Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

Recombinant lactic acid bacteria secreting an anti-inflammatory compound

- Construction and evaluation of anti-inflammatory strains
In this part of the work emphasis was on engineering different Lactobacillus strains to secrete murine interleukin-10. Five strains, namely Lb. casei LMG6904, Lb. casei BL23, Lb. plantarum NCIMB 8826, Lb. plantarum LMG 9211 and Lb. rhamnosus LMG 10770 could be transformed and secreted mIL10 . In all cases, the expression module consisted of the constitutive lactococcal P1 promoter and the gene usp-45 secretion leader sequence fused in frame to the encoding mature mIL10 and was located on the high copy number plasmid, pT1mIL10, which specifies resistance to erythromycin. Initial tests with intragastric administration of recombinant Lb. plantarum NCIMB 8826 in the DSS model for chronic colitis set up in Balb/c and C57Bl6 mice did not result in a clear improvement of the anti-inflammatory properties.

In collaboration with IPL we have introduced the trinitrobenzene sulfonic acid (TNBS)-induced colitis model for evaluation in our animal facility. Interestingly, while the recombinant Lc. lactis strain ssequence ecreting mIL-10 protected against colitis and was thus largely superior to its wild type counterpart, this was not observed in the case of Lb. plantarum. This result can best be explained by the fact that the protective effect of IL-10 delivery is very dose dependent. Optimisation of the treatment regimen with Lb. plantarum/mIL-10 is thus necessary before making any firm conclusion. The strains Lb. casei BL 23, Lb. plantarum NCIMB 8826 and Lb. rhamnosus LMG 10770 were further analyzed in greater molecular detail. Using PCR-based detection of the pT1mIL10 plasmid we established that all three strains stably maintained the plasmid after storage at -70°C in MRS medium containing 20% glycerol. The plasmid copy number was essentially the same in all three strains and comparable to that observed in Lactococcus lactis. All three strains secreted biologically active IL-10, albeit in lower amounts than L.lactis.

- In vivo analysis of the recombinant strains
With the aim of following the fate and transit of a bacterial inoculum trough the intestine we constructed a plasmid that expresses the Green Fluorescent Protein (GFP). The GFP gene was cloned under control of the lactococcal P1 promoter. Commercially available variants of GFP, which should give improved fluorescence, could not be successfully used in L. lactis. We then constructed a synthetic GFP gene (GFPma), in which the codons were adapted to the preferred A/T rich codon usage of lactobacilli. Using fluorescence microscopy we could now clearly detect individual bacterial cells from overnight cultures of L. lactis MG1363, transformed with plasmid pP1GFPma. With support from the EU officer, it was decided to extend the collaboration with IPL to the micro array analysis of the colon samples of mice receiving anti-inflammatory LAB strains prior to TNBS colitis induction. Experiments are still in progress.

- Construction of a biologically contained strain
The original aim was the elimination of antibiotic selection markers from the recombinant strains by targeting the expression units to non-essential genomic regions of the lactobacilli (in collaboration with UNISI). In the course of the project it was decided to adapt a different strategy that had already been initiated within another framework at VIB. Rather than merely removing the antibiotic resistance marker, we had set out to construct a GMO that incorporated biological containment features. The target strain was L. lactis MG1363, secreting human IL-10.

Our approach was to exchange the chromosomal thymidylate synthase gene, thyA, for the expression cassette encoding hIL-10. The rationale for choosing the thyA gene derives from the observation that starvation for thymine in a thyA mutant is bactericidal rather than bacteriostatic (as commonly observed for most other auxotrophies). We cloned and sequenced the thyA gene from L. lactis MG1363 together with its upstream and downstream flanking regions. We further constructed conditional-replicative plasmids carrying a series of hIL10 expression cassettes flanked by 1 kb of the regions upstream and downstream of the MG1363 thyA gene. These were used to obtain L. lactis strains in which the thyA gene was replaced by the hIL10 gene following double homologous crossover. A representative strain, designated Thy12, contains the usp45-hIL10 sequence downstream of the MG1363 thyA promoter and secretes hIL-10. Survival of the strain is strictly dependent on the availability of thymine or thymidine. Accumulation of the genetically modified strain in the environment is very unlikely, as rapid death occurs after thymidine starvation.

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