Introduction of Modified BglBrick System in Lactococcus lactis for Straightforward Assembly of Multiple Gene Cassettes

Plavec, Tina Vida; Ključevšek, Tim; Berlec, Aleš

doi:10.3389/fbioe.2021.797521

Cited by 6 publications

(7 citation statements)

References 29 publications

(29 reference statements)

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“…The expression of two or more anti-cytokine modalities on L. lactis can produce an additive or synergistic effect [ 32 ]. To facilitate construction of multifunctional bacteria, we have devised a novel engineering tool (modified Bglbrick system) that enables straightforward cloning and efficient expression of multiple proteins in L. lactis [ 52 ] and can be used for the generation of bacteria that target several cytokines.…”

Section: Discussionmentioning

confidence: 99%

Targeting IL-6 by engineered Lactococcus lactis via surface-displayed affibody

Zahirović

Berlec

2022

Microb Cell Fact

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Background Dysregulated production of interleukin (IL)-6 is implicated in the pathology of inflammatory bowel disease (IBD). Neutralization of IL-6 in the gut by safe probiotic bacteria may help alleviate intestinal inflammation. Here, we developed Lactococcus lactis with potent and selective IL-6 binding activity by displaying IL-6-specific affibody on its surface. Results Anti-IL-6 affibody (designated as ZIL) was expressed in fusion with lactococcal secretion peptide Usp45 and anchoring protein AcmA. A high amount of ZIL fusion protein was detected on bacterial surface, and its functionality was validated by confocal microscopy and flow cytometry. Removal of IL-6 from the surrounding medium by the engineered L. lactis was evaluated using enzyme-linked immunosorbent assay. ZIL-displaying L. lactis sequestered recombinant human IL-6 from the solution in a concentration-dependent manner by up to 99% and showed no binding to other pro-inflammatory cytokines, thus proving to be highly specific for IL-6. The removal was equally efficient across different IL-6 concentrations (150–1200 pg/mL) that were found to be clinically relevant in IBD patients. The ability of engineered bacteria to capture IL-6 from cell culture supernatant was assessed using immunostimulated human monocytic cell lines (THP-1 and U-937) differentiated into macrophage-like cells. ZIL-displaying L. lactis reduced the content of IL-6 in the supernatants of both cell lines in a concentration-dependent manner by up to 94%. Dose response analysis showed that bacterial cell concentrations of 107 and 109 CFU/mL (colony forming units per mL) were required for half-maximal removal of recombinant and macrophage-derived IL-6, respectively. Conclusion The ability of ZIL-displaying L. lactis to bind pathological concentrations of IL-6 at common bacterial doses suggests physiological significance.

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Section: Discussionmentioning

confidence: 99%

Targeting IL-6 by engineered Lactococcus lactis via surface-displayed affibody

Zahirović

Berlec

2022

Microb Cell Fact

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“…Zil gene was cloned into a lactococcal plasmid pSDBA3b in the same way as evasin 3 following the protocol described in Škrlec et al (2017). For visualization of engineered bacteria, IRFP-encoding gene was subsequently inserted into the plasmid along with the protein binders, using BglBrick cloning, which allowed easier assembly of multiple gene cassettes (Plavec et al, 2021a).…”

Section: Construction Of Dual Protein Expression Plasmidsmentioning

confidence: 99%

“…The feasibility of tumor targeting with AFFI co-expressing bacteria was assessed using HEK293 cells transfected with the EpCAM receptor fused to fluorescent protein superfolder GFP (sfGFP). To visualize bacterial adhesion by fluorescence microscopy, we used L. lactis that co-expressed the IRFP together with the tumor antigen binders and cytokine binders (Plavec et al, 2021a). Surface presentation of the protein binders and intracellular IRFP expression were confirmed by flow cytometry and fluorescence measurements, respectively (Plavec et al, 2021a).…”

Section: Lactis Co-expressing Il-6 Binder Zil and The Tumor Antigen Binder Affi Or Zher Removes Il-6 Released By Thp-1 And U-937 Monocytementioning

confidence: 99%

“…To visualize bacterial adhesion by fluorescence microscopy, we used L. lactis that co-expressed the IRFP together with the tumor antigen binders and cytokine binders (Plavec et al, 2021a). Surface presentation of the protein binders and intracellular IRFP expression were confirmed by flow cytometry and fluorescence measurements, respectively (Plavec et al, 2021a). Confocal microscopy showed that the engineered bacteria L. lactis-AFFI-EVA-IRFP and L. lactis-AFFI-ZIL-IRFP strongly and selectively adhered to EpCAMoverexpressing HEK293 cells, in contrast to the control strain L. lactis-IRFP, which showed no adhesion ability (Figure 7A).…”

Section: Lactis Co-expressing Il-6 Binder Zil and The Tumor Antigen Binder Affi Or Zher Removes Il-6 Released By Thp-1 And U-937 Monocytementioning

confidence: 99%

“…The feasibility of tumor targeting with ZHER co-expressing bacteria was investigated using HEK293 cells transfected with HER2 receptor fused to fluorescent protein monovalent variant of GFP mEmerald. The attached bacteria were visualized by confocal fluorescence microscopy via the IRFP reporter protein that was co-expressed in L. lactis with the protein binders (Plavec et al, 2021a). Confocal microscopy showed that the engineered bacteria selectively adhered to HER2-overexpressing HEK293 cells and showed no reactivity towards nontransfected HEK293 cells (Figures 8A,B).…”

Section: Engineered L Lactis Selectively Adhere To Her2-overexpressing Hek293 Cellsmentioning

confidence: 99%

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Dual Functionalized Lactococcus lactis Shows Tumor Antigen Targeting and Cytokine Binding in Vitro

Zahirović

Plavec

Berlec

2022

Front. Bioeng. Biotechnol.

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Pro-inflammatory cytokines play an important role in the development and progression of colorectal cancer (CRC). Tumor-targeting bacteria that can capture pro-inflammatory cytokines in the tumor microenvironment and thus block their tumor-promoting effects might provide clinical benefits in inflammation-associated CRC. The aim of this study was to develop bacteria with dual functionality for selective delivery of cytokine-binding proteins to the tumor by targeting specific receptors on cancer cells. We engineered a model lactic acid bacterium, Lactococcus lactis, to co-display on its surface a protein ligand for tumor antigens (EpCAM-binding affitin; HER2-binding affibody) and a ligand for pro-inflammatory cytokines (IL-8-binding evasin; IL-6-binding affibody). Genes that encoded protein binders were cloned into a lactococcal dual promoter plasmid, and protein co-expression was confirmed by Western blotting. To assess the removal of IL-8 and IL-6 by the engineered bacteria, we established inflammatory cell models by stimulating cytokine secretion in human colon adenocarcinoma cells (Caco-2; HT-29) and monocyte-like cells (THP-1; U-937). The engineered L. lactis removed considerable amounts of IL-8 from the supernatant of Caco-2 and HT-29 cells, and depleted IL-6 from the supernatant of THP-1 and U-937 cells as determined by ELISA. The tumor targeting properties of the engineered bacteria were evaluated in human embryonic kidney epithelial cells HEK293 transfected to overexpress EpCAM or HER2 receptors. Fluorescence microscopy revealed that the engineered L. lactis specifically adhered to transfected HEK293 cells, where the EpCAM-targeting bacteria exhibited greater adhesion efficiency than the HER2-targeting bacteria. These results confirm the concept that L. lactis can be efficiently modified to display two proteins simultaneously on their surface: a tumor antigen binder and a cytokine binder. Both proteins remain biologically active and provide the bacteria with tumor antigen targeting and cytokine binding ability.

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Brucella abortus antigen omp25 vaccines: Development and targeting based on Lactococcus lactis

Gouran,

Doosti,

Jami

2023

Veterinary Medicine & Sci

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Background Most Brucella infections take place on mucosal membranes. Therefore, creating vaccinations delivered through the mucosa may be crucial for managing brucellosis. Consequently, we assessed the efficacy of a recombinant oral antigen delivery system based on Lactococcus lactis for Brucella abortus omp25 antigen. Method Oral vaccinations with L. lactis transformed with pNZ8148 variants encoding for omp25 (pNZ8148:omp25) and free‐pNZ8148 were administered to mice. On day 30, following immunization in animal groups, anti‐omp25‐specific IgG1 antibodies were assessed by the ELISA test. Additionally, nasal and bronchoalveolar lavages containing omp25‐specific secretory IgA (sIgA) were analysed by ELISA. ELISA test and real‐time PCR were also used to analyse cytokine responses up to 28 days following the last boost. In addition, the protective potential of L. lactis pNZ8148:omp25 vaccines was assessed in BALB/c mice by exposing them to the B. abortus strain. Results Based on the initial screening results, the omp25 protein was identified for immunogenicity because it had the maximum solubility and flexibility and antigenic values of 0.75. The produced plasmid was digested using KpnI and XbaI. By electrophoretic isolation of the digestion fragments at 786 bp, the omp25 gene, the successful production of the recombinant plasmid, was confirmed. Antigen expression at the protein level revealed that the target group generated the 25 kDa‐sized omp25 protein, but there was no protein expression in the control group. Fourteen days after priming, there was a considerable amount of omp25‐specific IgG1 in the sera of mice vaccinated with pNZ8148–Usp45–omp25–L. lactis (p < 0.001 in target groups compared to the phosphate‐buffered saline control group). IFN‐γ and TNF‐α levels were more significant in samples from mice that had been given the pNZ8148–Usp45–omp25–L. lactis and IRBA vaccinations, in samples taken on days 14 and 28, respectively (p < 0.001). The pNZ8148–Usp45–omp25–L. lactis and IRBA immunization groups had significantly greater IL‐4 and IL‐10 transcription levels than the other groups. The spleen portions from the pNZ8148–Usp45–omp25–L. lactis and IRIBA vac group had less extensive spleen injuries, alveolar oedema, lymphocyte infiltration and morphological damage due to the inflammatory process. Conclusion Our study offers a novel method for using the food‐grade, non‐pathogenic and noncommercial bacterium L. lactis as a protein cell factory to produce the novel immunogenic fusion candidate romp25. This method offers an appealing new approach to assessing the cost‐effective, safe, sustainable, simple pilot development of pharmaceutical products.

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Introduction of Modified BglBrick System in Lactococcus lactis for Straightforward Assembly of Multiple Gene Cassettes

Cited by 6 publications

References 29 publications

Targeting IL-6 by engineered Lactococcus lactis via surface-displayed affibody

Targeting IL-6 by engineered Lactococcus lactis via surface-displayed affibody

Dual Functionalized Lactococcus lactis Shows Tumor Antigen Targeting and Cytokine Binding in Vitro

Brucella abortus antigen omp25 vaccines: Development and targeting based on Lactococcus lactis

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