Generation of food-grade recombinant Lactobacillus casei delivering Myxococcus xanthus prolyl endopeptidase

Abstract

Prolyl endopeptidases (PEP), a family of serine proteases with the ability to hydrolyze the peptide bond on the carboxyl side of an internal proline residue, are able to degrade immunotoxic peptides responsible for celiac disease (CD), such as a 33-residue gluten peptide (33-mer). Oral administration of PEP has been suggested as a potential therapeutic approach for CD, although delivery of the enzyme to the small intestine requires intrinsic gastric stability or advanced formulation technologies. We have engineered two food-grade Lactobacillus casei strains to deliver PEP in an in vitro model of small intestine environment. One strain secretes PEP into the extracellular medium, whereas the other retains PEP in the intracellular environment. The strain that secretes PEP into the extracellular medium is the most effective to degrade the 33-mer and is resistant to simulated gastrointestinal stress. Our results suggest that in a future, after more studies and clinical trials, an engineered food-grade Lactobacillus strain may be useful as a vector for in situ production of PEP in the upper small intestine of CD patients.

Identification and characterization of intestinal lactobacilli strains capable of degrading immunotoxic peptides present in gluten

“…) and genetically engineered bacteria (Alvarez‐Sieiro et al . ) in that they are naturally adapted to the conditions of the small intestine and therefore are likely more physiologically active and functional. However, ultimately, only clinical trials can determine which strains work best in humans.…”

“…plantarum 299v), that could be readily applied, also degrade the immunotoxic peptides to some extent. However, the four porcine stains identified during our strain selection might have advantages to human colonic (Laparra and Sanz 2010;Caminero et al 2014), oral (Zamakhchari et al 2011;Fernandez-Feo et al 2013) and genetically engineered bacteria (Alvarez-Sieiro et al 2014) in that they are naturally adapted to the conditions of the small intestine and therefore are likely more physiologically active and functional. However, ultimately, only clinical trials can determine which strains work best in humans.…”

Implementation of the agmatine-controlled expression system for inducible gene expression in Lactococcus lactis

“…To produce PEP using the ACE system, the pep gene was PCR-amplified from pNZ8048- pep and cloned into the Nco I- Xba I sites in the pACE vector, resulting in the pACE- pep plasmid. To produce PEP using the NICE system, the pep gene was released from plasmid pUC57- pep [ 36 ] as a Nco I- Xba I fragment and cloned into the same sites in pNZ8048 under the control of the nisA promoter [ 29 ], thus generating plasmid pNZ8048- pep .…”

“…The present work reports the adaptation of the ACE system—an inducible gene expression system that involves aguR and the P aguB promoter (the latter with its natural ribosome binding site)—to L. lactis . This was successfully tested via the production of green fluorescent protein (GFP) and Myxococcus xanthus prolyl-endopeptidase (PEP) [ 35 , 36 ].…”

Celiac Treatments, Adjuvant Therapies and Alternatives to the Gluten-Free Diet

“…A number of studies from different groups have described substantial differences in the intestinal microbiota of patients with CD [55][56][57] . Bifidobacterium longum CECT7347 is a probiotic bacterial strain isolated from a healthy breastfed child with anti-inflammatory effects and proteolytic activity toward gliadin peptides in vitro [58][59][60] 65 . This may raise discussions on the public acceptability of GMO's, despite the fact these have been shown to be safe for administration to mice and humans 66,67 .…”