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Cell wall mutants of lactic acid bacteria with enhanced immunomodulatory properties

The fact that probiotics may influence the intestinal physiology through modulation of the endogenous flora or the immune system is presently well recognised. For example, numerous observations support the claim that given LAB strains may act as immunomodulators (i.e. may stimulate the immune system or reduce abnormal situations such as inflammation), a property that seem to vary substantially between species and perhaps between strains belonging to the same species. As for other probiotic traits, there is virtually no understanding of the mechanisms accounting for the observed effect and of the bacterial components involved in the cross-talk between a probiotic and the host. As a consequence it is almost impossible today to propose a rational screening procedure to select new and efficient probiotic strains. As the bacterial cell wall is probably recognized at first by the intestinal immune system, mutants affected in their cell wall structure were constructed in order to verify if modification of the lipoteichoic acids leads to altered immunomodulation capacity.

Mutations in the alanine racemase encoding gene (Alr- mutant) or in the D-alanine incorporation operon (Dlt- mutant) were studied in two genetic backgrounds, i.e. Lactobacillus plantarum and Lactococcus lactis. These strains were chosen because they differ substantially in their capacity to survive in the mammal gastro-intestinal tract. The cell wall mutants were first compared in vitro to their wild type counterpart, mainly for their capacity to induce secretion of specific cytokines from human peripheral mononuclear cells (PBMC). This led to the identification of two types of interesting candidates that presented somehow opposite immune properties. Thus strains with opposite cytokine stimulation profile were selected to analyse their in vivo effect in mouse models mimicking human diseases. First, the Dlt- mutant of L. plantarum (defective in D-Ala incorporation in teichoic acids (TA)) turned out to be much more anti-inflammatory than the parent strain, both in vitro and in vivo.

Experiments conducted in the mouse TNBS colitis model proved it to be equally protective as a L. lactis secreting mIL-10. The second L. plantarum cell wall mutant selected for its altered immunological properties was the Alr- mutant that produces modified TA and peptidoglycan (PG) as a results of a defect in Alanine racemase. This mutant was used as live vaccine delivery vehicle, first with a potent immunogen, i.e. the C subunit of Tetanus toxin (TTFC). Mice immunisation experiments by the intragastric, intravaginal and rectal routes proved that it behaved as a substantially improved antigen delivery system as compared to the WT strain, especially leading to enhanced mucosal immune responses. The Alr- mutant of L. plantarum and L. lactis behave similarly. This remarkable property was confirmed by the construction and immunological evaluation of an L. plantarum Alr- recombinant strain producing the UreB antigen of H. pylori. From all the candidate anti-Helicobacter strains constructed within the project, only the Alr-/UreB strain was shown to partially protect against a Helicobacter challenge, upon intragastric immunization and in absence of mucosal adjuvants. Quite surprisingly, the L. lactis Dlt- mutant exhibited different properties than the equivalent mutant in L. plantarum, as it turned out to be much more immunogenic than its wild type counterpart.

In summary, cell wall mutants have been engineered that exhibit significantly enhanced immune properties as compared to the wild type strains. One strain corresponds to a much more potent anti-inflammatory candidate that induced equivalent protection in experimental colitis as a strain producing a heterologous immune modulator, i.e. IL-10. The three other ones present a remarkably enhanced capacity to deliver antigens at mucosal surfaces, thus representing improved candidates to develop live mucosal vaccines. This work allowed us to highlight the importance of the TA and PG composition in the immune modulation properties of given strains, and to identify host factors involved in their recognition. In addition, the Alr- mutants which are depending on D-Alanine for growth, should behave as biologically contained strains.

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Institut Pasteur de Lille
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