Dextranos de bacterias lácticas aisladas de productos cárnicos: caracterización y aplicaciones

Algunas bacterias del ácido láctico (BAL) sintetizan exopolisacáridos (EPS) que mejoran las propiedades reológicas de alimentos fermentados, lo que despierta su interés industrial. Además, algunos de ellos han demostrado poseer propiedades beneficiosas para la salud (antitumorales, inmunomoduladoras, hipocolesterolémicas o prebióticas) y, por tanto, son buenos candidatos para la elaboración de alimentos funcionales. Entre los EPS, los dextranos son sintetizados por especies de los géneros Lactobacillus, Leuconostoc, Oenococcus, Streptococcus y Weisella de forma constitutiva o inducible. Sin embargo, poco se conoce sobre la regulación de la expresión de los genes dsr, que codifican las proteínas que los sintetizan, las dextransacarasas. El presente trabajo de tesis doctoral se ha centrado en la caracterización a nivel molecular, fisiológico, metabólico y fisicoquímico de los EPS producidos por Lactobacillus sakei MN1 (EPS-LS) y Leuconostoc mesenteroides RTF10 (EPS-LM), dos BAL aisladas de productos cárnicos. También ha contemplado el estudio de la funcionalidad biológica de dichos polímeros, como agentes antivirales e inmunoestimulantes y el análisis del potencial de Lb. sakei MN1 como probiótico frente a patógenos de peces. En primer lugar, se ha determinado que ambas cepas, en presencia de sacarosa como fuente de carbono, producen homopolisacáridos tipo dextrano con enlaces α-(1-6) en su cadena principal y un 3 % (EPS-LS) o un 9 % (EPS-LM) de ramificaciones con enlaces α-(1-3). Dichos polisacáridos se encuentran asociados a la pared celular o rodeando a las células, según se ha observado por microscopía electrónica. Como resultado del estudio genético, se ha demostrado que los determinantes genéticos responsables de la producción del EPS-LS y el EPS-LM, se encuentran localizados en los plásmidos denominados pMN1 (13,7 kpb) y pRTF10 (20,6 kpb), respectivamente. Además, la determinación de la secuencia completa de nucleótidos de pMN1 (11.126 pb) ha revelado que pertenece a una familia de plásmidos que replican por el mecanismo de tipo theta, cuyo prototipo es pUCL287, y que contiene un replicón (el origen de replicación y los genes repA y repB), el gen dsrLS y 7 marcos de lectura abierta. También, se ha estudiado la expresión del gen dsrLS a nivel transcripcional y los resultados obtenidos indican que la dextransacarasa DsrLS se sintetiza a partir de dos transcritos: uno monocistrónico y otro policistrónico, que incluye los genes repA, repB y dsrLS. Además, se ha comprobado que la expresión de ambos mRNAs no se ve incrementada en presencia de sacarosa en el medio de cultivo. Por tanto, esta hexosa no es un agente inductor de la expresión de DsrLS siendo este el primer caso descrito de expresión sincronizada de una dextransacarasa y de la maquinara replicativa de un plásmido. El análisis bioinformático del producto de dsrLS ha revelado que codifica un péptido de 1.767 aminoácidos con una masa molecular de 190.039 Da y que contiene un péptido líder en su extremo amino terminal, lo que indica que DsrLS debe ser una proteína extracelular. Los modelos de la estructura 3D de DsrLS, basados en homología de aminoácidos y de conformación estructural, indican que es un enzima dimérico cuyo sustrato es la sacarosa y que posee actividad dextransacarasa. Cultivos de Lb. sakei MN1 y Lc. mesenteroides RTF10 crecidos en medio definido han sido utilizados para la producción de EPS-LS y EPS-LM, respectivamente. Los polímeros han sido purificados a partir de los sobrenadantes de los cultivos por precipitación con etanol, dialisis y fraccionamiento cromatográfico con una recuperación de los mismos del 80 % y con un grado de pureza del 99 %. Ambos EPS han mostrado actividad antiviral in vitro frente a la infección por dos virus de salmónidos: el virus de la necrosis hematopoyética infecciosa (VNHI) y el virus de la necrosis pancreática infecciosa (VNPI). Además, se ha comprobado que el EPS-LS in vivo posee actividad antiviral y actúa como un agente estimulante de la respuesta inmune de truchas arcoíris (Oncorhynchus mykiss). Por otro lado, Lb. sakei MN1 ha mostrado in vitro la capacidad de agregarse y formar biopelículas cuando crece en presencia de glucosa (no produce dextrano), pero no en presencia de sacarosa, cuando el dextrano es sintetizado por la bacteria. Este comportamiento ha sido validado in vivo utilizando un modelo de larvas gnotobióticas de pez cebra (Danio rerio). Además, este modelo ha permitido demostrar que Lb. sakei MN1 colonizada el intestino de las larvas y es capaz de competir con el patógeno de peces Vibrio anguillarum. Por todo lo expuesto, tanto Lb. sakei MN1 por su potencial probiótico, como el EPS-LS que produce, por su acción inmunomoduladora y antiviral, resultan muy prometedores para su aplicación en el desarrollo de piensos funcionales para salmónidos.Some lactic acid bacteria (LAB) synthesize exopolysaccharides (EPS) that improve the rheological properties of fermented foods, which is of interest to the food industries. Moreover, some EPS have demonstrated beneficial health properties, such as antitumor, immunomodulatory, hypocholesterolemic or prebiotic activities. Thus, LAB are good candidates for the development of functional foods. Among the EPS, dextrans have various industrial applications outside of the food sector. It is known that these EPS are synthesized by species of the genera Lactobacillus, Leuconostoc, Oenococcus, Streptococcus and Weisella. However, little is known about the regulation of expression of the dsr genes, which encode the dextransucrases that synthesise dextrans. The present doctoral work has characterized at the molecular, physiological, metabolic and physicochemical levels, two EPS: EPS-LS and EPS-LM produced respectively by Lactobacillus sakei MN1 and Leuconostoc mesenteroides RTF10, both isolated from meat products. The biological functionality of these polymers as antiviral agents and immunostimulants, as well as the potential of the Lb. sakei MN1 as a probiotic against fish pathogens, has been investigated. The physiological and physicochemical studies revealed that in the presence of sucrose as carbon source Lb. sakei MN1 and Lc. mesenteroides RTF10 produce dextrans with α-(1-6) linkages in their main chain and 3% (EPS-LS) or 9% (EPS-LM) of branches with α- (1-3) linkages. Analysis by electron microscopy showed that the EPS are associated with the cell wall or cells surrounding. A further study showed that the genetic determinants for the production of EPS-LS and EPS-LM, are located respectively in the pMN1 (13.7 kbp) and the pRTF10 (20.6 kbp) plasmids. Furthermore, the determination of the complete nucleotide sequence of pMN1 (11,126 bp) revealed that it belongs to a family of plasmids which replicate by the theta type mechanism and whose prototype is pUCL287. The plasmid carries a replicon (the origin of replication and the repA and repB genes), the dsrLS gene and 7 open reading frames. Analysis of the dsrLS gene expression at the transcriptional level showed that the dextransucrase DsrLS is synthesized from two transcripts: one monocistronic and other polycistronic including repA, repB and dsrLS. As far as we know, this is the first instance of synchronised expression of a dextransucrase and the machinery of plasmid replication. In addition, the expression of both mRNAs does not increase when sucrose is present in the growth medium, revealing that this hexose is not an inducer of DsrLS expression. Bioinformatic analysis of the dsrLS gene product revealed that it is a peptide composed of 1,767 amino acids with a molecular mass of 190,039 Da and that contains a leader peptide at its amino terminus, indicating that DsrLS is an extracellular protein. 3D models of DsrLS structure based on amino acid homology and structural conformation indicate that the protein is a dimeric enzyme, whose substrate is sucrose and which has dextransucrase activity. Lb. sakei MN1 and Lc. mesenteroides RTF10 cultures grown in defined medium have been used for production of EPS-LS and EPS-LM. The polymers have been purified from the culture supernatants by ethanol precipitation, dialysis and chromatographic fractionation, with a recovery of 80% and with a purity of 99%. Both dextrans have shown in vitro antiviral activity against infection by two salmonid viruses: infectious hematopoietic necrosis virus (IHNV) and Infectious pancreatic necrosis virus (IPNV). Furthermore, it has been demonstrated that EPS-LS has antiviral activity in vivo, and acts as an immunostimulant of the immune response of rainbow trout (Oncorhynchus mykiss). Furthermore, it was shown that in vitro Lb. sakei MN1 has the ability to aggregate and to form biofilms when grown in the presence of glucose but not in the presence of sucrose, condition in which produces dextran. This behaviour has been validated by use of a gnotobiotic zebrafish (Danio rerio) larva model. Moreover, this model has demonstrated that Lb. sakei MN1 colonizes the larval intestine and it is able to compete in this habitat with the fish pathogen Vibrio anguillarum. For all the above mentioned reasons, both Lb. sakei MN1 for its probiotic potential as competitor with bacterial pathogens, and the EPS-LS for its immunomodulatory and antiviral activities, have potential application for the development of functional feed for salmonids

Development of a novel peptide nucleic acid probe for the detection of Legionella spp. in water samples

Legionella are opportunistic intracellular pathogens that are found throughout the environment. The Legionella contamination of water systems represents a serious social problem that can lead to severe diseases, which can manifest as both Pontiac fever and Legionnaires’ disease (LD) infections. Fluorescence in situ hybridization using nucleic acid mimic probes (NAM-FISH) is a powerful and versatile technique for bacterial detection. By optimizing a peptide nucleic acid (PNA) sequence based on fluorescently selective binding to specific bacterial rRNA sequences, we established a new PNA-FISH method that has been successfully designed for the specific detection of the genus Legionella. The LEG22 PNA probe has shown great theoretical performance, presenting 99.9% specificity and 96.9% sensitivity. We also demonstrated that the PNA-FISH approach presents a good signal-to-noise ratio when applied in artificially contaminated water samples directly on filtration membranes or after cells elution. For water samples with higher turbidity (from cooling tower water systems), there is still the need for further method optimization in order to detect cellular contents and to overcome interferents’ autofluorescence, which hinders probe signal visualization. Nevertheless, this work shows that the PNA-FISH approach could be a promising alternative for the rapid (3–4 h) and accurate detection of Legionella.This work was financially supported by: LA/P/0045/2020 (ALiCE), UIDB/00511/2020 and UIDP/00511/2020 (LEPABE), funded by national funds through the FCT/MCTES (PIDDAC); Projects POCI-01-0145-FEDER-029961, POCI-01-0145-FEDER-031011 and POCI-01-0145-FEDER-030431 funded by FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacional ização (POCI) and by national funds (PIDDAC) through FCT/MCTES. Montserrat Nácher-Vázquez and Laura Cerqueira were also financed by Project POCI-01-0145-FEDER-029961.info:eu-repo/semantics/publishedVersio

Probióticos, Prebióticos y Salud: Evidencia Científica

24 páginas.-- Resúmenes de las ponencias presentadas al II Workshop Científico: Probióticos, prebióticos y salud: evidencia científica, celebrado en Madrid del 16 al 17 de Diciembre de 2010.-- et al.Peer reviewe

Estudio de la producción de exopolisacáridos por bacterias lácticas y su aplicación en el desarrollo de alimentos funcionales

Trabajo presentado en la 4º Reunión de la Red Temática (Participación de las bacterias lácticas en la salud humana y en la calidad alimentaria), celebrada en Granada (España) del 12 al 14 de noviembre de 2009

Evaluation of yoghurt as a carrier of ß-D-glucan producing lactic acid and bacteria

Trabajo presentado en el 3rd Congress of European Microbiologists (FEMS 2009), celebrado en Gotemburgo (Suecia), del 28 de junio al 2 de julio de 200

Comparative analysis of production and purification of homo- and hetero-polysaccharides produced by lactic acid bacteria

2 figuras, 2 tablasLactic acid bacteria (LAB) produce homopolysaccharides (HoPS) and heteropolysaccharides (HePS) with potential functional properties. In this work, we have performed a comparative analysis of production and purification trials of these biopolymers from bacterial culture supernatants. LAB strains belonging to four different genera, both natural as well as recombinant, were used as model systems for the production of HoPS and HePS. Two well characterized strains carrying the gft gene were used for β-glucan production, Pediococcus parvulus 2.6 (P. parvulus 2.6) isolated from cider, and the recombinant strain Lactococcus lactis NZ9000[pGTF] (L. lactis NZ9000[pGTF]). In addition, another cider isolate, Lactobacillus suebicus CUPV225 (L. suebicus CUPV225), and Leuconostoc mesenteroides RTF10 (L. mesenteroides RTF10), isolated from meat products were included in the study. Chemical analysis of the EPS revealed that L. mesenteroides produces a dextran, L. suebicus a complex heteropolysaccharide, and the β-glucan producing-strains the expected 2-substituted (1,3)-β-glucanThis study was supported by grants AGL2009-12998-C03 and CSD2007-00063 from the Spanish Ministry of Science and Innovation, ACOMP/2009/257 from the Generalitat Valenciana, and IT335-10 from the Basque Government. M. N-V and S. N. are the recipients of JAE pre-doctor grants from the “Consejo Superior de Investigaciones Científicas” (CSIC)Peer reviewe

Dextran production by Lactobacillus sakei MN1 coincides with reduced autoagglutination, biofilm formation and epithelial cell adhesion

40 p.-7 fig.-4 fig.supl.In this work we have investigated two dextran-producing lactic acid bacteria, Lactobacillus sakei MN1 and Leuconostoc mesenteroides RTF10, isolated from fermented meat products. These bacteria synthesise dextran when sucrose, but not glucose, is present in the growth medium. The influence of dextran on bacterial aggregation, adhesion and biofilm formation was investigated in cultures challenged with sucrose or glucose. For Lb. sakei MN1, the synthesis of the dextran drastically impaired the three processes; in contrast it had no effect on Lc. mesenteroides RTF10. Therefore, the influence of dextran on probiotic properties of Lb. sakei MN1 was tested in vivo using gnotobiotic zebrafish models. The bacterium efficiently colonised the fish gut and inhibited the killing activity of Vibrio anguillarum NB10[pOT11]. Furthermore, under conditions of dextran synthesis, the adhesion of Lb. sakei MN1 to the epithelial cells decreased, without greatly affecting its anti V. anguillarum activity.This work was supported by the Spanish Ministry of Economics and Competitiveness [grants AGL2012-40084C03-01 and AGL2015-65010-C3-1-R] and by the Agriculture and Fisheries Department of the Basque Government [project VIVAQUA], and II is the recipient of a PhD fellowship from Technological centres foundation-Iñaki Goenaga.Peer reviewe

Influence of the side chain structure on the anti-leishmanial effect of methotrexate conjugates with polymeric branched chain polypeptides

1 p. 4 fig. 1 tab.Peer reviewe

Dextransucrase Expression Is Concomitant with that of Replication and Maintenance Functions of the pMN1 Plasmid in Lactobacillus sakei MN1

The exopolysaccharide synthesized by Lactobacillus sakei MN1 is a dextran with antiviral and immunomodulatory properties of potential utility in aquaculture. In this work we have investigated the genetic basis of dextran production by this bacterium. Southern blot hybridization experiments demonstrated the plasmidic location of the dsrLS gene, which encodes the dextransucrase involved in dextran synthesis. DNA sequencing of the 11,126 kbp plasmid (pMN1) revealed that it belongs to a family which replicates by the theta mechanism, whose prototype is pUCL287. The plasmid comprises the origin of replication, repA, repB, and dsrLS genes, as well as seven open reading frames of uncharacterized function. Lb. sakei MN1 produces dextran when sucrose, but not glucose, is present in the growth medium. Therefore, plasmid copy number and stability, as well as dsrLS expression, were investigated in cultures grown in the presence of either sucrose or glucose. The results revealed that pMN1 is a stable low-copy-number plasmid in both conditions. Gene expression studies showed that dsrLS is constitutively expressed, irrespective of the carbon source present in the medium. Moreover, dsrLS is expressed from a monocistronic transcript as well as from a polycistronic repA-repB-orf1-dsrLS mRNA. To our knowledge, this is the first report of a plasmid-borne dextransucrase-encoding gene, as well as the first time that co-transcription of genes involved in plasmid maintenance and replication with a gene encoding an enzyme has been established.This work was supported by the Spanish Ministry of Economy and Competitiveness (grant AGL2015-65010-C3-1-R).Peer reviewe

Mode of action of a formulation containing hydrazones and saponins against leishmania spp. Role in mitochondria, proteases and reinfection process

13 p.-8 fig.-3 tab.Toxicity and poor adherence to treatment that favors the generation of resistance in the Leishmania parasites highlight the need to develop better alternatives. Here, we evaluated the in vitro effectiveness of hydrazone derived from chromanes 2-(2,3-dihydro-4H-1-benzothiopyran-4-ylidene) hydrazide (TC1) and 2-(2,3-dihydro- 4H-1-benzopyran-4-ylidene) hydrazide (TC2) and the mixture of triterpene saponin hederagenin-3-O-(3,4-Odiacetyl-ß-D-xylopyranosyl-(1à3)-a-L- rhamnopyranosyl-(1à2)-a-L-arabinofuranoside, hederagenin-3-O-(3,4-Odiacetyl-a-L- arabinopyranosyl-(1à3)-a-L-rhamnopyranosyl-(1à2)-a-L-arabinofuranoside and, hederagenin-3-O-(4-O-acetyl-ß-D-xylopyranosyl-(1à3)-a-L-rhamnopyranosyl-(1à2)-a-L-arabinofuranoside from Sapindus saponaria(SS) on L. braziliensis and L. pifanoi. Mixtures of TC1 or TC2 with saponin were formulated for topical application and the therapeutic effectiveness was evaluated in the model for cutaneous leishmaniasis (CL) in golden hamster.The mode of action of these compounds was tested on various parasite processes and ultrastructural parasite modifications. TC1, TC2 and SS showed moderate cytotoxicity when tested independently but toxicity was improved when tested in combination. The compounds were more active against intracellular Leishmania amastigotes. In vivo studies showed that combinations of TC1 or TC2 with SS in 1:1 ratio (w/w) cured 100% of hamsters with no signs associated with toxicity. The compounds did cause changes in the mitochondrial activity of the parasite with a decrease in ATP levels and depolarization of membrane potential and overproduction of reactive oxygen species; nevertheless, these effects were not related to alterations in membrane permeability.The phagolysosome ultrastructure was also affected impacting the survival of Leishmania but the function of the lysosome nor the pH inside the phagolysosome did not change.Lastly, there was a protease inhibition which was directly related to the decrease in the ability of Leishmania to infect and multiply inside the macrophage. The results suggest that the combination of TC1 and TC2 with SS in a 1:1 ratio is capable of curing CL in hamsters.This effect may be due to the ability of these compounds to affect parasite survival and the ability to infect new cells.The work with Sapindus saponaria had “Permission of Access to Genetic Resources and its Derived Products” issued by ANLA-Colombia (Framework Contract No.154RGE0237).Peer reviewe