Expression of a streptococcal glucosyltransferase as a fusion to a solute-binding protein in Lactobacillus fermentum BR11

A locus encoding two repetitive proteins that have LPXTG cell wall anchoring signals from Lactobacillus fermentum BR11 has been identified by using an antiserum raised against whole L. fermentum BR11 cells. The first protein, Rlp, is similar to the Rib surface protein from Streptococcus agalactiae, while the other protein, Mlp, is similar to the mucus binding protein Mub from Lactobacillus reuteri. It was shown that multiple copies of mlp exist in the genome of L. fermentum BR11. Regions of Rlp, Mlp, and the previously characterized surface protein BspA were used to surface display or secrete heterologous peptides in L. fermentum. The peptides tested were 10 amino acids of the human cystic fibrosis transmembrane regulator protein and a six-histidine epitope (His 6 ). The BspA promoter and secretion signal were used in combination with the Rlp cell wall sorting signal to express, export, and covalently anchor the heterologous peptides to the cell wall. Detection of the cell surface protein fusions revealed that Rlp was a significantly better surface display vector than BspA despite having lower cellular levels (0.7 mg per liter for the Rlp fusion compared with 4 mg per liter for the BspA fusion). The mlp promoter and encoded secretion signal were used to express and export large (328-kDa at 10 mg per liter) and small (27-kDa at 0.06 mg per liter) amino-terminal fragments of the Mlp protein fused to the His 6 and CFTR peptides or His 6 peptide, respectively. Therefore, these newly described proteins from L. fermentum BR11 have potential as protein production and targeting vectors.Over the past several years investigation into the healthpromoting, or probiotic, activity of certain strains of lactic acid bacteria in humans has been the subject of substantial research. Of particular interest are lactobacilli, which due to their nonpathogenic and mucosal surface-colonizing properties have been developed for recombinant protein delivery because they have the potential to express proteins at specific mucosal sites. For the most part, lactobacilli have been investigated as recombinant mucosal vaccines. There have been recent developments in the engineering of lactobacilli and other colonizing lactic acid bacteria so that they secrete or surface display single-chain Fv fragments with direct therapeutic effects (4, 16).Lactobacillus secretion vectors have mainly utilized the secretion signals of proteins derived from bacteria other than lactobacilli, with examples including those of the M protein from Streptococcus pyogenes, Usp45 and PrtP from Lactococcus lactis, and protein A from Staphylococcus aureus (13,31,35). Lactobacillus secretion signals that have been used for extracellular heterologous protein production include the S-layer secretion signal from Lactobacillus brevis (33) and the ␣-amylase protein from Lactobacillus casei (16,17). Protein surface display systems using lactobacilli can be divided into two classes, covalent and noncovalent anchoring systems. The covalent anchor system uses proteins that contain LP...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Peptide Surface Display and Secretion Using Two LPXTG-Containing Surface Proteins from Lactobacillus fermentum BR11

Turner

Hafner

Walsh

et al. 2003

“…The proteins with the LPXTG motif are cleaved after translocation of the plasma membrane and are amide linked to a free amino group of the peptide cross-bridge in the cell wall by a postulated sortase (18). Other reported types of display system have made use of S-layer subunits (3,13), BspA anchor protein (12,29), and AcmA anchor protein (4,25,30). S-layer subunits and BspA are anchored via charge interactions and can be extracted from the cell surface with chargeoccupying agents such as lithium chloride.…”

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confidence: 99%

Display of α-Amylase on the Surface of Lactobacillus casei Cells by Use of the PgsA Anchor Protein, and Production of Lactic Acid from Starch

Narita¹,

Okano

Kitao

et al. 2006

106

We developed a new cell surface engineering system based on the PgsA anchor protein from Bacillus subtilis. In this system, the N terminus of the target protein was fused to the PgsA protein and the resulting fusion protein was expressed on the cell surface. Using this new system, we constructed a novel starch-degrading strain of Lactobacillus casei by genetically displaying ␣-amylase from the Streptococcus bovis strain 148 with a FLAG peptide tag (AmyAF). Localization of the PgsA-AmyA-FLAG fusion protein on the cell surface was confirmed by immunofluorescence microscopy and flow cytometric analysis. The lactic acid bacteria which displayed AmyAF showed significantly elevated hydrolytic activity toward soluble starch. By fermentation using AmyAF-displaying L. casei cells, 50 g/liter of soluble starch was reduced to 13.7 g/liter, and 21.8 g/liter of lactic acid was produced within about 24 h. The yield in terms of grams of lactic acid produced per gram of carbohydrate utilized was 0.60 g per g of carbohydrate consumed at 24 h. Since AmyA was immobilized on the cells, cells were recovered after fermentation and used repeatedly. During repeated utilization of cells, the lactic acid yield was improved to 0.81 g per g of carbohydrate consumed at 72 h. These results indicate that efficient simultaneous saccharification and fermentation from soluble starch to lactic acid were carried out by recombinant L. casei cells with cell surface display of AmyA.

“…To date, we have characterized a number of different surface proteins from L. fermentum BR11 and tested them as heterologous protein fusion partners. These include the noncovalently anchored cystine binding surface protein, BspA, and the covalently anchored LPXTG-containing proteins Mlp and Rlp (10,29,30,32). Here we report the identification and characterization of a novel abundant small exported protein (Sep) from L. fermen-tum BR11.…”

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confidence: 99%

Identification and Characterization of the Novel LysM Domain-Containing Surface Protein Sep from Lactobacillus fermentum BR11 and Its Use as a Peptide Fusion Partner in Lactobacillus and Lactococcus

Turner

Hafner

Walsh

et al. 2004

Examination of supernatant fractions from broth cultures ofLactobacilli have been engineered to express a variety of heterologous proteins of interest, including antigens, enzymes, and more recently therapeutic single-chain antibodies and pathogen receptors (5,8,12,30). Certain features of lactobacilli make them attractive as recombinant protein delivery vehicles. These include their generally regarded as safe (GRAS) status; their presence in dairy, meat, and vegetable food fermentation processes; and their ability to reside naturally and persist on mucosal surfaces where they can exert certain beneficial effects to the host.A number of protein expression and targeting systems in lactobacilli have been developed which allow accumulation of the protein either intracellularly, anchored to the cell surface, or in the extracellular environment. Protein secretion and surface anchoring systems rely on the utilization of functional elements of extracellular proteins to efficiently secrete and/or anchor the heterologous protein to the cell surface. The majority of extracellular proteins in gram-positive bacteria are at some stage anchored to the cell wall either via covalent or noncovalent cell wall binding domains. Covalent attachment to the cell wall or cytoplasmic membrane occurs via the carboxyterminal LPXTG-type or amino-terminal LXXC sorting signals, respectively (18). Noncovalent attachment to the cell wall or cell wall components occurs either via specific repetitive LysM, YG, or choline-binding GW or S-layer homology domains (3, 7, 18) or via nonspecific cationic domains (2, 31). Varying amounts of both covalent and noncovalent surface anchored proteins may be released into the environment due to cell wall turnover, cell lysis, or proteolytic events (4,21,23,24).Our research is geared towards the development of heterologous protein expression systems using the targeting regions of native lactobacilli surface proteins. Our model organism is Lactobacillus fermentum BR11, which is a vaginal tract isolate of a guinea pig that has been shown to be amenable to genetic manipulation (25). Members of the L. fermentum-Lactobacillus reuteri group are commonly found inhabitants of mucosal surfaces of mammals and are one of the predominant Lactobacillus species found in the intestines of humans. To date, we have characterized a number of different surface proteins from L. fermentum BR11 and tested them as heterologous protein fusion partners. These include the noncovalently anchored cystine binding surface protein, BspA, and the covalently anchored LPXTG-containing proteins Mlp and Rlp (10,29,30,32). Here we report the identification and characterization of a novel abundant small exported protein (Sep) from L. fermen-