Silvia Llopis

A cocoa peptide protects Caenorhabditis elegans from oxidative stress and β-amyloid peptide toxicity.

Background Cocoa and cocoa-based products contain different compounds with beneficial properties for human health. Polyphenols are the most frequently studied, and display antioxidant properties. Moreover, protein content is a very interesting source of antioxidant bioactive peptides, which can be used therapeutically for the prevention of age-related diseases. Methodology/Principal Findings A bioactive peptide, 13L (DNYDNSAGKWWVT), was obtained from a hydrolyzed cocoa by-product by chromatography. The in vitro inhibition of prolyl endopeptidase (PEP) was used as screening method to select the suitable fraction for peptide identification. Functional analysis of 13L peptide was achieved using the transgenic Caenorhabditis elegans strain CL4176 expressing the human Aβ1–42 peptide as a pre-clinical in vivo model for Alzheimers disease. Among the peptides isolated, peptide 13L (1 µg/mL) showed the highest antioxidant activity (P≤0.001) in the wild-type strain (N2). Furthermore, 13L produced a significant delay in body paralysis in strain CL4176, especially in the 24–47 h period after Aβ1–42 peptide induction (P≤0.0001). This observation is in accordance with the reduction of Aβ deposits in CL4176 by western blot. Finally, transcriptomic analysis in wild-type nematodes treated with 13L revealed modulation of the proteosomal and synaptic functions as the main metabolic targets of the peptide. Conclusions/Significance These findings suggest that the cocoa 13L peptide has antioxidant activity and may reduce Aβ deposition in a C. elegans model of Alzheimers disease; and therefore has a putative therapeutic potential for prevention of age-related diseases. Further studies in murine models and humans will be essential to analyze the effectiveness of the 13L peptide in higher animals.

In vivo virulence of commercial Saccharomyces cerevisiae strains with pathogenicity-associated phenotypical traits.

Two commercial Saccharomyces cerevisiae strains, a bakers strain and the bio-therapeutic agent Ultralevure, have been proposed as a possible exogenous source of human colonization (de Llanos et al., 2004, 2006a). Moreover, these strains express phenotypical traits associated to pathogenicity (de Llanos et al., 2006b). Taking into account that both commercial preparations represent an important source of living S. cerevisiae cells we have performed an in vivo study to evaluate whether there is a potential safety risk to humans. Their virulence was compared with that of other commercial strains with less virulent traits, and with clinical isolates, using two murine models (BALB/c and DBA/2N mice). Burden determination in the brain and kidneys showed that the ability to disseminate, colonize and persist was manifested not only by clinical isolates but also by commercial strains. Among these, the bakers strain and Ultralevure were able to cause the death of BALB/c mice at rates similar to those shown by two of the clinical isolates. These results highlight the pathogenic potential of these strains and show that four-week-old BALB/c mice are an appropriate murine model to study the virulence of yeasts with low or moderate pathogenicity. Furthermore, we have shown the positive effect of an immunosuppressive therapy with cyclophosphamide in the virulence of the bakers strains and Ultralevure but not in the rest of the commercial strains under study. The data suggest that although S. cerevisiae has always been considered a GRAS microorganism, commercial preparations should include only those strains shown to be safe in order to minimize complications in risk groups.

In Vivo and In Vitro Studies on Virulence and Host Responses to Saccharomyces cerevisiae Clinical and Non-Clinical Isolates

We have studied the virulence and host responses to several clinical and non-clinical Saccharomyces cerevisiae isolates: two vaginal isolates (60, 61), one isolate from faeces (20), a brewers yeast isolate used in dietetics (D14), one S. boulardii isolate from a commercial probiotic product, and a reference natural wine yeast (CECT 10431). Hematogenously disseminated infection in a mouse model demonstrated that four isolates (all, except 20 and 10431) were able to colonize preferentially the brain, as well as kidney and spleen, to a lesser extent, of immunocompetent mice. In vitro adhesion assays to epithelial and endothelial cell lines also showed an increased adherence ability of strains 60, 61, D14 and S. boulardii. In vitro cytokine production assays by RAW 264.7 murine macrophages challenged with yeasts showed a relative increased production of TNF- in response to the 20 and 10431 strains; viability of RAW cells after coculture was similar in all cases (2-5% non-viable cells) except for 60 strain (11% non-viable cells). In vitro phagocytosis assays of yeasts by RAW cells showed that two isolates (D14 and particularly S. boulardii) were engulfed less efficiently. These results point out that S. cerevisiae isolates, from both clinical and non-clinical (dietetic and probiotic) origin, may vary in the expression of putative virulence factors contributing to their ability to develop the infectious process.

Probiotic Strain Bifidobacterium animalis subsp. lactis CECT 8145 Reduces Fat Content and Modulates Lipid Metabolism and Antioxidant Response in Caenorhabditis elegans

Recently, microbial changes in the human gut have been proposed as a possible cause of obesity. Therefore, modulation of microbiota through probiotic supplements is of great interest to support obesity therapeutics. The present study examines the functional effect and metabolic targets of a bacterial strain, Bifidobacterium animalis subsp. lactis CECT 8145, selected from a screening in Caenorhabditis elegans. This strain significantly reduced total lipids (40.5% ± 2.4) and triglycerides (27.6% ± 0.5), exerting antioxidant effects in the nematode (30% ± 2.8 increase in survival vs control); activities were also preserved in a final food matrix (milk). Furthermore, transcriptomic and metabolomic analyses in nematodes fed with strain CECT 8145 revealed modulation of the energy and lipid metabolism, as well as the tryptophan metabolism (satiety), as the main metabolic targets of the probiotic. In conclusion, our study describes for the first time a new B. animalis subsp. lactis strain, CECT 8145, as a promising probiotic for obesity disorders. Furthermore, the data support future studies in obesity murine models.

Clinical Saccharomyces cerevisiae isolates cannot cross the epithelial barrier in vitro

Saccharomyces cerevisiae is generally considered to be a safe organism and is essential to produce many different kinds of foods as well as being widely used as a dietary supplement. However, several isolates, which are genetically related to brewing and baking yeasts, have shown virulent traits, being able to produce human infections in immunodeficient patients. Previously it has been shown that the administration of S. cerevisiae clinical isolates can lead to systemic infections, reaching several organs in murine systems. In this work, we studied S. cerevisiae clinical isolates in an in vitro intestinal epithelial barrier model, comparing their behaviour with that of several strains of the related pathogens Candida glabrata and Candida albicans. The results showed that, in contrast to C. glabrata and C. albicans, S. cerevisiae was not able to cross the intestinal barrier. We concluded that S. cerevisiae can only perform opportunistic or passive crossings when epithelial barrier integrity is previously compromised.

Transcriptomics in human blood incubation reveals the importance of oxidative stress response in Saccharomyces cerevisiae clinical strains.

BackgroundIn recent years an increasing number of yeast infections in humans have been related to certain clinical isolates of Saccharomyces cerevisiae. Some clinical strains showed in vivo and in vitro virulence traits and were able to cause death in mice whereas other clinical strains were avirulent.ResultsIn this work, we studied the transcriptional profiles of two S. cerevisiae clinical strains showing virulent traits and two control non-virulent strains during a blood incubation model and detected a specific transcriptional response of clinical strains. This response involves an mRNA levels increase of amino acid biosynthesis genes and especially oxidative stress related genes. We observed that the clinical strains were more resistant to reactive oxygen species in vitro. In addition, blood survival of clinical isolates was high, reaching similar levels to pathogenic Candida albicans strain. Furthermore, a virulent strain mutant in the transcription factor Yap1p, unable to grow in oxidative stress conditions, presented decreased survival levels in human blood compared with the wild type or YAP1 reconstituted strain.ConclusionsOur data suggest that this enhanced oxidative stress response in virulent clinical isolates, presumably induced in response to oxidative burst from host defense cells, is important to increase survival in human blood and can help to infect and even produce death in mice models.

Pathogenic potential of Saccharomyces strains isolated from dietary supplements.

Saccharomyces cerevisiae plays a beneficial role in health because of its intrinsic nutritional value and bio-functional properties, which is why it is also used as a dietary supplement. However, the perception that S. cerevisiae is harmless has changed due to an increasing number of infections caused by this yeast. Given this scenario, we have tested whether viable strains contained in dietary supplements displayed virulence-associated phenotypic traits that could contribute to virulence in humans. We have also performed an in vivo study of the pathogenic potential of these strains using a murine model of systemic infection by intravenous inoculation. A total of 5 strains were isolated from 22 commercial products and tested. Results highlight one strain (D14) in terms of burden levels in brains and kidneys and ability to cause death, whereas the other two strains (D2 and D4) were considered of low virulence. Our results suggest a strong relationship between some of the virulence-associated phenotypic traits (ability to grow at 39°C and pseudohyphal growth) and the in vivo virulence in a mouse model of intravenous inoculation for isolates under study. The isolate displaying greatest virulence (D14) was evaluated in an experimental murine model of gastrointestinal infection with immunosuppression and disruption of mucosal integrity, which are common risk factors for developing infection in humans, and results were compared with an avirulent strain (D23). We showed that D14 was able to spread to mesenteric nodes and distant organs under these conditions. Given the widespread consumption of dietary supplements, we recommend only safe strains be used.

Comparative Genomic Analysis Reveals a Critical Role of De Novo Nucleotide Biosynthesis for Saccharomyces cerevisiae Virulence

In recent years, the number of human infection cases produced by the food related species Saccharomyces cerevisiae has increased. Whereas many strains of this species are considered safe, other ‘opportunistic’ strains show a high degree of potential virulence attributes and can cause infections in immunocompromised patients. Here we studied the genetic characteristics of selected opportunistic strains isolated from dietary supplements and also from patients by array comparative genomic hybridization. Our results show increased copy numbers of IMD genes in opportunistic strains, which are implicated in the de novo biosynthesis of the purine nucleotides pathway. The importance of this pathway for virulence of S. cerevisiae was confirmed by infections in immunodeficient murine models using a GUA1 mutant, a key gene of this pathway. We show that exogenous guanine, an end product of this pathway in its triphosphorylated form, increases the survival of yeast strains in ex vivo blood infections. Finally, we show the importance of the DNA damage response that activates dNTP biosynthesis in yeast cells during ex vivo blood infections. We conclude that opportunistic yeasts may use an enhanced de novo biosynthesis of the purine nucleotides pathway to increase survival and favor infections in the host.

Molecular Identification and Characterization of Wine Yeasts

The transformation of grape must into wine is a complex microbiological process involving the sequential growth of bacteria and yeasts, although only the yeasts are responsible for alcoholic fermentation. In the past, winemaking was purely empirical, but it is now a well-understood, controlled process that has been gradually improved over time. Advances have largely been made possible by the development of molecular techniques to identify and characterize wine yeasts based on analysis of their DNA. These methods are rapid, reproducible, and sensitive, and continue to be used for a variety of purposes, such as analyzing variation in naturally occurring and inoculated yeast populations, monitoring the dynamics of inoculated strains, characterizing wine yeasts, and detecting spoilage yeasts.

A nutritional supplement containing lactoferrin stimulates the immune system, extends lifespan, and reduces amyloid β peptide toxicity in Caenorhabditis elegans

Abstract Lactoferrin is a highly multifunctional glycoprotein involved in many physiological functions, including regulation of iron absorption and immune responses. Moreover, there is increasing evidence for neuroprotective effects of lactoferrin. We used Caenorhabditis elegans as a model to test the protective effects, both on phenotype and transcriptome, of a nutraceutical product based on lactoferrin liposomes. In a dose‐dependent manner, the lactoferrin‐based product protected against acute oxidative stress and extended lifespan of C. elegans N2. Furthermore, Paralysis of the transgenic C. elegans strain CL4176, caused by Aβ1‐42 aggregates, was clearly ameliorated by treatment. Transcriptome analysis in treated nematodes indicated immune system stimulation, together with enhancement of processes involved in the oxidative stress response. The lactoferrin‐based product also improved the protein homeostasis processes, cellular adhesion processes, and neurogenesis in the nematode. In summary, the tested product exerts protection against aging and neurodegeneration, modulating processes involved in oxidative stress response, protein homeostasis, synaptic function, and xenobiotic metabolism. This lactoferrin‐based product is also able to stimulate the immune system, as well as improving reproductive status and energy metabolism. These findings suggest that oral supplementation with this lactoferrin‐based product could improve the immune system and antioxidant capacity. Further studies to understand the molecular mechanisms related with neuronal function would be of interest.