Designing a Microbial Prolyl Peptidase Delivery System for the Treatment of Celiac Disease

Abstract

Indigestible gluten-derived peptides rich in proline residues are known to be responsible for eliciting the inappropriate immune response characteristic of the disease. In this investigation, surface level expression of prolyl peptidase activity by genetically engineered probiotic lactobacilli was postulated to be a possible treatment for this disease. Plasmid-based reporter vectors were constructed utilizing a novel, homologybased cloning technique to assess the expression and localization signals from the S-layer protein gene of Lactobacillus acidophilus. These plasmids were mutated during construction due to toxicity associated with the cloned cassettes. The toxicity of the Slayer secretion and/or anchoring domains in E. coli was confirmed by cloning the fused components into an inducible expression system. When the prolyl peptidase, Xaa-Pro, from L. reuteri was incorporated into the S-layer expression cassette, the full-length protein was efficiently expressed but was not active, likely due to protein aggregation and inclusion body formation. Future research directions are discussed and a modified experimental design strategy is presented. This work provides a foundation for continued investigation into the feasibility of utilizing genetically engineered lactobacilli as a potential treatment strategy for celiac disease.v ACKNOWLEDGMENTS

Development of a Novel Plasmid-based Gene Integration System for Lactobacillus reuteri for the Persistent Treatment of Celiac Disease

“…the SlpA expression vector developed by Matthew Shurtluff was used. Primer sequences are defined in [65]. Briefly, Pro-Dic-F and ProL-R were used to amplify up the Slp-A promoter and leader(PL) with an expected size of 375 bp.…”

“…Two hypotheses were proposed to explain the loss of toxicity: 1) the inclusion bodies were no longer capable of exerting the toxic effect, or 2) the addition of the peptidase may cause a conformational change in the Slp-A leader sequence or the truncated anchor sequence which negates the toxicity. For a more complete explanation of the Slp-A gene structure and the difficulties in protein expression using Slp-A genetic elements, see the Masters Thesis of Matthew Shurtleff [65].…”

“…The resulting amplicon was 2,573 bp long. To add the Slp-A terminator to the P-Clp-P expression system, an existing plasmid pGK (MCS)PGT [65] …”

“…The lab has a pET30-GFP plasmid built with AscI sites flanking the ORF of GFP which is in frame with the C terminal His-Tag of the plasmid. For the construction of pET30-GFP see [65]. GusA was inserted into pET30 by replacing the GFP coding region with GusA at the AscI sites.…”

“…When construction of the integration plasmid started, we planned on using the SlpA expression system to drive peptidase expression. Therefore, constructs were built using PLST (from Slp-A) as inserts to blunt end clone into pCR4 [65]. PLST was used as a place holder in the TOPO Cloning site while the remaining parts of the integration vector were inserted into pCR4.…”