magnetic nanotools offer significant improvements and advantages over conventional methods for biofilm eradication, yet their successful future applications depend on addressing and overcoming critical material, biological, and engineering challenges.

Bacterial biofilms attach to various surfaces and represent an important clinical and public health problem, as they are highly recalcitrant and are often associated with chronic, nonhealing diseases and healthcare-associated infections. Antibacterial agents are often not sufficient for their elimination and have to be combined with mechanical removal. Mechanical forces can be generated by actuating nonspherical (anisotropic) magnetically responsive nanoparticles in a rotating magnetic field. We have thus prepared anisotropic superparamagnetic nanochains in the size range of 0.5-1 μm by magnetically assembling several iron oxide nanoparticle clusters and coating them with a layer of silica with different shell morphologies: smooth, moderately rough, and highly rough. The silica surface was additionally functionalized with carboxylic groups to increase colloidal stability. The efficacy of the nanochains in biofilm removal was studied systematically with three different model nonpathogenic bacterial species Escherichia coli, Lactococcus lactis, and Pseudomonas fragi; two different magnetic field strengths; two stirring speeds; and two treatment durations. All bacterial species were engineered to express fluorescent proteins to enable quantification of biofilm removal by colony-forming unit count and fluorescence measurements. Nanochains removed >90% of Gram-negative E. coli and P. fragi with a stronger magnetic field, and L. lactis with a weaker magnetic field. Surface roughness of nanochains, duration, and stirring speed also affected removal, but the effect could not be generalized. In contrast to their effects on biofilms, the functionalized nanochains showed no toxicity to Caco-2 intestinal epithelial cells, regardless of whether magnetomechanical force was employed or not. In summary, we demonstrated that remotely controlled spatial movement of nanoparticles can generate sufficient mechanical forces to disperse attached biofilms while retaining safety in an epithelial cell model.

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.

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.