EXPLORING LACTOBACILLUS PARACASEI PROBIOSIS AND METABOLIC POTENTIAL

Probiotics have been used so far for the prevention and treatment of various medical conditions and to support general wellness: for this reason, they are currently the subject of significant microbiological and clinical research. In fact, a body of literature suggests that including probiotics in the diet can be a strategy to reduce host-related immune diseases and, in general, modulate the intestinal microbiota composition. Although the mechanisms of action of probiotics are still largely unknown, particularly at molecular level, it is well understood that they can act in different ways, through interaction with the other bacteria residing the same niche, as well as the host, at both local and systemic levels. The genus Lactobacillus, which has important industrial applications as fermented food starter and probiotic adjunct, is a taxonomically broad and heterogeneous group and includes the species Lactobacillus paracasei, which is generally associated with habitats rich in nutrients, such as dairy food and human ecosystems, like gut and vagina. The main purpose of my PhD activity, concerned the study of two L. paracasei strains, named DG and LPC-S01, isolated from two different ecological niches (human gut and vagina, respectively). They are already available on the market, included in two products as food supplements, but their characterization is still incomplete, particularly for LPC-S01. In order to deepen the knowledge about these strains, the first part of the work focused on exploring L. paracasei DG and LPC-S01 essential characteristics to define potential probiotics, by using L. paracasei Shirota as reference strain. The comparative genomic analyses evidenced that strain LPC-S01, a bacterium isolated from human vagina, but plausibly having its origin in the gut, resulted having the genetic features of a niche-generalist member of its species. Similarly, strain DG exhibited the potential ability to adapt to a wide range of environmental conditions if compared with other strains of dairy origin. In vitro tests conventionally used to evidence probiotic properties revealed that strains LPC-S01 and DG possessed comparable ability to resist to gastro-intestinal transit, as evidenced by tolerance to bile, and to decrease NF-κB activation in Caco-2 cells, with respect to strain Shirota. Moreover, LPC-S01 displayed higher tolerance to gastric juice and higher capacity to adhere to Caco-2 epithelial cells (whereas Shirota showed inability to adhere on Caco-2-cells). The in vitro observations were confirmed by setting up a pilot intervention trial on healthy adult volunteers, that demonstrated that LPC-S01 and DG can transiently colonize the gastrointestinal tract of the host, persisting for at least 5 days after the end of a 7-days oral consumption (corresponding to an average of 7 evacuations). Thanks to the comparative genomic analysis on L. paracasei strains DG and LPC-S01, we identified two gene clusters putatively coding for exopolysaccharides biosynthesis related enzymes. Exopolysaccharides (EPSs), apart from their industrial applications, are found to be associated with many physiological functions, although their mechanism of action has not been fully clarified yet. In collaboration with Prof. Andy Laws, University of Huddersfield (United Kingdom), the second part of the work focused on the identification of the potential EPSs matrix from both strains DG and LPC-S01, and their structural characterization. Nonetheless, in the experimental conditions tested so far, only DG resulted able to synthetize EPSs. After performing its purification, we characterized DG derived EPS repeating unit by NMR spectroscopy based approaches. DG EPS structure resulted peculiar and unique compared to those identified in other lactic acid bacteria, prompting us to investigate its immunomodulatory potential, by using it as stimulus on phagocytes. Interestingly, THP-1 macrophages were highly responsive to the EPS stimulus, particularly through the activation of COX-2 expression. Moreover, COX-2 expression was also subjected to an additive effect due to the combination of EPS with the pro-inflammatory stimulus of lipopolysaccharides (LPS). This results strongly suggest a role of DG and its secreted polysaccharidic molecule in triggering stimulatory immune responses together with the activation of protective mechanisms of the intestinal mucosa. Starting by previous observations on other lactic acid bacteria, particularly on Lactococcus lactis, which demonstrated the ability to switch from fermentation to respiratory metabolism, and thanks to the identification of the operon cydABCD in L. paracasei, the third part of the work focused on evaluating if respiration was activated also in the strains under study. The respiratory metabolism, activated by the addition of heme and menaquinone, two essential co-factors not synthetized by the cell, typically results in two main advantages for the cell: increased biomass and long-term survival. Our preliminary data, however, indicated that only one of the two phenotypes occurred in L. paracasei, namely a very strong robustness achieved only upon addition of both heme and menaquinone, that resulted in the maintenance of viability for long periods of storage at 4° C (>200 days). The robustness phenotype was present also when the two co-factors were added during the storage time, and not during growth. This fact, along with (i) the detection of the added heme only outside the cells, (ii) the under regulation of cydA evaluated by gene expression analyses, and (iii) the decrease of ATP reservoir, suggested that the phenotype observed was not a consequence of a respiratory metabolism. Our data are instead more consistent with the presence of an external electron transfer, as already described for other intestinal bacteria. A deeper investigation is required to clarify the molecular basis of L. paracasei acquired resistance. However, our collected evidences, i.e. the robustness phenotype, can potentially be exploited in industrial applications to ameliorate the technological performances, but also in the niche colonized by the bacterium, where it can counteract environmental stresses and exert its probiotic potential by reducing and neutralizing damaging chemicals in the surrounding environment. In conclusion, this PhD work evidenced the multiple properties of two strains belonging to L. paracasei species to colonize the human gut (and potentially the vagina) upon oral administration, and to interact with the host immune system. Moreover, L. paracasei possesses very interesting metabolic abilities that may be exploited both in in vivo conditions and in the industrial processes.