A food-grade fimbrial adhesin FaeG expression system inLactococcus lactisandLactobacillus casei

2016 ◽  
Vol 62 (3) ◽  
pp. 241-248 ◽  
Author(s):  
W.W. Lu ◽  
T. Wang ◽  
Y. Wang ◽  
M. Xin ◽  
J. Kong
Keyword(s):  
Genetically Modified ◽  
Expression System ◽  
Enterotoxigenic Escherichia Coli ◽  
Selection Marker ◽  
Delivery Vehicle ◽  
Food Grade ◽  
Neonatal Piglets ◽  
Extracellular Secretion ◽  
Etec Infection ◽  
Fimbrial Adhesin

Enterotoxigenic Escherichia coli (ETEC) infection is the major cause of diarrhea in neonatal piglets. The fimbriae as colonizing factor in the pathogenesis of ETEC constitute a primary target for vaccination against ETEC. Lactic acid bacteria (LAB) are attractive tools to deliver antigens at the mucosal level. With the safety of genetically modified LAB in mind, a food-grade secretion vector (pALRc or pALRb) was constructed with DNA entirely from LAB, including the replicon, promoter, signal peptide, and selection marker alanine racemase gene (alr). To evaluate the feasibility of the system, the nuclease gene (nuc) from Staphylococcus aureus was used as a reporter to be expressed in both Lactococcus lactis and Lactobacillus casei. Subsequently, the extracellular secretion of the fimbrial adhesin FaeG of ETEC was confirmed by Western blot analysis. These results showed that this food-grade expression system has potential as the delivery vehicle for the safe use of genetically modified LAB for the development of vaccines against ETEC infection.

scholarly journals Cell Wall Anchoring of a Bacterial Chitosanase in Lactobacillus plantarum Using a Food-Grade Expression System and Two Versions of an LP × TG Anchor

2020 ◽  
Vol 21 (11) ◽  
pp. 3773 ◽  
Author(s):  
Mai-Lan Pham ◽  
Anh-Minh Tran ◽  
Geir Mathiesen ◽  
Hoang-Minh Nguyen ◽  
Thu-Ha Nguyen
Keyword(s):  
Cell Wall ◽  
Cell Surface ◽  
Lactobacillus Plantarum ◽  
Expression System ◽  
Surface Display ◽  
Cell Surface Display ◽  
Expression Vectors ◽  
Selection Marker ◽  
Food Grade ◽  
Cell Factories

Lactic acid bacteria (LAB) have attracted increasing interest recently as cell factories for the production of proteins as well as a carrier of proteins that are of interest for food and therapeutic applications. In this present study, we exploit a lactobacillal food-grade expression system derived from the pSIP expression vectors using the alr (alanine racemase) gene as the selection marker for the expression and cell-surface display of a chitosanase in Lactobacillus plantarum using two truncated forms of a LP × TG anchor. CsnA, a chitosanase from Bacillus subtilis 168 (ATCC23857), was fused to two different truncated forms (short-S and long-L anchors) of an LP × TG anchor derived from Lp_1229, a key-protein for mannose-specific adhesion in L. plantarum WCFS1. The expression and cell-surface display efficiency driven by the food-grade alr-based system were compared with those obtained from the erm-based pSIP system in terms of enzyme activities and their localisation on L. plantarum cells. The localization of the protein on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest enzymatic activity of CsnA-displaying cells was obtained from the strain carrying the alr-based expression plasmid with short cell wall anchor S. However, the attachment of chitosanase on L. plantarum cells via the long anchor L was shown to be more stable compared with the short anchor after several repeated reaction cycles. CsnA displayed on L. plantarum cells is catalytically active and can convert chitosan into chito-oligosaccharides, of which chitobiose and chitotriose are the main products.

Production of Recombinant β-Galactosidase in Lactobacillus plantarum, Using a pSIP-Based Food-Grade Expression System

2014 ◽  
Vol 931-932 ◽  
pp. 1518-1523 ◽  
Author(s):  
Numphon Thaiwong ◽  
Siwatt Thaiudom ◽  
Dietmar Haltrich ◽  
Montarop Yamabhai
Keyword(s):  
Enzyme Production ◽  
Expression Vector ◽  
Expression System ◽  
Selection Marker ◽  
Expression Systems ◽  
Recombinant Enzymes ◽  
Food Grade ◽  
Food Industries ◽  
Specific Enzymatic Activity ◽  
Food Applications

Food-grade expression systems based on using food-grade microorganisms have been developed for the production of recombinant enzymes used in food applications. Lactic acid bacteria (LAB), especially Lactobacilli, have been widely used for various purposes in food and recognized as a promising host of food-grade enzyme production. In this study, the pSIP409 vectors, originally containing the erm gene, were used to replace this selection marker by the alr gene resulting in the production of the pSIP609 expression vector in L. planatarum. This vector could express high amounts of β-galactosidases, showing both high volumetric as well a specific enzymatic activity. Thus, the food-grade recombinant enzyme production in L. planatarum harboring pSIP609 was very fruitful and useful for food industries.

scholarly journals Identification of the Propionicin F Bacteriocin Immunity Gene (pcfI) and Development of a Food-Grade Cloning System for Propionibacterium freudenreichii

2007 ◽  
Vol 73 (23) ◽  
pp. 7542-7547 ◽  
Author(s):  
Dag Anders Brede ◽  
Sheba Lothe ◽  
Zhian Salehian ◽  
Therese Faye ◽  
Ingolf F. Nes
Keyword(s):  
Selection Marker ◽  
Multiple Cloning Site ◽  
Propionibacterium Freudenreichii ◽  
Expression Plasmid ◽  
Physiochemical Properties ◽  
Food Grade ◽  
Cloning System ◽  
Immunity Gene ◽  
Membrane Bound ◽  
High Level

ABSTRACT This report describes the first functional analysis of a bacteriocin immunity gene from Propionibacterium freudenreichii and its use as a selection marker for food-grade cloning. Cloning of the pcfI gene (previously orf5 [located as part of the pcfABC propionicin F operon]) rendered the sensitive host 1,000-fold more tolerant to the propionicin F bacteriocin. The physiochemical properties of the 127-residue large PcfI protein resemble those of membrane-bound immunity proteins from bacteriocin systems found in lactic acid bacteria. The high level of immunity conferred by pcfI allowed its use as a selection marker for plasmid transformation in P. freudenreichii. Electroporation of P. freudenreichii IFO12426 by use of the pcfI expression plasmid pSL102 and propionicin F selection (200 bacteriocin units/ml) yielded 107 transformants/μg DNA. The 2.7-kb P. freudenreichii food-grade cloning vector pSL104 consists of the pLME108 replicon, a multiple cloning site, and pcfI expressed from the constitutive PpampS promoter for selection. The pSL104 vector efficiently facilitated cloning of the propionicin T1 bacteriocin in P. freudenreichii. High-level propionicin T1 production (640 BU/ml) was obtained with the IFO12426 strain, and the food-grade propionicin T1 expression plasmid pSL106 was maintained by ∼91% of the cells over 25 generations in the absence of selection. To the best of our knowledge this is the first report of an efficient cloning system that facilitates the generation of food-grade recombinant P. freudenreichii strains.

scholarly journals Immunogenicity and protective efficacy of enterotoxigenic Escherichia coli (ETEC) total RNA against ETEC challenge in a mouse model

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Mandi Liu ◽  
Yue Zhang ◽  
Di Zhang ◽  
Yun Bai ◽  
Guomei Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mouse Model ◽  
Immune Responses ◽  
Protective Efficacy ◽  
Enterotoxigenic Escherichia Coli ◽  
Economic Losses ◽  
Th2 Response ◽  
Total Rna ◽  
Etec Infection

AbstractEnterotoxigenic Escherichia coli (ETEC), an essential cause of post-weaning diarrhea (PWD) in piglets, leads to significant economic losses to the pig industry. The present study aims to identify the role of ETEC total RNA in eliciting immune responses to protect animals against ETEC infection. The results showed that the total RNA isolated from pig-derived ETEC K88ac strain effectively stimulated the IL-1β secretion of porcine intestinal epithelial cells (IPEC-J2). The mouse model immunized with ETEC total RNA via intramuscular injection (IM) or oral route (OR) was used to evaluate the protective efficiency of the ETEC total RNA. The results suggested that 70 μg ETEC total RNA administered by either route significantly promoted the production of the serum IL-1β and K88ac specific immunoglobulins (IgG, IgM, and IgA). Besides, the ETEC RNA administration augmented strong mucosal immunity by elevating K88ac specific IgA level in the intestinal fluid. Intramuscularly administered RNA induced a Th1/Th2 shift toward a Th2 response, while the orally administered RNA did not. The ETEC total RNA efficiently protected the animals against the ETEC challenge either by itself or as an adjuvant. The histology characterization of the small intestines also suggested the ETEC RNA administration protected the small intestinal structure against the ETEC infection. Particularly of note was that the immunity level and protective efficacy caused by ETEC RNA were dose-dependent. These findings will help understand the role of bacterial RNA in eliciting immune responses, and benefit the development of RNA-based vaccines or adjuvants.

scholarly journals Enterotoxigenic Escherichia coli infection and intestinal thiamin uptake: studies with intestinal epithelial Caco-2 monolayers

2013 ◽  
Vol 305 (11) ◽  
pp. C1185-C1191 ◽  
Author(s):  
Abhisek Ghosal ◽  
Nabendu S. Chatterjee ◽  
Tristan Chou ◽  
Hamid M. Said
Keyword(s):  
Escherichia Coli ◽  
Significant Inhibition ◽  
Water Soluble ◽  
Enterotoxigenic Escherichia Coli ◽  
Adenylate Cyclase System ◽  
Mrna Levels ◽  
Intestinal Epithelial ◽  
E Coli ◽  
Heat Labile ◽  
Etec Infection

Infections with enteric pathogens like enterotoxigenic Escherichia coli ( ETEC) is a major health issue worldwide and while diarrhea is the major problem, prolonged, severe, and dual infections with multiple pathogens may also compromise the nutritional status of the infected individuals. There is almost nothing currently known about the effect of ETEC infection on intestinal absorptions of water-soluble vitamins including thiamin. We examined the effect of ETEC infection on intestinal uptake of the thiamin using as a model the human-derived intestinal epithelial Caco-2 cells. The results showed that infecting confluent Caco-2 monolayers with live ETEC (but not with boiled/killed ETEC or nonpathogenic E. coli) or treatment with bacterial culture supernatant led to a significant inhibition in thiamin uptake. This inhibition appears to be caused by a heat-labile and -secreted ETEC component and is mediated via activation of the epithelial adenylate cyclase system. The inhibition in thiamin uptake by ETEC was associated with a significant reduction in expression of human thiamin transporter-1 and -2 (hTHTR1 and hTHTR2) at the protein and mRNA levels as well as in the activity of the SLC19A2 and SLC19A3 promoters. Dual infection of Caco-2 cells with ETEC and EPEC (enteropathogenic E. coli) led to compounded inhibition in intestinal thiamin uptake. These results show for the first time that infection of human intestinal epithelial cells with ETEC causes a significant inhibition in intestinal thiamin uptake. This inhibition is mediated by a secreted heat-labile toxin and is associated with a decrease in the expression of intestinal thiamin transporters.

scholarly journals Enterotoxigenic Escherichia coli in Developing Countries: Epidemiology, Microbiology, Clinical Features, Treatment, and Prevention

2005 ◽  
Vol 18 (3) ◽  
pp. 465-483 ◽  
Author(s):  
Firdausi Qadri ◽  
Ann-Mari Svennerholm ◽  
A. S. G. Faruque ◽  
R. Bradley Sack
Keyword(s):  
Clinical Symptoms ◽  
Enterotoxigenic Escherichia Coli ◽  
Traveler’S Diarrhea ◽  
Treatment And Prevention ◽  
Protection Strategies ◽  
Enteric Infections ◽  
Colonization Factors ◽  
Traveler's Diarrhea ◽  
Etec Infection

SUMMARY ETEC is an underrecognized but extremely important cause of diarrhea in the developing world where there is inadequate clean water and poor sanitation. It is the most frequent bacterial cause of diarrhea in children and adults living in these areas and also the most common cause of traveler's diarrhea. ETEC diarrhea is most frequently seen in children, suggesting that a protective immune response occurs with age. The pathogenesis of ETEC-induced diarrhea is similar to that of cholera and includes the production of enterotoxins and colonization factors. The clinical symptoms of ETEC infection can range from mild diarrhea to a severe cholera-like syndrome. The effective treatment of ETEC diarrhea by rehydration is similar to treatment for cholera, but antibiotics are not used routinely for treatment except in traveler's diarrhea. The frequency and characterization of ETEC on a worldwide scale are inadequate because of the difficulty in recognizing the organisms; no simple diagnostic tests are presently available. Protection strategies, as for other enteric infections, include improvements in hygiene and development of effective vaccines. Increases in antimicrobial resistance will dictate the drugs used for the treatment of traveler's diarrhea. Efforts need to be made to improve our understanding of the worldwide importance of ETEC.

scholarly journals The fimbrial adhesin F17-G of enterotoxigenic Escherichia coli has an immunoglobulin-like lectin domain that binds N-acetylglucosamine

2004 ◽  
Vol 49 (3) ◽  
pp. 705-715 ◽  
Author(s):  
Lieven Buts ◽  
Julie Bouckaert ◽  
Erwin De Genst ◽  
Remy Loris ◽  
Stefan Oscarson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enterotoxigenic Escherichia Coli ◽  
Lectin Domain ◽  
Fimbrial Adhesin

scholarly journals Use of the alr Gene as a Food-Grade Selection Marker in Lactic Acid Bacteria

2002 ◽  
Vol 68 (11) ◽  
pp. 5663-5670 ◽  
Author(s):  
Peter A. Bron ◽  
Marcos G. Benchimol ◽  
Jolanda Lambert ◽  
Emmanuelle Palumbo ◽  
Marie Deghorain ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Competitive Inhibitor ◽  
Selection Marker ◽  
Wild Type ◽  
Gene Encoding ◽  
Food Grade ◽  
Selection For ◽  
Isogenic Mutants ◽  
Type Strains

ABSTRACT Both Lactococcus lactis and Lactobacillus plantarum contain a single alr gene, encoding an alanine racemase (EC 5.1.1.1), which catalyzes the interconversion of d-alanine and l-alanine. The alr genes of these lactic acid bacteria were investigated for their application as food-grade selection markers in a heterologous complementation approach. Since isogenic mutants of both species carrying an alr deletion (Δalr) showed auxotrophy for d-alanine, plasmids carrying a heterologous alr were constructed and could be selected, since they complemented d-alanine auxotrophy in the L. plantarum Δalr and L. lactis Δalr strains. Selection was found to be highly stringent, and plasmids were stably maintained over 200 generations of culturing. Moreover, the plasmids carrying the heterologous alr genes could be stably maintained in wild-type strains of L. plantarum and L. lactis by selection for resistance to d-cycloserine, a competitive inhibitor of Alr (600 and 200 μg/ml, respectively). In addition, a plasmid carrying the L. plantarum alr gene under control of the regulated nisA promoter was constructed to demonstrate that d-cycloserine resistance of L. lactis is linearly correlated to the alr expression level. Finally, the L. lactis alr gene controlled by the nisA promoter, together with the nisin-regulatory genes nisRK, were integrated into the chromosome of L. plantarum Δalr. The resulting strain could grow in the absence of d-alanine only when expression of the alr gene was induced with nisin.

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