Variabilitat de la xarxa LexA en els bacteris

Consultable des del TDXTítol obtingut de la portada digitalitzadaEl sistema SOS és una xarxa multigènica sota el control negatiu del repressor LexA, amb importància vital per a la supervivència cel·lular ja que la seva desrepressió es dóna quan la cèl·lula presenta danys en el material genètic que poden ser transmesos a la descendència. Aquest fet, junt amb la seva amplia distribució en el domini Bacteria ens presenten el coneixement del seu funcionament en els diferents grups com una eina filogenètica idònia. Per a tal fi, i com a fil conductor d'aquest treball, és va procedir a l'estudi del reguló LexA a Dehalococcoides ethenogenes, un bacteri verd no sulfurós; Magnetococcus sp. soca MC-1,un bacteri magnetotàctic; i Leptospira interrogans serovar Lai, la primera espiroqueta amb un lexA identificat. Per a tal es va procedir a la clonació i purificació del gen lexA de cada un d'ells i a la purificació del seu producte. En el primer d'ells es va definir la caixa d'unió de LexA (AGAACN4GTTCT), comprovant a partir d'ella els seus vincles amb els bacteris grampositius, així com la no regulació del gen recA, considerat com a canònic, pel mateix. A Magnetococcus sp. soca MC-1, la caixa SOS descrita fou GTTCN7GTTC. Fins el moment, aquest microorganisme es trobava ubicat dins la subclasse Alfa del Proteobacteris, però tant el coneixement del motiu d'unió del seu LexA com els gens que integraven el reguló, ens el mostren com una branca estretament relacionada amb aquesta classe, però clarament independent. A Leptospira interrogans serovar Lai, el resultat fou sorprenent, ja que la caixa SOS d'aquest microorganisme, la varem haver d'identificar a partir de la regió promotora de recA, ja que no era present en la de lexA, esdevenint així el primer exemple en el que LexA no autoregula la seva expressió. Aquesta dada així com el fet de no poder detectar cap altre gen sota el seu control, ens mostren Leptospira, com un pas entremig en la tendència evolutiva seguida en les espiroquetes de perdre el seu gen lexA. Els següents articles es presenten a la secció d'annexes, i en ells es descriuen i discuteixen els resultats sobre els quals està basada aquesta tesis.: I. Fernandez de Henestrosa, A.R., J. Cuñé, I. Erill, J.K. Magnuson, i J. Barbé. 2002. A green nonsulfur bacterium, Dehalococcoides ethenogenes, with the LexA binding sequence found in gram-positive organisms. J. Bacteriol. 184(21): 6073 - 6080. II. Fernandez de Henestrosa, A.R., J. Cuñé, G. Mazón, B.L. Dubbels, D.A. Bazylinski, i J. Barbé. 2003. Characterization of a new LexA binding motif in the marine magnetotactic bacterium strain MC-1. J. Bacteriol. 185: 4471 - 4482. III. Cuñé, J., P. Cullen, G. Mazon, S. Campoy, B. Adler, i J. Barbé. 2005. The Leptospira interrogans lexA gene is not autoregulated. J. Bacteriol. 187: 5841 - 5845

Reduction of ureteral stent encrustation by modulating the urine pH and inhibiting the crystal film with a new oral composition: a multicenter, placebo controlled, double blind, randomized clinical trial

Background: Encrustation of ureteral double J stents is a common complication that may affect its removal. The aim of the proposed study is to evaluate the efficacy and safety of a new oral composition to prevent double J stent encrustation in indwelling times up to 8 weeks. Methods: A double-blinded, multicenter, placebo-controlled trial was conducted with 105 patients with indwelling double J stents enrolled across 9 public hospitals in Spain. The patients were randomly assigned (1:1) into intervention (53 patients) or placebo (52 patients) groups for 3 to 8 weeks and both groups self-monitored daily their morning urine pH levels. The primary outcome of analysis was the degree of stent ends encrustation, defined by a 4-point score (0 - none; 3 - global encrustation) using macroscopic and electron microscopy analysis of crystals, after 3 to 8-w indwelling period. Score was exponentially transformed according to calcium levels. Secondary endpoints included urine pH decrease, stent removal, and incidence of adverse events. Results: The intervention group benefits from a lower global encrustation rate of stent ends than placebo group (1% vs 8.2%; p < 0.018). Mean encrustation score was 85.12 (274.5) in the placebo group and 18.91 (102.27) in the intervention group (p < 0.025). Considering the secondary end points, treated patients reported greater urine pH decreases (p = 0.002). No differences in the incidence of adverse events were identified between the groups. Conclusions: Our data suggest that the use of this new oral composition is beneficial in the context of ureteral double J indwelling by decreasing mean, as well as global encrustation

Potent induction of trained immunity by Saccharomyces cerevisiae β-glucans

Candida albicans cell wall component β-glucan has been extensively studied for its ability to induce epigenetic and functional reprogramming of innate immune cells, a process termed trained immunity. We show that a high-complexity blend of two individual β-glucans from Saccharomyces cerevisiae possesses strong bioactivity, resulting in an enhanced trained innate immune response by human primary monocytes. The training required the Dectin-1/CR3, TLR4, and MMR receptors, as well as the Raf-1, Syk, and PI3K downstream signaling molecules. By activating multiple receptors and downstream signaling pathways, the components of this β-glucan preparation are able to act synergistically, causing a robust secondary response upon an unrelated challenge. In in-vivo murine models of melanoma and bladder cell carcinoma, pre-treatment of mice with the β-glucan preparation led to a significant reduction in tumor growth. These insights may aid in the development of future therapies based on β-glucan structures that induce an effective trained immunity response

Anti-Inflammatory Effects, Protection of Gut Barrier Integrity and Stimulation of Phagocytosis of Postbiotic Combination ABB C1

This study evaluated the anti-inflammatory effects, the protection of gut barrier integrity, and the stimulation of phagocytosis in peripheral cells of a nutritional supplement based on a synergistic combination of yeast-based ingredients with a unique 1,3/1,6-glucan complex and a consortium of postbiotic Saccharomyces cerevisiae rich in selenium and zinc. The anti-inflammatory effect in caco-2 cells in the presence and absence of a pro-inflammatory challenge (tumour necrosis factor alpha [TNF-&alpha;]/interferon gamma [IFN-&#611;]) showed statistically significant reductions in IFN-&#611; induced protein-10 (IP-10), and monocyte chemoattractant protein-1 (MCP-1) levels vs. controls (p &lt; 0.001). Disruption of the gut integrity in the presence or absence of Escherichia coli (ETEC H10407) showed transepithelial electrical resistance (TEER) values higher in the ABB C1&reg; group after 6 h of testing. Spontaneous build-up of the gut epithelium monolayer over 22 days was also greater in the ABB C1&reg; condition vs. a negative control. ABB C1&reg; showed a significantly higher capacity to stimulate phagocytosis as compared with controls of algae &beta;-1,3-glucan and yeast &beta;-1,3/1,6 glucan (p &lt; 0.001). This study supports the mechanism of action by which ABB C1&reg; may improve the immune response and be useful to prevent infection and allergy in clinical practice

The Leptospira interrogans lexA Gene Is Not Autoregulated

Footprinting and mutagenesis experiments demonstrated that Leptospira interrogans LexA binds the palindrome TTTGN(5)CAAA found in the recA promoter but not in the lexA promoter. In silico analysis revealed that none of the other canonical SOS genes is under direct control of LexA, making the leptospiral lexA gene the first described which is not autoregulated

A Green Nonsulfur Bacterium, Dehalococcoides ethenogenes, with the LexA Binding Sequence Found in Gram-Positive Organisms

Dehalococcoides ethenogenes is a member of the physiologically diverse division of green nonsulfur bacteria. Using a TBLASTN search, the D. ethenogenes lexA gene has been identified, cloned, and expressed and its protein has been purified. Mobility shift assays revealed that the D. ethenogenes LexA protein specifically binds to both its own promoter and that of the uvrA gene, but not to the recA promoter. Our results demonstrate that the D. ethenogenes LexA binding site is GAACN(4)GTTC, which is identical to that found in gram-positive bacteria. In agreement with this fact, the Bacillus subtilis DinR protein binds specifically to the D. ethenogenes LexA operator. This constitutes the first non-gram-positive bacterium exhibiting a LexA binding site identical to that of B. subtilis

Characterization of a New LexA Binding Motif in the Marine Magnetotactic Bacterium Strain MC-1

MC-1 is a marine, magnetotactic bacterium that is phylogenetically associated with the alpha subclass of the Proteobacteria and is the first and only magnetotactic coccus isolated in pure culture to date. By using a TBLASTN search, a lexA gene was identified in the published genome of MC-1; it was subsequently cloned, and the protein was purified to >90% purity. Results from reverse transcription-PCR analysis revealed that the MC-1 lexA gene comprises a single transcriptional unit with two open reading frames encoding proteins of unknown function and with a rumA-like gene, a homologue of the Escherichia coli umuD gene. Mobility shift assays revealed that this LexA protein specifically binds both to its own promoter and to that of the umuDC operon. However, MC-1 LexA does not bind to the promoter regions of other genes, such as recA and uvrA, that have been previously reported to be regulated by LexA in bacterial species belonging to the alpha subclass of the Proteobacteria. Site-directed mutagenesis of both the lexA and umuDC operator regions demonstrated that the sequence CCTN(10)AGG is the specific target motif for the MC-1 LexA protein

Effect of the degree of polymerization of fructans on ex vivo fermented human gut microbiome

Prebiotic supplements are used to promote gastrointestinal health by stimulating beneficial bacteria. The aim of this study was to compare the potential prebiotic effects of fructans with increasing degrees of polymerization, namely fructooligosaccharides (FOS) and inulins with a low and high polymerization degree (LPDI and HPDI, respectively), using an ex vivo fermentation system to simulate the colonic environment. The system was inoculated with pooled feces from three healthy donors with the same baseline enterotype. Changes in microbiota composition were measured by 16S metagenomic sequencing after 2, 7, and 14 days of fermentation, and acid production was measured throughout the experiment. Alpha-diversity decreased upon inoculation of the ex vivo fermentation under all treatments. Composition changed significantly across both treatments and time (ANOSIM p < 0.005 for both factors). HPDI and LPDI seemed to be similar to each other regarding composition and acidification activity, but different from the control and FOS. FOS differed from the control in terms of composition but not acidification. HDPI restored alpha-diversity on day 14 as compared to the control (Bonferroni p < 0.05). In conclusion, the prebiotic activity of fructans appears to depend on the degree of polymerization, with LPDI and especially HPDI having a greater effect than FOS

JOURNAL OF BACTERIOLOGY, Nov. 2002, p. 6073--6080 Vol. 184, No. 21 0021-9193/02/$04.000 DOI: 10.1128/JB.184.21.6073--6080.2002

this report, there were no data available pertaining to the motif recognized by LexA in this bacterial division. Finally, * Corresponding author. Mailing address: Universitat Autonoma de Barcelona, Departament de Genetica i Microbiologia, Unitat de Microbiologia, Edifici C, Bellaterra, 08193 Barcelona, Spain. Phone: 34 93 581 1837. Fax: 34 93 581 23 87. E-mail for Antonio R. Fernandez de Henestrosa: [email protected]. E-mail for Jordi Barbe: [email protected]

DataSheet_1_Potent induction of trained immunity by Saccharomyces cerevisiae β-glucans.docx

Candida albicans cell wall component β-glucan has been extensively studied for its ability to induce epigenetic and functional reprogramming of innate immune cells, a process termed trained immunity. We show that a high-complexity blend of two individual β-glucans from Saccharomyces cerevisiae possesses strong bioactivity, resulting in an enhanced trained innate immune response by human primary monocytes. The training required the Dectin-1/CR3, TLR4, and MMR receptors, as well as the Raf-1, Syk, and PI3K downstream signaling molecules. By activating multiple receptors and downstream signaling pathways, the components of this β-glucan preparation are able to act synergistically, causing a robust secondary response upon an unrelated challenge. In in-vivo murine models of melanoma and bladder cell carcinoma, pre-treatment of mice with the β-glucan preparation led to a significant reduction in tumor growth. These insights may aid in the development of future therapies based on β-glucan structures that induce an effective trained immunity response.</p