Adaptation of the Nisin-Controlled Expression System in Lactobacillus plantarum : a Tool To Study In Vivo Biological Effects

Abstract

The potential of lactic acid bacteria as live vehicles for the production and delivery of therapeutic molecules is being actively investigated today. For future applications it is essential to be able to establish dose-response curves for the targeted biological effect and thus to control the production of a heterologous biopeptide by a live lactobacillus. We therefore implemented in Lactobacillus plantarum NCIMB8826 the powerful nisin-controlled expression (NICE) system based on the autoregulatory properties of the bacteriocin nisin, which is produced by Lactococcus lactis. The original two-plasmid NICE system turned out to be poorly suited to L. plantarum. In order to obtain a stable and reproducible nisin dose-dependent synthesis of a reporter protein (␤-glucuronidase) or a model antigen (the C subunit of the tetanus toxin, TTFC), the lactococcal nisRK regulatory genes were integrated into the chromosome of L. plantarum NCIMB8826. Moreover, recombinant L. plantarum producing increasing amounts of TTFC was used to establish a dose-response curve after subcutaneous administration to mice. The induced serum immunoglobulin G response was correlated with the dose of antigen delivered by the live lactobacilli.Lactic acid bacteria (LAB) are used worldwide in the preparation of fermented foods, including dairy products. They are also known for the potentially beneficial effects they may exert on the health of humans and animals (see, for example, reference 25). Their "generally recognized as safe" status (1), linked to their metabolic and technological properties, has recently led to their development as potential live-vaccine vehicles. Lactobacillus plantarum NCIMB8826 (17, 33) has been chosen for this purpose in our laboratory on the basis of its capability to persist in the mouse gastrointestinal and urogenital tracts (38). The ability to control the expression level of foreign proteins in LAB may offer certain advantages. However, while several controlled expression systems have been developed for Lactococcus lactis (9, 23), very few inducible promoters are available for lactobacilli: the xylR promoter from Lactobacillus pentosus (29), the ␣-amylase promoter from L. amylovorus (31), and the p-coumarate decarboxylase promoter from L. plantarum (4). One of the most promising lactococcal controlled expression systems is based on the autoregulatory properties of the L. lactis nisin gene cluster (7,23). Nisin is an antimicrobial peptide belonging to the family of lantibiotics (19) and is used as a natural preservative in the food industry (5). Nisin induces the transcription of the genes under control of the nisA and nisF promoters, via a two-component regulatory system (34, 37) consisting of the histidine protein kinase NisK and the response regulator NisR (14,21,22). A transferable nisin-controlled expression (NICE) system (24) based on the combination of the nisA promoter and the nisRK regulatory genes has recently been developed (7,20). It consists of two compatible replicons, a plasmid carrying the nisRK regulato...

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

“…Another example is pSIP411, which yielded GUS levels that were five to ten times higher than the levels obtained with the best prototype vectors (Sørvig et al, 2003) and that were comparable to the levels obtained with the NICE system (Table 3). Previous studies have shown that nisinbased plasmid expression systems used in lactobacilli are poorly regulated (Pavan et al, 2000;Sørvig et al, 2003), and that integration of the regulatory genes into the chromosome may be necessary to sustain the inducibility of the vectors and to avoid basal activity (Pavan et al, 2000). The work presented in the present study demonstrates that it is possible to obtain controlled, high-level expression with minor basal activities with a one-plasmid system and to sustain inducibility without integrating genes into the chromosome of the host.…”

“…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).…”

“…While being efficient and well regulated in lactococci, the plasmid-based nisin-controlled expression (NICE) systems often exhibit significant basal activity (i.e. activity without induction) in lactobacilli (Pavan et al, 2000;Sørvig et al, 2003). This problem can be circumvented by integrating the histidine kinase and responseregulator genes in the chromosome (Pavan et al, 2000), thus limiting the expression systems to specially designed host strains.…”

“…In both cases, the activated response regulator enhances transcription of all operons involved in bacteriocin production. gene expression systems for lactococci (de Ruyter et al, 1996), lactobacilli (Pavan et al, 2000;Sørvig et al, 2003) and other Gram-positive bacteria (Eichenbaum et al, 1998;Bryan et al, 2000). While being efficient and well regulated in lactococci, the plasmid-based nisin-controlled expression (NICE) systems often exhibit significant basal activity (i.e.…”

“…activity without induction) in lactobacilli (Pavan et al, 2000;Sørvig et al, 2003). This problem can be circumvented by integrating the histidine kinase and responseregulator genes in the chromosome (Pavan et al, 2000), thus limiting the expression systems to specially designed host strains.Recently, we have developed one-plasmid inducible expression systems based on promoters and regulatory genes involved in the production of the class II bacteriocins sakacin A (sap gene cluster) and sakacin P (spp gene cluster) Sørvig et al, 2003). Sørvig et al (2003) constructed prototypes of the modular so-called pSIP expression vectors (Fig.…”