[30] Lactobacilli as vehicles for targeting antigens to mucosal tissues by surface exposition of foreign antigens

Pouwels, Peter H.; Vriesema, Aldwin; Martı́nez, Beatriz; Tielen, Frans; Seegers, Jos F. M. L.; Leer, R.J.; Jore, J.p.m.; Smit, Elize

doi:10.1016/s0076-6879(01)36602-8

Cited by 38 publications

(36 citation statements)

References 27 publications

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“…The present study went into greater depth by using four L. casei promoters suspected of exhibiting different degrees of effectiveness in vitro. The activity ranking determined in vitro (ldhp Ͼ lacTp* Ͼ ccpAp Ͼ dltp) was consistent with that reported in previous studies (16,25,26). Moreover, a molecular approach consisting of RT-QPCR was applied to the transcriptional fusion constructs to clearly show de novo mRNA synthesis by bacteria during GI transit.…”

Section: Discussionsupporting

confidence: 85%

“…Four L. casei promoters involved in different metabolic pathways were selected among promoter sequences available in GenBank: the deregulated lactose operon promoter (lacTp*) (1,18), the promoter of L-(ϩ)-lactate dehydrogenase (ldhp) (25), the ccpA promoter (ccpAp) (16), and the dlt operon promoter (dltp) (17).…”

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confidence: 99%

“…In Bacillus subtilis, peak expression of the dlt operon occurs during logarithmic growth prior to down-regulation by the SpoOA and AbrB global regulators (23). In L. casei, the promoter ldhp is known to be a highly efficient and constitutive promoter (25). In Lactobacillus helveticus, the ldh gene is strongly expressed in the logarithmic phase, but in the stationary phase, the level of ldh transcripts decreases rapidly (30).…”

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confidence: 99%

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Differential Activities of FourLactobacillus caseiPromoters during Bacterial Transit through the Gastrointestinal Tracts of Human-Microbiota-Associated Mice

Oozeer

Furet

Goupil-Feuillerat

et al. 2005

Appl Environ Microbiol

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In a previous study using fusion of the deregulated lactose promoter lacTp* and reporter genes, we suggested that Lactobacillus casei could initiate de novo protein synthesis during intestinal transit. In order to confirm this finding and extend it to other promoters, we adopted a reverse transcriptase quantitative PCR (RT-QPCR) approach combined with a transcriptional fusion system consisting of luciferase genes under the control of four promoters (ccpA, dlt, ldh, and lacT*) from L. casei DN-114 001. Promoter expression was monitored during cell growth, and variable luciferase activities were detected. In 3-day cultures, all the genetically modified strains survived but without exhibiting luciferase activity. Luciferase mRNA levels determined by RT-QPCR analysis (RNA/CFU) were not significant. The cultures were administered to human-microbiota-associated mice, and the feces were collected 6 h later. L. casei promoters lacTp* and ldhp initiated mRNA synthesis during gastrointestinal transit. The promoters, ccpAp and dltp, exhibited no luciferase activity, nor was de novosynthesized luciferase mRNA detected in the feces. L. casei seems to adapt its physiology to the gastrointestinal tract environment by modulating promoter activities. The approach (fecal transcriptional analysis) described herein may, moreover, be of value in studying gene expression of transiting bacteria in human fecal specimens.Lactobacillus casei has attracted considerable interest as a probiotic over the last few years (15,19). The strain L. casei DN-114 001 has been shown to alleviate acute diarrhea in healthy children (21,22), to modulate the production of proinflammatory cytokines in Crohn's disease ex vivo (3), and to enhance the immune system during bacterial gastrointestinal (GI) transit (20). However, little is known about the survival of L. casei species and their physiological status in the environment of the GI tract.Probiotics are usually ingested with fermented milk during the late stationary phase of the bacterial cycle. At the onset of the stationary phase, the overall rate of protein synthesis has been shown to drop precipitously to a rate equivalent to approximately 0.2% of the exponential growth rate (13). These changes are concomitant with a decrease in overall mRNA and DNA synthesis (13). The behavior of such metabolically inactive cells in the GI environment was thus investigated with a view to identifying the mechanisms underlying their action on health.To carry out this work, we developed a genetic approach using transcriptional fusion of luciferase genes (luxA and luxB) and selected promoters to study the response of lactic acid bacteria to the GI tract environment (5). The same approach was then extended to a probiotic microorganism, L. casei DN-114 001, to study its response to the GI tract in the humanmicrobiota-associated (HMA) mouse model using transcriptional fusion of the modified L. casei lacTEGF operon promoter and luxA and luxB genes from Photorhabdus luminescens. Resulting data suggest that L. casei can init...

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Section: Discussionsupporting

confidence: 85%

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confidence: 99%

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confidence: 99%

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Differential Activities of FourLactobacillus caseiPromoters during Bacterial Transit through the Gastrointestinal Tracts of Human-Microbiota-Associated Mice

Oozeer

Furet

Goupil-Feuillerat

et al. 2005

Appl Environ Microbiol

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“…Some L. plantarum strains have probiotic effects on human health (Alander et al, 1999;Schultz et al, 2002;Mercenier et al, 2003). Lactobacilli have great potential as food-grade cell factories and as delivery vehicles for interesting proteins, such as antigens, antibodies and growth factors (Pouwels et al, 1996(Pouwels et al, , 2001Pavan et al, 2000; Krüger et al, 2002;Scheppler et al, 2002). Thus, there is considerable interest in the development of genetic tools for efficient and controllable gene expression in lactobacilli (de Vos, 1999;Mercenier et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors

Sørvig

Mathiesen

Naterstad³

et al. 2005

Microbiology

182

185

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Vectors have been developed for inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum in which expression of the gene of interest is driven by strong, regulated promoters from bacteriocin operons found in L. sakei strains. The activity of these promoters is controlled via a two-component signal transduction system, which responds to an externally added peptide pheromone. The vectors have a modular design, permitting easy exchange of all essential elements: the inducible promoter, the cognate regulatory system, the gene of interest, the antibiotic resistance marker and the replicon. Various variants of these so-called 'pSIP' vectors were constructed and tested, differing in terms of the bacteriocin regulon from which the regulatory elements were derived (sakacin A or sakacin P), the regulated promoter selected from these regulons, and the replicon (derived from p256 or pSH71). Using b-glucuronidase (GusA) and aminopeptidase N (PepN) as reporters, it was shown that the best vectors permitted inducible, pheromone-dose-dependent gene expression at very high levels, while displaying moderate basal activities when not induced. The most effective set-up was obtained using a vector containing the pSH71 replicon, the orfX promoter from the sakacin P regulon, and the cognate regulatory genes, in a L. sakei host. GusA levels obtained with this set-up were approximately ten times higher than the levels obtained with prototype pSIP versions, whereas PepN levels amounted to almost 50 % of total cellular protein. INTRODUCTIONMany Lactobacillus species are important in the food industry, where they are used in a variety of products. Lactobacillus sakei is used in meat and vegetable fermentations and has a broad range of possible habitats. The versatility of L. sakei can partly be explained by its ability to survive and grow under adverse conditions, such as low temperature and pH, high salt concentration, smoke, ethanol, low water activity and radiation. Lactobacillus plantarum is also found in many habitats, including dairy and meat products, plant and vegetable fermentations, and in the gastrointestinal tract and oral cavity of humans (Axelsson & Ahrné, 2000). Some L. plantarum strains have probiotic effects on human health (Alander et al., 1999;Schultz et al., 2002;Mercenier et al., 2003). Lactobacilli have great potential as food-grade cell factories and as delivery vehicles for interesting proteins, such as antigens, antibodies and growth factors (Pouwels et al., 1996(Pouwels et al., , 2001Pavan et al., 2000; Krüger et al., 2002;Scheppler et al., 2002). Thus, there is considerable interest in the development of genetic tools for efficient and controllable gene expression in lactobacilli (de Vos, 1999;Mercenier et al., 2000).Many lactic acid bacteria (LAB) produce antimicrobial peptides called bacteriocins, and their production is often regulated via quorum-sensing mechanisms based on a secreted peptide pheromone (Eijsink et al., 2002;Quadri, 2002). The pheromone activates a two-component regulatory s...

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“…Gilliland, 1990;Goldin, 1998;Vaughan et al, 1999;Gorbach, 2000;Rolfe, 2000;Mercenier et al, 2003). Also, in recent years, selected Lactobacillus species have been used as vaccine delivery systems, which can be administered orally and can effectively induce protective immune responses (Mercenier et al, 1996;Wells et al, 1996;Pouwels et al, 2001;Seegers, 2002;Scheppler et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

Construction of a combined physical and genetic map of the chromosome of Lactobacillus acidophilus ATCC 4356 and characterization of the rRNA operons

2005

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The combination of PFGE and hybridization approaches was used to study the genome of Lactobacillus acidophilus neotype strain ATCC 4356. PFGE analysis of chromosomal DNA after digestion with each of the rare-cutting restriction enzymes I-CeuI, NotI, CspI, SmaI, ApaI and SgrAI allowed the size of the circular chromosome of L. acidophilus to be estimated at 2?061 Mbp. The physical map contained 86 restriction sites for the six enzymes employed, with intervals between the sites varying from 1 to 88 kbp (~0?05-4?3 % of the chromosome). Based on the physical map, a genetic map was constructed via Southern blot analyses of L. acidophilus DNA using specific gene probes. A total of 73 probes representing key genes, including 12 rRNA (rrn) genes, were positioned on the latter map. Mapping analysis also indicated the presence of four rrn operons (rrnA-D) on the chromosome, each containing a single copy of each of the three rrn genes 16S (rrl ), 23S (rrs) and 5S (rrf ). Operon rrnD was inverted in orientation with respect to the others and contained a long 16S-23S intergenic spacer region with tRNA Ile and tRNA Ala genes, whereas the other operons contained a short spacer lacking any tRNA genes. The high-resolution physical/genetic map constructed in this study provides a platform for genomic and genetic studies of Lactobacillus species and for improving industrial and probiotic strains.

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[30] Lactobacilli as vehicles for targeting antigens to mucosal tissues by surface exposition of foreign antigens

Cited by 38 publications

References 27 publications

Differential Activities of FourLactobacillus caseiPromoters during Bacterial Transit through the Gastrointestinal Tracts of Human-Microbiota-Associated Mice

Differential Activities of FourLactobacillus caseiPromoters during Bacterial Transit through the Gastrointestinal Tracts of Human-Microbiota-Associated Mice

High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors

Construction of a combined physical and genetic map of the chromosome of Lactobacillus acidophilus ATCC 4356 and characterization of the rRNA operons

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