An important criterion for the selection of a probiotic bacterial strain is its ability to adhere to the mucosal surface. Adhesion is usually mediated by proteins or other components located on the ...outer cell surface of the bacterium. In the present study we characterized the adhesive properties of two classical intracellular enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase (ENO) isolated from the outer cell surface of the probiotic bacterium Lactobacillus plantarum 299v. None of the genes encoded signal peptides or cell surface anchoring motifs that could explain their extracellular location on the bacterial surface. The presence of the glycolytic enzymes on the outer surface was verified by western blotting using polyclonal antibodies raised against the specific enzymes. GAPDH and ENO showed a highly specific binding to plasminogen and fibronectin whereas GAPDH but not ENO showed weak binding to mucin. Furthermore, a pH dependent and specific binding of GAPDH and ENO to intestinal epithelial Caco-2 cells at pH 5 but not at pH 7 was demonstrated. The results showed that these glycolytic enzymes could play a role in the adhesion of the probiotic bacterium L. plantarum 299v to the gastrointestinal tract of the host. Finally, a number of probiotic as well non-probiotic Lactobacillus strains were analyzed for the presence of GAPDH and ENO on the outer surface, but no correlation between the extracellular location of these enzymes and the probiotic status of the applied strains was demonstrated.
In a previous study, we described the use of transposon Tn917‐LTV1 for identification of environmentally regulated promoters in Lactococcus lactis. Here, we report the molecular analysis of one of ...these promoters, P170, that is upregulated at low pH during the transition to stationary phase. The minimal DNA region required for both promoter activity and pH regulation was mapped to a 51 bp fragment located 7 bp upstream of the transcriptional start site. This fragment lacked the consensus −35 promoter region, but it contained an ‘extended’−10 promoter region. When a 28 bp segment, containing the consensus −35 region and 22 bp upstream of this in a constitutive promoter, was replaced with the corresponding sequence of P170, the hybrid promoter became regulated by pH and growth phase. This demonstrates that the P170 segment contains a cis‐acting sequence involved in the control of promoter regulation. Transcriptional analysis showed that P170 is responsible for the transcription of a monocistronic gene orfX encoding a polypeptide homologous to a hypothetical protein from Bacillus subtilis. Analysis of total RNA from L. lactis grown at constant pH confirmed that transcription from P170 was induced between pH 6.5 and pH 6.0, but only when the culture entered stationary phase. Deletion analysis and chemical mutagenesis of P170 defined a specific region within the untranslated mRNA leader that is able to modulate the expression level directed by the P170 promoter. Deletion of a 72 bp HaeIII fragment from this leader region resulted in a 150‐ to 200‐fold increase in the level of gene expression, without affecting the regulation. The functionality was confirmed by introducing this modulating element downstream of other lactococcal promoters.
Some lactic acid bacteria, especially
spp., possess adhesive properties enabling colonization of the human gastrointestinal tract. Two probiotic
strains, WCSF1 and 299v, display highly different ...mannose-specific adhesion, with
299v being superior to
WCFS1 based on a yeast agglutination assay. A straightforward correlation between the mannose adhesion capacity and domain composition of the mannose-specific adhesin (Msa) in the two strains has not been demonstrated previously. In this study, we analyzed the promoter regions upstream of the
gene encoding a mannose-specific adhesin in these two strains. The promoter region was mapped by primer extension and DNA sequence analysis, and only a single nucleotide change was identified between the two strains. However, Northern blot analysis showed a stronger
transcript band in 299v than in WCFS1 correlating with the different adhesion capacities. During the establishment of a high-throughput yeast agglutination assay, we isolated variants of WCFS1 that displayed a very strong mannose-specific adhesion phenotype. The region upstream of the
gene in these variants showed an inversion of a 104-bp fragment located between two perfectly inverted repeats present in the untranslated leader region. The inversion disrupts a strong hairpin structure that otherwise most likely would terminate the
transcript. In addition, the ribosome binding site upstream of the
gene, which is also masked within this hairpin structure, becomes accessible upon inversion, thereby increasing the frequency of translation initiation in the variant strains. Furthermore, Northern blot analysis showed a higher abundance of the
transcript in the variants than in the wild type, correlating with a strong-Msa phenotype.
Probiotic strains possess adhesive properties enabling colonization of the human intestinal tract through interactions between molecules present on the probiotic bacteria and components of the epithelial surface. In
, interaction is mediated through bacterial surface proteins like Msa, which binds to mannose residues present on the intestinal cells. Such interactions are believed to be important for the health-promoting effects of probiotics, including displacement of pathogens, immunomodulation, and protective effects on the intestinal barrier function. In this study, we have identified a new molecular switch controlling expression of the
gene in
strain WCFS1. Strains with increased
expression could be valuable in the development and manufacture of improved probiotic products.
Abstract
The use of the Gram-positive bacterium Lactococcus lactis in recombinant protein production has several advantages, including the organism's long history of safe use in food production and ...the fact that it does not produce endotoxins. Furthermore the current non-dairy L. lactis production strains contain few proteases and can secrete stable recombinant protein to the growth medium. The P170 expression system used for recombinant protein production in L. lactis utilizes an inducible promoter, P170, which is up-regulated as lactate accumulates in the growth medium. We have optimised the components of the expression system, including improved promoter strength, signal peptides and isolation of production strains with increased productivity. Recombinant proteins are produced in a growth medium with no animal-derived components as a simple batch fermentation requiring minimal process control. The accumulation of lactate in the growth medium does, however, inhibit growth and limits the yield from batch and fed-batch processes. We therefore combined the P170 expression system with the REED™ technology, which allows control of lactate concentration by electro-dialysis during fermentation. Using this combination, production of the Staphylococcus aureus nuclease reached 2.5 g L−1.
This study describes the development of a simple production system for manufacturing recombinant proteins and enzymes in a safe bacterial host.
Purpose
Production and characterization of a chimeric fusion protein (GMZ2’.10C) which combines epitopes of key malaria parasite antigens: glutamate-rich protein (GLURP), merozoite surface protein 3 ...(MSP3), and the highly disulphide bonded
Pf
s48/45 (10C). GMZ2’.10C is a potential candidate for a multi-stage malaria vaccine that targets both transmission and asexual life-cycle stages of the parasite.
Methods
GMZ2’.10C was produced in
Lactococcus lactis
and purified using either an immunoaffinity purification (IP) or a conventional purification (CP) method. Protein purity and stability was analysed by RP-HPLC, SEC-HPLC, 2-site ELISA, gel-electrophoresis and Western blotting. Structural characterization (mass analysis, peptide mapping and cysteine connectivity mapping) was performed by LC-MS/MS.
Results
CP-GMZ2’.10C resulted in similar purity, yield, structure and stability as compared to IP-GMZ2’.10C. CP-GMZ2’.10C and IP-GMZ2’.10C both elicited a high titer of transmission blocking (TB) antibodies in rodents. The intricate disulphide-bond connectivity of C-terminus
Pf
s48/45 was analysed by tandem mass spectrometry and was established for GMZ2’.10C and two reference fusion proteins encompassing similar parts of
Pf
s48/45.
Conclusion
GMZ2’.10C, combining GMZ2’ and correctly-folded
Pf
s48/45 can be produced by the
Lactoccus lactis
P170 based expression system in purity and quality for pharmaceutical development and elicit high level of TB antibodies. The cysteine connectivity for the 10C region of
Pf
s48/45 was revealed experimentally, providing an important guideline for employing the
Pf
s48/45 antigen in vaccine design.
The authors have previously reported the identification of novel signal peptides (SPs) from Lactococcus lactis using transposon insertion. Of these, SP310 caused the highest level of secretion. ...However, the levels were lower than those obtained using the signal peptide from Usp45 (SPUSP), the major secreted lactococcal protein. In this study, site-directed mutagenesis of signal peptide SP310 was used to investigate the effect of amino acid alterations on lactococcal secretion and to improve secretion efficiency. Several mutated SPs caused higher secretion. This increase in secretion was due to modifications in the cleavage region. In fermenter experiments, the signal peptide SP310mut2 resulted in an extracellular Staphylococcus aureus nuclease (Nuc) yield which was 45 % higher than that with the natural SP310. Surprisingly, increasing the hydrophobicity of the hydrophobic core or increasing the number of positively charged amino acids in the N-terminal region of SP310 decreased secretion. High extracellular yields of Nuc resulted from more efficient secretion, as strains with less efficient SPs accumulated more intracellular SP-Nuc precursor.
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