Host-probiotic interaction: in viltro analyses

ABSTRACT Functional foods can positively influence functions of the body, by improving the health or reducing the risk of disease. Some functional foods contain ‘probiotics’, defined as ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’. The development and use of in vitro and in vivo protocols to assess the probiotic efficacy of microorganisms are highly encouraged by FAO and WHO. In this thesis, the probiotic potential of the lactic acid bacterium Lactobacillus plantarum, wild type and derivative mutant strains, was investigated. The distinctive cell surface features exhibited by stress gene mutants prompted us to produce, by gene knockout, other L. plantarum defective strains and led us to investigate whether these characteristics could affect host-microbe interaction. The bacterial survival of L. plantarum strains and commercial probiotics was evaluated by designing an in vitro system simulating the transit along the human oro-gastrointestinal tract. Different carrier matrices were assayed in relation to possible prebiotic effects. The bacterial molecular response to such stresses was monitored by analysing the expression of stress, adhesion and probiosis genes. Interaction with the host was studied in vitro by i) assessing bacterial adhesive ability to gut epithelial cells; ii) investigating anti-inflammatory properties and induction of innate immunity genes in human host cells. L. plantarum strains were resistant to the combined stress at the various steps of the simulated oro-gastrointestinal tract. Major decreases in viability were observed mainly under drastic acidic conditions (pH ≤ 2.0) of the gastric compartment. Abiotic stresses associated to the intestinal environment (small intestine) poorly affected bacterial vitality. The protective effect of vehicle matrices correlated with composition and bacterial nutritional needs. A relationship was found between bacterial survival and stress gene pattern. All strains significantly adhered to human intestinal epithelial cells, with the ΔctsR L. plantarum mutant exhibiting the highest adhesion. Colonization ability was improved by addition of prebiotics. Supernatants from all strains of L. plantarum reduced proinflammatory cytokine secretion by activated human immune cells. Induction of immune-related genes resulted generally higher upon incubation with heat-inactivated bacteria, rather than with live ones. For specific genes, a differential transcriptional pattern was observed upon stimulation with the different L. plantarum strains, pointing to a possible role of the knocked out bacterial genes in modulation of host cells response. Particularly, cells from Δhsp18.55 and ΔftsH mutants strongly triggered immune defence genes. This study highlights the relevance of the microbial genetic background in host-probiotic interaction and might contribute to: i) define selection criteria and/or conditions for probiotic screening and delivery; ii) identify candidate bacterial genes and/or molecules involved in probiosis, so to tailor probiotics for specific clinical applications.