Effects of Electrospinning on the Viability of Ten Species of Lactic Acid Bacteria in Poly(Ethylene Oxide) Nanofibers.

Lactic acid bacteria can have beneficial health effects and be used for the treatment of various diseases. However, there remains the challenge of encapsulating probiotics into delivery systems with a high viability and encapsulation efficacy. The electrospinning of bacteria is a novel and little-studied method, and further investigation of its promising potential is needed. Here, the morphology, zeta potential, hydrophobicity, average cell mass, and growth characteristics of nine different species of Lactobacillus and one of Lactococcus are characterized. The electrospinning of polymer solutions containing ~10 log colony forming units (CFU)/mL lactic acid bacteria enabled the successful incorporation of all bacterial species tested, from the smallest (0.74 µm; Lactococcus lactis) to the largest (10.82 µm; Lactobacillus delbrueckii ssp. bulgaricus), into poly(ethylene oxide) nanofibers with an average diameter of ~100 nm. All of these lactobacilli were viable after incorporation into nanofibers, with 0 to 3 log CFU/mg loss in viability, depending on the species. Viability correlated with the hydrophobicity and extreme length of lactic acid bacteria, whereas a horizonal or vertical electrospinning set-up did not have any role. Therefore, electrospinning represents a promising method for the incorporation of lactic acid bacteria into solid delivery systems, while drying the bacterial dispersion at the same time.

Development of electrospun nanofibers that enable high loading and long-term viability of probiotics.

The interest in probiotics has grown in recent years due to increased awareness of the importance of microbiota for human health. We present the development of monolithic poly(ethylene oxide) and composite poly(ethylene oxide)/lyoprotectant nanofibers loaded with the probiotic Lactobacillus plantarum ATCC 8014. High loading was achieved for L. plantarum cells (up to 7.6 × 108 colony-forming unit/mg) that were either unmodified or expressing mCherry fluorescent protein. The initial concentration of L. plantarum in poly(ethylene oxide) solution was reported, for the first time, as the most critical parameter for its high viability after electrospinning, whereas the applied electric voltage and relative humidity during electrospinning did not vitally impact upon L. plantarum viability. The presence of amorphous lyoprotectant (especially trehalose) in the nanofibers promoted L. plantarum survival due to lyoprotectant interactions with L. plantarum cells. L. plantarum cells in nanofibers were stable over 24 weeks at low temperature, thereby achieving stability comparable with that in lyophilizates. The poly(ethylene oxide) nanofibers released almost all of the L. plantarum cells over 30 min, which will be adequate for their local administration. Our integrated approach enabled development of a promising nanodelivery system that provides high loading and long-term viability of L. plantarum in nanofibers, for local delivery to re-establish the microbiota balance e.g. in vagina.

Engineering recombinant Lactococcus lactis as a delivery vehicle for BPC-157 peptide with antioxidant activities.

Lactic acid bacteria (LAB) are attractive hosts for the expression of heterologous proteins and can be engineered to deliver therapeutic proteins or peptides to mucosal surfaces. The gastric stable pentadecapeptide BPC-157 is able to prevent and treat gastrointestinal inflammation by reducing the production of reactive oxygen species (ROS). In this study, we used LAB Lactococcus lactis as a vector to deliver BPC-157 by surface display and trypsin shedding or by secretion to the growth medium. Surface display of BPC-157 was achieved by fusing it with basic membrane protein A (BmpA) or with the peptidoglycan binding domain of AcmA and Usp45 secretion signal. While the expression of BmpA-fusion proteins was higher than that of AcmA/Usp45-fusion protein, the surface display ability of BPC-157 was approximately 14-fold higher with AcmA/Usp45-fusion protein. Release of BPC-157 from the bacterial surface or from isolated fusion proteins by trypsinization was demonstrated with anti-BPC-157 antibodies or by mass spectrometry. The concentration of BPC-157 delivered by surface display via AcmA/Usp45-fusion was 30 ng/ml. This increased to 117 ng/ml by Usp45 signal-mediated secretion, making the latter the most effective lactococcal delivery approach for BPC-157. Secreted BPC-157 significantly decreased ROS production in 149BR fibroblast cell model, suggesting its potential benefit in the treatment of intestinal inflammations. Additionally, a comparison of different modes of small peptide delivery by L. lactis, performed in the present study, will facilitate the future use of L. lactis as peptide delivery vehicle.

p19-Targeting ILP Protein Blockers of IL-23/Th-17 Pro-Inflammatory Axis Displayed on Engineered Bacteria of Food Origin.

IL-23-mediated Th-17 cell activation and stimulation of IL-17-driven pro-inflammatory axis has been associated with autoimmunity disorders such as Inflammatory Bowel Disease (IBD) or Crohn's Disease (CD). Recently we developed a unique class of IL-23-specific protein blockers, called ILP binding proteins that inhibit binding of IL-23 to its cognate cell-surface receptor (IL-23R) and exhibit immunosuppressive effect on human primary blood leukocytes ex vivo. In this study, we aimed to generate a recombinant Lactococcus lactis strain which could serve as in vivo producer/secretor of IL-23 protein blockers into the gut. To achieve this goal, we introduced ILP030, ILP317 and ILP323 cDNA sequences into expression plasmid vector containing USP45 secretion signal, FLAG sequence consensus and LysM-containing cA surface anchor (AcmA) ensuring cell-surface peptidoglycan anchoring. We demonstrate that all ILP variants are expressed in L. lactis cells, efficiently transported and secreted from the cell and displayed on the bacterial surface. The binding function of AcmA-immobilized ILP proteins is documented by interaction with a recombinant p19 protein, alpha subunit of human IL-23, which was assembled in the form of a fusion with Thioredoxin A. ILP317 variant exhibits the best binding to the human IL-23 cytokine, as demonstrated for particular L.lactis-ILP recombinant variants by Enzyme-Linked ImmunoSorbent Assay (ELISA). We conclude that novel recombinant ILP-secreting L. lactis strains were developed that might be useful for further in vivo studies of IL-23-mediated inflammation on animal model of experimentally-induced colitis.

Single plasmid systems for inducible dual protein expression and for CRISPR-Cas9/CRISPRi gene regulation in lactic acid bacterium Lactococcus lactis.

Lactococcus lactis is a food-grade lactic acid bacterium that is used in the dairy industry as a cell factory and as a host for recombinant protein expression. The nisin-controlled inducible expression (NICE) system is frequently applied in L. lactis; however new tools for its genetic modification are highly desirable. In this work NICE was adapted for dual protein expression. Plasmid pNZDual, that contains two nisin promoters and multiple cloning sites (MCSs), and pNZPolycist, that contains a single nisin promoter and two MCSs separated by the ribosome binding site, were constructed. Genes for the infrared fluorescent protein and for the human IgG-binding DARPin were cloned in all possible combinations to assess the protein yield. The dual promoter plasmid pNZDual enabled balanced expression of the two model proteins. It was exploited for the development of a single-plasmid inducible CRISPR-Cas9 system (pNZCRISPR) by using a nisin promoter, first to drive Cas9 expression and, secondly, to drive single guide RNA transcription. sgRNAs against htrA and ermR directed Cas9 against genomic or plasmid DNA and caused changes in bacterial growth and survival. Replacing Cas9 by dCas9 enabled CRISPR interference-mediated silencing of the upp gene. The present study introduces a new series of plasmids for advanced genetic modification of lactic acid bacterium L. lactis.

Evasin-displaying lactic acid bacteria bind different chemokines and neutralize CXCL8 production in Caco-2 cells.

Chemokines are key signals in the immune system and play an important role as proinflammatory mediators in the pathology of inflammatory bowel disease and colorectal cancer, making them an important target for therapy. Recombinant lactic acid bacteria (LAB) were engineered to bind CC and CXC chemokines by displaying chemokine-binding proteins evasin-1, evasin-3 and evasin-4 on their surface. Evasin genes were cloned into lactococcal surface display vector and overexpressed in L. lactis NZ9000 and NZ9000ΔhtrA in fusion with secretion signal and surface anchor. Evasin-displaying bacteria removed from 15% to 90% of 11 different chemokines from the solution as determined with ELISA and Luminex multiplexing assays, whereby L. lactis NZ9000ΔhtrA proved more efficient. Lactobacillus salivarius ATCC 11741 was coated with L. . lactis-expressed evasin fusion protein, and its ability to bind chemokines was also confirmed. Evasin-3-displaying L. lactis removed 76.0% of IL-1ß-induced CXCL8 from the supernatant of Caco-2 epithelial cells. It also prevented secretion of CXCL8 from Caco-2 cells in a time-dependent manner when added before induction with IL-1ß. Evasin-displaying LAB have the ability to bind multiple chemokines simultaneously and exert synergistic activity. This innovative treatment approach therefore has the potential for mucosal therapy of inflammatory bowel disease or colorectal cancer.

Non-immunoglobulin scaffolds: a focus on their targets.

Non-immunoglobulin (non-Ig) scaffolds are, in contrast to antibodies, small single-domain proteins that require no post-translational modification, often lack disulfide bonds, and can undergo straightforward multimerization. Among the 20 different types of non-Ig scaffolds, Adhirons, Alphabodies, Centyrins, Pronectins, Repebodies, Affimers, and Obodies have been introduced in the past 4 years. 102 proteins have been specifically targeted by 139 different non-Ig scaffold binders. The most frequent application of non-Ig scaffolds is in the treatment and diagnosis of cancer and inflammatory diseases, and 10 non-Ig scaffolds have already been tested in clinical trials. Recently, non-Ig scaffolds have often been used in research as structure determination chaperones, for intracellular monitoring of post-translational modifications, and as antibody alternatives for microscopy, flow cytometry, and Western blotting.