Delivery Of Heterologous Proteins Research Articles

Bacillus subtilis spores displaying RBD domain of SARS-CoV-2 spike protein

Bacillus subtilis spores are considered to be efficient and useful vehicles for the surface display and delivery of heterologous proteins. In this study, we prepared recombinant spores with the receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein displayed on their surface in fusion with the CotZ or CotY spore coat proteins as a possible tool for the development of an oral vaccine against the SARS-CoV-2 virus. The RBD was attached to the N-terminus or C-terminus of the coat proteins. We also directly adsorbed non-recombinantly produced RBD to the spore surface. SDS-PAGE, western blot and fluorescence microscopy were used to analyze RBD surface expression on purified spores. Results obtained from both display systems, recombinant and non-recombinant, demonstrated that RBD was present on the spore surfaces.

Delivery of Heterologous Proteins, Enzymes, and Antigens via the Bacterial Type III Secretion System.

The Type III Secretion System (T3SS) is a multimeric protein complex composed of over 20 different proteins, utilized by Gram-negative bacteria to infect eukaryotic host cells. The T3SS has been implicated as a virulence factor by which pathogens cause infection and has recently been characterized as a communication tool between bacteria and plant cells in the rhizosphere. The T3SS has been repurposed to be used as a tool for the delivery of non-native or heterologous proteins to eukaryotic cells or the extracellular space for a variety of purposes, including drug discovery and drug delivery. This review covers the methodology of heterologous protein secretion as well as multiple cases of utilizing the T3SS to deliver heterologous proteins or artificial materials. The research covered in this review will serve to outline the scope and limitations of utilizing the T3SS as a tool for protein delivery.

A new lactobacilli in vivo expression system for the production and delivery of heterologous proteins at mucosal surfaces.

Food-grade lactic acid bacteria, such as lactobacilli, represent good candidates for the development of mucosal vectors. Indeed, they are generally recognized as safe microorganisms and some strains display beneficial effects (probiotics). In this study, we described a new lactobacilli in vivo expression (LIVE) system for the production and delivery of therapeutic molecules at mucosal surfaces. The versatility and functionality of this system was successfully validated in several lactobacilli species; furthermore, we assessed in vivo LIVE system in two different mouse models of human pathologies: (i) a model of therapy against intestinal inflammation (inflammatory bowel diseases) and (ii) a model of vaccination against dental caries. We demonstrated that Lactobacillus gasseri expressing the anti-inflammatory cytokine IL-10 under LIVE system efficiently delivered the recombinant protein at mucosal surfaces and display anti-inflammatory effects. In the vaccination model against caries, LIVE system allowed the heterologous expression of Streptococcus mutans antigen GbpB by L. gasseri, leading to a stimulation of the host immune response.

Immunomodulation using genetically engineered bacteria for type III-mediated delivery of heterologous antigens and cytokines: Potential application in vaccine and therapeutical developments

The rational design of new approaches aiming to improve antigen or drug delivery remains a major challenge in numerous vaccine and therapeutical strategies. Studies on genetically engineered bacteria have led to significant progress in the delivery of heterologous proteins. Yet, the efficiency of such recombinant bacteria relies particularly on their ability to deliver sufficient amount of the drug to intended cell tissues. Gram-negative bacteria like Salmonella, Shigella, Yersinia and Pseudomonas have evolved powerful secretion machinery called type III secretion system (T3SS) that is used to optimize their virulence effect by delivering bacterial effectors to the membrane or into the host cell cytoplasm. This review highlights recent studies unraveling whether T3SS of attenuated bacteria could be an efficient route for delivery of vaccine antigens or bioactive proteins such as cytokines for immunomodulation that could find a broad range of application purposes. These current experimental investigations can be considered proofs of concept that need to be translated readily to effective vaccines and therapeutics in humans.

Microencapsulated Engineered Lactococcus lactis Cells for Heterologous Protein Delivery: Preparation and In Vitro Analysis

This article demonstrates the potential of encapsulated, engineered Lactococcus lactis as a vehicle for the oral delivery of therapeutic proteins. Using alginate-poly-l-lysine-alginate membrane-encapsulated L. lactis engineered to secrete the reporter protein Staphylococcal aureus nuclease, we show comparable viability and protein secretion between free and immobilized cells. After 12 h, microcapsules with a cell density of 4.8 x 10(5) colony forming unit (CFU) ml(-1) grew to 2.2 x 10(8) CFU ml(-1) and released 0.24 arbitrary unit (AU) ml(-1) of nuclease, producing similar results as free cells, which grew from 3.4 x 10(5) to 1.9 x 10(8) CFU ml(-1) and secreted 0.21 AU ml(-1) of nuclease. Moreover, encapsulated cells at a density of 4.4 x 10(7) CFU ml(-1) grew to 2.2 x 10(10) CFU ml(-1) in 12 h and secreted 15.3 AU ml(-1) of nuclease although 3.1 x 10(7) CFU ml(-1)of free cells reached only 2.3 x 10(9) CFU ml(-1) and released 5.6 AU ml(-1) of nuclease. We also show the sustained stability of the microcapsules during storage at 4 degrees C over 8 weeks.

유산균을 이용한 겸구용 항원 단백질 수송능 연구

A promising application of Lactococcus lactis is its use as live vehicles for production and delivery of heterologous proteins of vaccines and therapeutic substances. Because L. lactis has GRAS ('generally regarded as safe') status, we tested whether L. lactis could function as the carrier of the Ll protein of human papillomavirus (HPV) type 16. The RNA level expression of Ll gene was detected in L. Lactis. The Ll protein was expressed in L. lactis with Ll gene. The growth of strains L. lactis with an empty plasmid (pAMJ328) and L. lactis with Ll-encoding plasmid (pAMJ328-Ll) was slightly decreased in comparison with the growth of strains L. lactis (wild type). However, all the three strains of L. lactis maintained the ability to ferment sugars primarily into lactic acid, indicating that Ll protein did not affect the biochemical property of L. lactis. These results suggest that L. lactis, capable of carrying Ll protein, might be further developed as a biocompatible oral protein delivery system.

Recombinant Streptococcus gordonii for Mucosal Delivery of a scFv Microbicidal Antibody

The gram-positive bacterium Streptococcus gordonii was engineered to express the microbicidal molecule H6, which is an antiidiotypic single chain antibody mimicking a yeast killer toxin. S. gordonii is a human commensal which we developed as a model system for mucosal delivery of heterologous proteins. The in vivo candidacidal activity of both H6-secreting and H6-surface-displaying streptococcal strains were assayed in a well-established rat model of vaginal candidiasis. At day 21 full clearance of Candida albicans infection was observed in 75% of animals treated with the H6-secreting strain, and in 37.5% of animals treated with the strain expressing H6 on the surface, while all animals treated with the control strain were still infected. The observed candidacidal effect was comparable with that observed with the antimycotic drug fluconazole. These data confirm the potential of H6 as a candidacidal agent and show how promising is the approach of using recombinant bacteria for mucosal delivery of biologically active molecules.