Full Transcriptomic Response of Pseudomonas aeruginosa to an Inulin-Derived Fructooligosaccharide

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2020.00202/full#supplementary-materialPseudomonas aeruginosa is an ubiquitous gram-negative opportunistic human pathogen which is not considered part of the human commensal gut microbiota. However, depletion of the intestinal microbiota (Dysbiosis) following antibiotic treatment facilitates the colonization of the intestinal tract by Multidrug-Resistant P. aeruginosa. One possible strategy is based on the use of functional foods with prebiotic activity. The bifidogenic effect of the prebiotic inulin and its hydrolyzed form (fructooligosaccharide: FOS) is well established since they promote the growth of specific beneficial (probiotic) gut bacteria such as bifidobacteria. Previous studies of the opportunistic nosocomial pathogen Pseudomonas aeruginosa PAO1 have shown that inulin and to a greater extent FOS reduce growth and biofilm formation, which was found to be due to a decrease in motility and exotoxin secretion. However, the transcriptional basis for these phenotypic alterations remains unclear. To address this question we conducted RNAsequence analysis. Changes in the transcript level induced by inulin and FOS were similar, but a set of transcript levels were increased in response to inulin and reduced in the presence of FOS. In the presence of inulin or FOS, 260 and 217 transcript levels, respectively, were altered compared to the control to which no polysaccharide was added. Importantly, changes in transcript levels of 57 and 83 genes were found to be specific for either inulin or FOS, respectively, indicating that both compounds trigger different changes. Gene pathway analyses of differentially expressed genes (DEG) revealed a specific FOS-mediated reduction in transcript levels of genes that participate in several canonical pathways involved in metabolism and growth, motility, biofilm formation, b-lactamase resistance, and in the modulation of type III and VI secretion systems; results that have been partially verified by real time quantitative PCR measurements. Moreover, we have identified a genomic island formed by a cluster of 15 genes, encoding uncharacterized proteins, which were repressed in the presence of FOS. The analysis of isogenic mutants has shown that genes of this genomic island encode proteins involved in growth, biofilm formation and motility. These results indicate that FOS selectively modulates bacterial pathogenicity by interfering with different signaling pathways.This work was supported by grants from the Spanish Ministry for Economy and Competitiveness (AGL2017-85270-R). CS is funded by the program Juan de la Cierva-Formación (FJCI-2015-23810)

A Standardized Extract of Lentinula edodes Cultured Mycelium Inhibits Pseudomonas aeruginosa Infectivity Mechanisms

This work was supported by grants from FEDER project of Junta de Andalucia, Spain (30B572F301), Ministry of Economy and Competitivity, partly with Fondo Europeo de Desarrollo Regional FEDER funds (SAF2017-88457-R and AGL2017-85270-R), and by Junta de Andalucia (CTS235 and CTS164). MT-G was supported by the University of the Ministry of Education (Spain). CIBERehd is funded by Instituto de Salud Carlos III.The priority pathogen list of the World Health Organization classified Pseudomonas aeruginosa as the second top critical pathogen. Hence, the development of novel antibacterial strategies to tackle this bacterium is highly necessary. Herein we explore the potential antibacterial effect of a standardized extract of cultured mycelium of Lentinula edodes (AHCCR ) on P. aeruginosa. AHCCR was found to inhibit the growth rate and biofilm formation of strain PAO1. No change in swarming was observed, but AHCCR hampered swimming and twitching motility. In accordance, a decreased expression of metabolism, growth, and biofilm formation genes was shown. AHCCR also diminished the levels of exotoxin A and bacteria inside IEC18 cells and the secretion of IL-6, IL-10 and TNF by infected macrophages. This effect was related to a reduced phosphorylation of MAPKs and to bacteria internalization. Taken together, our data suggest that AHCCR has a potential role to prevent P. aeruginosa infections and may lead to the development of new therapies.FEDER project of Junta de Andalucia, Spain 30B572F301Ministry of Economy and CompetitivityEuropean Commission SAF2017-88457-R AGL2017-85270-RUniversity of the Ministry of Education (Spain)Junta de Andalucia CTS235 CTS164Instituto de Salud Carlos III European Commissio

Characterization of an extremophile bacterial acid phosphatase derived from metagenomics analysis

Acid phosphatases are enzymes that play a crucial role in the hydrolysis of various organophosphorous molecules. A putative acid phosphatase called FS6 was identified using genetic profiles and sequences from different environments. FS6 showed high sequence similarity to type C acid phosphatases and retained more than 30% of consensus residues in its protein sequence. A histidine-tagged recombinant FS6 produced in Escherichia coli exhibited extremophile properties, functioning effectively in a broad pH range between 3.5 and 8.5. The enzyme demonstrated optimal activity at temperatures between 25 and 50°C, with a melting temperature of 51.6°C. Kinetic parameters were determined using various substrates, and the reaction catalysed by FS6 with physiological substrates was at least 100-fold more efficient than with p-nitrophenyl phosphate. Furthermore, FS6 was found to be a decamer in solution, unlike the dimeric forms of crystallized proteins in its family.Grants from the Ministry of Science through grants from the Plan Nacional 2021 (PID2021-123469OBI00) and Transición Ecológica (TED2021129632BI00) and European Commission projects EJP soils (EJPSOIL862695) and PREPSOIL (HE/CL5SOILM/0102

Determination of Ligand Profiles for Pseudomonas aeruginosa Solute Binding Proteins

Solute binding proteins (SBPs) form a heterogeneous protein family that is found in all kingdoms of life. In bacteria, the ligand-loaded forms bind to transmembrane transporters providing the substrate. We present here the SBP repertoire of Pseudomonas aeruginosa PAO1 that is composed of 98 proteins. Bioinformatic predictions indicate that many of these proteins have a redundant ligand profile such as 27 SBPs for proteinogenic amino acids, 13 proteins for spermidine/putrescine, or 9 proteins for quaternary amines. To assess the precision of these bioinformatic predictions, we have purified 17 SBPs that were subsequently submitted to high-throughput ligand screening approaches followed by isothermal titration calorimetry studies, resulting in the identification of ligands for 15 of them. Experimentation revealed that PA0222 was specific for y-aminobutyrate (GABA), DppA2 for tripeptides, DppA3 for dipeptides, CysP for thiosulphate, OpuCC for betaine, and AotJ for arginine. Furthermore, RbsB bound D-ribose and D-allose, ModA bound molybdate, tungstate, and chromate, whereas AatJ recognized aspartate and glutamate. The majority of experimentally identified ligands were found to be chemoattractants. Data show that the ligand class recognized by SPBs can be predicted with confidence using bioinformatic methods, but experimental work is necessary to identify the precise ligand profile.This work was supported by FEDER funds and Fondo Social Europeo through a grant from the Spanish Ministry for Economy and Competitiveness to T. Krell (BIO2016-76779-P). This funding source was not involved in the design and conduct of this stud

Analysis of the pathogenic potential of nosocomial Pseudomonas putida strains

Pseudomonas putida strains are ubiquitous in soil and water but have also been reported as opportunistic human pathogens capable of causing nosocomial infections. In this study we describe the multilocus sequence typing of four P. putida strains (HB13667, HB8234, HB4184 and HB3267) isolated from in-patients at the Besançon Hospital (France). The four isolates (in particular HB3267) were resistant to a number of antibiotics. The pathogenicity and virulence potential of the strains was tested ex vivo and in vivo using different biological models: human tissue culture, mammalian tissues and insect larvae. Our results showed a significant variability in the ability of the four strains to damage the host; HB13667 did not exhibit any pathogenic traits, HB4184 caused damage only ex vivo in human tissue cultures, and HB8234 had a deleterious effect in tissue culture and in vivo on rat skin, but not in insect larvae. Interestingly, strain HB3267 caused damage in all the model systems studied. The putative evolution of these strains in medical environments is discussed

Analysis of the pathogenic potential of nosocomial Pseudomonas putida strains

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb.2015.00871Pseudomonas putida strains are ubiquitous in soil and water but have also been reported as opportunistic human pathogens capable of causing nosocomial infections. In this study we describe the multilocus sequence typing of four P. putida strains (HB13667, HB8234, HB4184, and HB3267) isolated from in-patients at the Besançon Hospital (France). The four isolates (in particular HB3267) were resistant to a number of antibiotics. The pathogenicity and virulence potential of the strains was tested ex vivo and in vivo using different biological models: human tissue culture, mammalian tissues, and insect larvae. Our results showed a significant variability in the ability of the four strains to damage the host; HB13667 did not exhibit any pathogenic traits, HB4184 caused damage only ex vivo in human tissue cultures, and HB8234 had a deleterious effect in tissue culture and in vivo on rat skin, but not in insect larvae. Interestingly, strain HB3267 caused damage in all the model systems studied. The putative evolution of these strains in medical environments is discussed.Work in this study was supported by the ERANET Pathogenomics Program through the ADHERS-Signature Project (reference: BIO2008-04419-E)Peer reviewe

Fructooligosacharides Reduce Pseudomonas aeruginosa PAO1 Pathogenicity through Distinct Mechanisms

Pseudomonas aeruginosa is ubiquitously present in the environment and acts as an opportunistic pathogen on humans, animals and plants. We report here the effects of the prebiotic polysaccharide inulin and its hydrolysed form FOS on this bacterium. FOS was found to inhibit bacterial growth of strain PAO1, while inulin did not affect growth rate or yield in a significant manner. Inulin stimulated biofilm formation, whereas a dramatic reduction of the biofilm formation was observed in the presence of FOS. Similar opposing effects were observed for bacterial motility, where FOS inhibited the swarming and twitching behaviour whereas inulin caused its stimulation. In co-cultures with eukaryotic cells (macrophages) FOS and, to a lesser extent, inulin reduced the secretion of the inflammatory cytokines IL-6, IL-10 and TNF- a . Western blot experiments indicated that the effects mediated by FOS in macrophages are associated with a decreased activation of the NF- k B pathway. Since FOS and inulin stimulate pathway activation in the absence of bacteria, the FOS mediated effect is likely to be of indirect nature, such as via a reduction of bacterial virulence. Further, this modulatory effect is observed also with the highly virulent ptxS mutated strain. Co-culture experiments of P. aeruginosa with IEC18 eukaryotic cells showed that FOS reduces the concentration of the major virulence factor, exotoxin A, suggesting that this is a possible mechanism for the reduction of pathogenicity. The potential of these compounds as components of antibacterial and anti-inflammatory cocktails is discussed.The authors acknowledge financial support from FEDER funds and Fondo Social Europeo through grants from the Spanish Ministry of Economy and Competitiveness (grants SAF2011-22922, SAF2011-22812) the Andalusian regional government Junta de Andalucía (grant CVI-7335) and the Centre of Networked Biomedical Research on Hepatic and Digestive Diseases (CIBERehd) which is funded by the Carlos III Health Institute and the Ramón Areces Foundation, Spain

Editorial 5(19) AyTBUAP. ¿Qué necesitamos saber sobre Oligosacáridos de la Leche Humana (HMOs)?

La leche humana contiene oligosacáridos (OSLH) con efecto prebiótico, son complejos y de amplia variedad estructural, habiéndose identificado más de 130 estructuras distintas formando parte de los alimentos funcionales (AF) utilizados tanto por la industria alimentaria como farmacéutica. Se ha descrito que estos OSLH poseen un papel único e importante en el crecimiento y desarrollo del niño. Especialmente, durante los primeros meses de vida contribuyen al establecimiento de la microbiota intestinal. En este contexto, por su estructura, los OSLH pueden actuar como receptores de virus y bacterias bloqueando la adherencia de éstos a células eucariotas y previniendo por tanto la infección. Adicionalmente, los OSLH son esenciales para el desarrollo del sistema inmune del recién nacido, pero también para la protección y modulación de la respuesta inmune en el adulto. En general, los oligosacáridos son fundamentales para el desarrollo saludable de las personas y en tiempos de COVID-19 las estrategias multidisciplinarias son las que salvaguardarán la calidad de vida, tratando de mantener un equilibrio para un estado de alimentación correcto y una inmunidad fortalecida. En este número de Alianzas y Tendencias BUAP 5(19) se publicaron 3 artículos originales y 3 revisiones. El conocimiento desarrollado a la fecha podría servirnos de base para una mejor producción agrícola, mejor alimentación y estimulación de la salud humana, pero si esa estrategia es rebasada por una infección viral se debe implementar una respuesta efectiva contra las enfermedades con el uso de fármacos y vacunas dirigidos contra el virus en cuestión

Regulación de la expresión del gen de la piruvato carboxilasa de rata por generación dediferentes isoformas de rna mensajero

La expresión de la piruvato carboxilasa es un punto de regulación del metabolismo intermediario. Este enzima se regula mediante el control de su síntesis. Hay que destacar que el gen de la piruvato carboxilasa se encuentra controlado por dos promotores, uno proximal a la que se le ha asignado un papel relevante en gluconeogénesis y lipogénesis, y un segundo promotor, en una posición distal del gen, al que se le ha atribuido un papel importante en el mantenimiento de unos niveles transcripcionales basales en las situaciones en que es necesario un relleno anaplerótico. Estudios previos mostraban un cierto grado de variabilidad en el mRNA de la piruvato carboxilasa. De nuestros resultados se desprende que mientras que existe un alto grado de variabilidad génica en la región 5'-UTR del enzima, no existen variantes ni en la región codificante del gen ni en el extremo 3'-UTR. El análisis de la variabilidad de la región 5'-UTR nos indica que existen 7 isoformas del mRNA, dos de ellas previamente no descritas. De estas nuevas isoformas, una se genera por combinación de sub-exones regulados por lo promotores proximal y distal del gen. La función de estas regiones 5'-UTR se ha establecido mediante transfecciones de células eucariótas en cultivo con construcciones que codifican para proteínas de fusión entre la secuencia de importación a la mitocondria de la piruvato carboxilasa y una proteína fluorescente verde. Estas construcciones incluyen además el extremo 5' no traducido de cada isoforma. Todas las isoformas presentan un patrón de expresión mitocondrial. No obstante, el nivel de expresión de estas isoformas es muy diferente. El análisis de los niveles de proteína producidos en estos ensayos y la cantidad de mRNA existente, indican que para cada isoforma existe un mecanismo de regulación. Estos mecanismos afectan a la estabilidad del mRNA o a la eficiencia de traducción. Al encontrar que todas las isoformas se expresan en todos los tejidos estudiados, tanto en situación de alimentación como durante el ayuno, no podemos todavía asignar una función exclusiva a los promtorres proximal y distal del gen, por lo que cabe concluir que la compleja regulación de la expresión del enzima corresponde de manera coordinada a ambos promotoresUniversidad de Granada, Departamento de Bioquímica y biología molecular. Leída el 14/07/0

Prebiotics and Probiotics: Healthy Biotools for Molecular Integrative and Modulation Approaches

The scope of this Special Issue is to highlight and expand our knowledge on the molecular mechanisms of prebiotics and probiotics, as well as to offer a broad overview of current advancements and future directions in this research field [...