A diverse range of human gut bacteria have the potential to metabolize the dietary component gallic acid

The human gut microbiota contains a broad variety of bacteria that possess functional genes, with resultant metabolites that affect human physiology and therefore health. Dietary gallates are phenolic components that are present in many foods and beverages and are regarded as having health-promoting attributes. However, the potential for metabolism of these phenolic compounds by the human microbiota remains largely unknown. The emergence of high-throughput sequencing (HTS) technologies allows this issue to be addressed. In this study, HTS was used to assess the incidence of gallate-decarboxylating bacteria within the gut microbiota of healthy individuals for whom bacterial diversity was previously determined to be high. This process was facilitated by the design and application of degenerate PCR primers to amplify a region encoding the catalytic C subunit of gallate decarboxylase (LpdC) from total metagenomic DNA extracted from human fecal samples. HTS resulted in the generation of a total of 3,261,967 sequence reads and revealed that the primary gallate-decarboxylating microbial phyla in the intestinal microbiota were Firmicutes (74.6%), Proteobacteria (17.6%), and Actinobacteria (7.8%). These reads corresponded to 53 genera, i.e., 47% of the bacterial genera detected previously in these samples. Among these genera, Anaerostipes and Klebsiella accounted for the majority of reads (40%). The usefulness of the HTS-lpdC method was demonstrated by the production of pyrogallol from gallic acid, as expected for functional gallate decarboxylases, among representative strains belonging to species identified in the human gut microbiota by this method. Importance: Despite the increasing wealth of sequencing data, the health contributions of many bacteria found in the human gut microbiota have yet to be elucidated. This study applies a novel experimental approach to predict the ability of gut microbes to carry out a specific metabolic activity, i.e., gallate metabolism. The study showed that, while gallate-decarboxylating bacteria represented 47% of the bacterial genera detected previously in the same human fecal samples, no gallate decarboxylase homologs were identified from representatives of Bacteroidetes. The presence of functional gallate decarboxylases was demonstrated in representative Proteobacteria and Firmicutes strains from the human microbiota, an observation that could be of considerable relevance to the in vivo production of pyrogallol, a physiologically important bioactive compound

Glicosil hidrolasas de Lactiplantibacillus plantarum WCFS1

Resumen del trabajo presentado a la 15ª Reunión de la Red Española de Bacterias Lácticas: Bacterias Lácticas en Alimentación y Salud. Valencia, 26 y 27 de mayo de 2022.AGL2017-84614-C2-1-R y AGL2017-84614-C2-2-RPeer reviewe

The commensal bacterium Lactiplantibacillus plantarum imprints innate memory-like responses in mononuclear phagocytes

Gut microbiota is a constant source of antigens and stimuli to which the resident immune system has developed tolerance. However, the mechanisms by which mononuclear phagocytes, specifically monocytes/macrophages, cope with these usually pro-inflammatory signals are poorly understood. Here, we show that innate immune memory promotes anti-inflammatory homeostasis, using as model strains of the commensal bacterium Lactiplantibacillus plantarum. Priming of monocytes/macrophages with bacteria, especially in its live form, enhances bacterial intracellular survival and decreases the release of pro-inflammatory signals to the environment, with lower production of TNF and higher levels of IL-10. Analysis of the transcriptomic landscape of these cells shows downregulation of pathways associated with the production of reactive oxygen species (ROS) and the release of cytokines, chemokines and antimicrobial peptides. Indeed, the induction of ROS prevents memory-induced bacterial survival. In addition, there is a dysregulation in gene expression of several metabolic pathways leading to decreased glycolytic and respiratory rates in memory cells. These data support commensal microbe-specific metabolic changes in innate immune memory cells that might contribute to homeostasis in the gut.Supported by grants from the Spanish Ministry of Science, Innovation and Universities (MCIU) co-financed with FEDER funds (RTI2018-096494-B-100 to JA; BFU2016-76872-R to EB; AGL2017-86757-R to LA; SAF2015-73549-JIN to HR; SAF2016–77433-R and PID2019-110240RB-I00 to RPR). AP is supported by a Postdoctoral Fellowship from the Basque Government. DB and TMM are recipients of MCIU FPI fellowships. APC is a recipient of a fellowship from the University of the Basque Country. LA and RPR are supported by the Ramon y Cajal program from the Spanish Ministry of Economy and Competitiveness. We thank the MCIU for the Severo Ochoa Excellence accreditation (SEV-2016-0644), the Basque Department of Industry, Tourism and Trade (Etortek and Elkartek programs) and the Innovation Technology Department of the Bizkaia Province. This work was further supported by grants from the Jesús de Gangoiti Barrera Foundation.Peer reviewe

Identificación y caracterización de glicosil hidrolasas en Lactobacillus plantarum WCFS1

Los carbohidratos son los compuestos orgánicos más abundantes en los alimentos después del agua, además de ser los más ampliamente distribuidos que se pueden presentar bajo las formas de monosacáridos, oligosacáridos o polisacáridos. Son compuestos frecuentes en alimentos de origen vegetal debido a que se originan en procesos derivados de la fotosíntesis y tienen gran importancia en alimentación debido a su carácter energético y a su influencia sobre algunas propiedades sensoriales de los alimentos. Lactobacillus plantarum es una especie de bacteria láctica que se encuentra en una amplia variedad de nichos con alto contenido en carbohidratos. El análisis de la secuencia del genoma de la cepa L. plantarum WCFS1 permitió identificar 55 proteínas anotadas en la base de datos CAZy como “glicosil hidrolasas” (GH) y que posiblemente estaban implicadas en el metabolismo de carbohidratos. Sin embargo, la mayoría de las proteínas presentes en las bases de datos no se han caracterizado experimentalmente..

Caracterización bioquímica de la proteína Lp_2777, una glicosidasa de la familia GH1 de Lactobacillus plantarum WCFS1

Póster presentado al IX Congreso CyTA-CESIA. celebrado en Madrid del 16 al 19 de mayo de 2017.La familia 1 de glicosil hidro/asas (GH1) incluye proteínas con actividades muy interesantes para la industria alimentaria, tales como beta-glucosidasas y beta-galactosidasas. En este trabajo se ha caracterizado bioquímicamente una proteína de L. plantarum WCFSI que presenta actividad beta-galactosidasa.Trabajo financiado por el MINEICO (proyecto AGL2014-52911-R).Peer Reviewe

Transcriptomic evidence of molecular mechanisms underlying the response of Lactobacillus plantarum WCFS1 to hydroxytyrosol

This article belongs to the Special Issue Olive Oil Antioxidants.This study was aimed to gain new insights into the molecular mechanisms used by Lactobacillus plantarum WCFS1 to respond to hydroxytyrosol (HXT), one of the main and health-relevant plant phenolics present in olive oil. To this goal, whole genome transcriptomic profiling was used to better understand the contribution of differential gene expression in the adaptation to HXT by this microorganism. The transcriptomic profile reveals an HXT-triggered antioxidant response involving genes from the ROS (reactive oxygen species) resistome of L. plantarum, genes coding for H2S-producing enzymes and genes involved in the response to thiol-specific oxidative stress. The expression of a set of genes involved in cell wall biogenesis was also upregulated, indicating that this subcellular compartment was a target of HXT. The expression of several MFS (major facilitator superfamily) efflux systems and ABC-transporters was differentially affected by HXT, probably to control its transport across the membrane. L. plantarum transcriptionally reprogrammed nitrogen metabolism and involved the stringent response (SR) to adapt to HXT, as indicated by the reduced expression of genes involved in cell proliferation or related to the metabolism of (p)ppGpp, the molecule that triggers the SR. Our data have identified, at genome scale, the antimicrobial mechanisms of HXT action as well as molecular mechanisms that potentially enable L. plantarum to cope with the effects of this phenolic compound.This research was funded by the Spanish Ministerio de Economía, Industria y Competitividad grant numbers AGL2017-84614-C2-2-R and AGL2014-52911 (AEI/FEDER, UE)

Biochemical characterization of Lp_2777, a family 1 glycoside hydrolase from Lactoibacillus plantarum WCFS1

Póster presentado a la VII International Conference on Environmental Industrial and Applied Microbiology, celebrada en Madrid (España) del 18 al 20 de octubre de 2017.Glycoside hydrolases (GHs) have been organized into 114 families in the CAZy database (http://www.cazy.org) based on amino acid sequence similarities. Among these families, glycoside hydrolase family 1 (GH1) enzymes are important members which hydrolyze β-glycosidic linkages. Carbohydrates are the major energy source for Lactic acid bacteria (LABs) and their metabolism utilizes a variety of sugars present in the environment. Carbohydrate uptake mostly occurs through the phosphoenolpyruvate-dependent phosphotranferase system (PEP-PTS). In olive (Olea europea), the glucoside oleuropein is the main phenolic component. Oleuropein is present in leaves as well as in olive fruits and is related to the bitterness of olive oil. Lactobacillus plantarum is a lactic acid bacteria frequently found in the fermentation of plant-derived food products like olives. It has been described that L. plantarum is able to transform oleuropein to hydroxytyrosol, the compound responsible of olive oil healthy properties. In order to identify the enzymes involved in oleuropein transformation, L. plantarum WCFS1 was exposed to oleuropein and the changes in gene expression analyzed. The transcriptomic study revealed the induction of the lp_2777 gene, encoding a putative 6 phospho β-glucosidase. Therefore, the lp_2777 gene was cloned and the recombinant protein was hiperproduced, purified, and biochemically characterized. Substrate specificity was assayed against a library of 4-nitrophenyl synthetic glycosides. The results showed that Lp_2777, in spite that it not exhibited β-glucosidase activity, it was able to hydrolyze 4-nitrophenyl-β-Dglucopyranoside-6-P, and 4-nitrophenyl-β-D-galactopyranoside-6-P. In addition, Lp_2777 protein showed thermal stability. This study constitutes the first characterization of a GH1 glycosidase from Lactobacillus plantarum WCFS1.This work was supported by grant AGL2014-52911-R (MINEICO).Peer Reviewe

Lp_3485, an α-D-galactosidase from Lactobacillus plantarum WCFs1: production and biochemical characterization

Póster presentado a la VII International Conference on Environmental Industrial and Applied Microbiology, celebrada en Madrid (España) del 18 al 20 de octubre de 2017.Glycosil hydrolases are classified into the sequence-based CAZY database (http://www.cazy.org) in families organized in mechanistically and structurally related clans. α-Galactosidases belong to families GH4, 27, 36, 57, 97 and 110. GH27 and 36 have been subjected to extensive biochemical characterization owing to their importance for industrial applications. Some foods, like legumes, contain α-galactosides possessing one or several galactose units, linked together or to the glucose moiety of sucrose though α-1, 6 linkages. Several microorganisms are known to produce α-galactosidases. Lactic acid bacteria, used in vegetable fermentations, can hydrolyze α-galactosides to digestible carbohydrates. The aim of this study was to characterize an α-galactosidase from the lactic acid bacterium Lactobacillus plantarum WCFS1, therefore the lp_3485 gene encoding a putative α-galactosidase was cloned and the recombinant protein was hiperproduced, purified, and biochemically characterized. Substrate specificity was assayed against a library of 4-nitrophenyl synthetic glycosides. As expected, the results confirmed that Lp_3485 was able to hydrolyze 4-nitrophenyl-α-Dgalactopyranoside. The use of Mn2+ and Fe2+ ions, triton-X-100, tween 20, DECP and EDTA greatly increased Lp_3485 activity. This α-galactosidase from L. plantarum could be used to enhance the digestibility of food α-galactosides.This work was supported by grant AGL2014-52911-R (MINEICO).Peer Reviewe

Transcriptomic-based analysis of the Lactobacillus plantarum WCFS1 response to oleuropein

Póster presentado a la VII International Conference on Environmental Industrial and Applied Microbiology, celebrada en Madrid (España) del 18 al 20 de octubre de 2017.Oleuropein is the main phenolic component of olive leaves, seed, pulp and peel of unripe olive fruits and is present in higher amounts in oils obtained from green olives. This compound confers natural resistance to Olea europeae against both gram positive and gram negative bacteria. On account of its anti-microbial activity oleuropein might play an important role to select the microbiota that colonizes the olive epidermis which is crucial for the quality of fermented table olives. Therefore it is important to increase knowledge on the oleuropein tolerance mechanisms of the olive microbiota. To this goal we have investigated how oleuropein affects the expression profile of Lactobacillus plantarum at genome scale since this microorganism colonizes the olive epidermis and plays an important role in the fermentation of olives. Whole-transcriptome analysis was based on customized microarray profiles. Differentially expressed genes (fold-changes { 2 (p < 0.05)) were used to perform a functional analysis by using the DAVID bioinformatics tool. The transcriptomic response revealed differential expression of genes involved in the transport and metabolism of several carbohydrates. Other set of genes whose expression was affected by the presence of oleuropein was that dedicated to the biosynthesis of fatty acids. In addition genes involved in the biosynthesis of membrane and cell wall components were also differentially transcribed respect to controls. Stress responses, including a specific oxidative stress response, were revealed by the transcriptomic datasets indicating the antimicrobial properties of this phenolic compound.Peer Reviewe

Enzymatic detoxification of ochratoxin A. Aspergillus niger vs. Alcaligenes faecalis ochratoxinases

Resumen del trabajo presentado al XXII EuroFoodChem Congress, celebrado en Belgrade, Serbia del 14 al 16 de junio de 2023Ochratoxin A (OTA) is one of the most important mycotoxins attending to its high prevalence and toxicity. It is the cause of a variety of toxicological effects both in humans and animals being the kidney its main target. To ensure food safety, it is necessary to reduce OTA presence to values as low as technologically possible. Once OTA is already contaminating food and feed there are different strategies to eliminate it, mainly: physical, chemical, and biological methods. In this context, biological methods arise as the most promising ones since they can be applied in mild conditions, are environmentally friendly, and more specific. To date, there are numerous examples in the literature of microorganisms capable of degrading OTA. In most cases, OTA detoxification is achieved through the hydrolysis of its amide bond rendering non-toxic ochratoxin α (OTα) and L-β-phenylalanine. Moreover, some enzymes from microbes have been studied for their OTA detoxification activity but few of them have been isolated and further characterized. Two of them are included in this study, the ochratoxinase from Aspergillus niger (which is the first reported microbial enzyme able to hydrolyse OTA) and an N-acyl-L-amino acid amidohydrolase from Alcaligenes faecalis. Since these enzymes could be good candidates for OTA-detoxification of food matrices, the aim of this study was to elucidate if their biochemical characteristics are compatible with their use in food technological processes. For this purpose, both proteins were recombinantly hyperproduced. Both enzymes possessed similar optimum pH, as both showed maximal activity around neutral pH values. Regarding its optimal temperature, A. niger ochratoxinase shows better activity at 65 ºC while amidohydrolase from A. faecalis is more active between 37 and 55 ºC. A. niger ochratoxinase maintains its activity after 6 hours of heat treatment at all the temperatures studied while amidohydrolase from A. faecalis shows a significant loss of activity. Enzyme hydrolytic activity was assayed against a set of 21 synthetic commercial carboxypeptidase substrates possessing different aminoacyl substituent residues at their C-terminal end. Ochratoxinase from A. niger was found to have a narrower substrate specificity, displaying a higher preference for substrates with phenylalanine in their C-terminal end (such as the mycotoxin OTA). Among the properties that an enzyme should have for its use in food matrices, an important one is to display a narrow substrate specificity to avoid altering the nutritional quality and organoleptic characteristics of the food substrates. Considering the results obtained in this study, ochratoxinase from A. niger possesses characteristics that might be perceived as more promising for its application in food matrices.Grant PID-2021-123291OB-I00 funded by MCIN/AEI/10.13039/501100011033 and by ERDF A way of making Europe. PRE2018-083862 FPI contract funded by MCIN/AEI/10.13039/501100011033 and by ESF Investing in your future (A. S-A.).Peer reviewe