Molecular screening and genetic diversity analysis of anticancer Azurin-encoding and Azurin-like genes in human gut microbiome deduced through cultivation-dependent and cultivation-independent studies

Azurin, a bacteriocin produced by a human gut bacterium Pseudomonas aeruginosa, can reveal selectively cytotoxic and induce apoptosis in cancer cells. After overcoming two phase I trials, a functional region of Azurin called p28 has been approved as a drug for the treatment of brain tumor glioma by FDA. The present study aims to improve a screening procedure and assess genetic diversity of Azurin genes in P. aeruginosa and Azurin-like genes in the gut microbiome of a specific population in Vietnam and global populations. Firstly, both cultivation-dependent and cultivation-independent techniques based on genomic and metagenomic DNAs extracted from fecal samples of the healthy specific population were performed and optimized to detect Azurin genes. Secondly, the Azurin gene sequences were analyzed and compared with global populations by using bioinformatics tools. Finally, the screening procedure improved from the first step was applied for screening Azurin-like genes, followed by the protein synthesis and NCI in vitro screening for anticancer activity. As a result, this study has successfully optimized the annealing temperatures to amplify DNAs for screening Azurin genes and applying to Azurin-like genes from human gut microbiota. The novelty of this study is the first of its kind to classify Azurin genes into five different genotypes at a global scale and confirm the potential anticancer activity of three Azurin-like synthetic proteins (Cnazu1, Dlazu11, and Ruazu12). The results contribute to the procedure development applied for screening anticancer proteins from human microbiome and a comprehensive understanding of their therapeutic response at a genetic level

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Identification of lactobacilli residing in chicken ceca with antagonism against Campylobacter

Bacteriocins produced by Lactobacillus salivarius have been recently recognized as a natural means to control Campylobacter and Salmonella in live poultry. This finding is of relevance since Campylobacter jejuni and Campylobacter coli are the predominant species isolated from poultry that are associated with human campylobacteriosis. In the present work, lactic acid bacteria (LAB) isolated from the cecum of twenty Tunisian chickens were identified and those isolates with antagonism against Campylobacter were further characterized. Following their preliminary confirmation as LAB, 150 strains were identified by combining morphological criteria, biochemical tests, and molecular methods, the latter inluding intergenic 16S- 23S PCR, specific lactobacilli PCR, and a biphasic approach. Most of the LAB isolated belonged to the genus Lactobacillus, among them Lb. sakei (33.3%), Lb. salivarius (19.4%), Lb. reuteri (8.6%), and Lb. curvatus (8.6%). The other LAB strains included those of the genus Weissella (16.7%), Enterococcus faecalis (5.3%), Leuconostoc mesenteroides (2.7%), Lactococcus graviae (2.7%), and Streptococcus sp. (2.7%). The Lactobacilli strains were tested for their antagonism against C. jejuni and C. coli. The activity of three of them, Lb. salivarius SMXD51, Lb. salivarius MMS122, and Lb. salivarius MMS151, against the aforementioned target strains could be ascribed to the production of bacteriocins (AU)

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Bacteriocinas de probióticos. Nuevos enfoques bioterapéuticos: PINHE / Bacteriocins of probiotics. New biotherapeutical approaches: PINHE

La prevención de enfermedades y la conservaciónde los alimentos han motivado que el mundo industrialy el científico se interese por conocer con mayordetalle el modo de acción de los probióticos como alimentosfuncionales y en particular las bacteriocinasque producen, por sus propiedades antimicrobianas.Cada día se descubren más bacteriocinas y se analizansus mecanismos de acción a nivel molecular. Nocabe duda, que en los próximos años se tendrá unmejor conocimiento de estos péptidos, que permitanaprovechar al máximo su potencial en beneficio delhombre, no sólo a nivel de la industria alimentariacomo conservadores, sino además como nuevas alternativasantibióticas y como bioterapéuticos en laprevención de ciertas patologías. En esté último sentido,Halocina H6 y en concreto PINHE (un péptidoderivado de esta bacteriocina procedente de haloarqueas),ha mostrado ser un buen candidato para prevenirlos daños ocasionados por isquemia y repercusiónen el infarto de miocardio (AU)

The prevention of the occurrence of diseases in theindividuals and the conservation of the foods has motivatedthat the industrial world and the scientist is interestedto know with greater detail the way actionthe probiotics and in particular those that producebacteriocins, for their antimicrobial properties. Everyday more bacteriocins are discovered and analyzed itsmechanisms of action at the molecular level. There isno doubt that in the next few years will have a betterunderstanding of these peptides, which allow to maximizetheir potential for the benefit of man, not onlyat the level of the food industry as a conservative, butalso as new antibiotic options and as bioterapeutics inthe prevention of certain diseases. In the latter regard,Halocina H6 and in particular PINHE (a peptidederived from this haloarchaeal bacteriocin) has beenshown to be a good candidate for preventing damagecaused by ischemia and reperfusion, en myocardialinfarction (AU)

Modeling the combined effects of enterocins A and B, lactate, and EDTA on the growth of Salmonella at different temperatures

The effects of enterocins A and B (produced by Enterococcus faecium CTC492), lactate, and ethylenediaminetetraacetic acid (EDTA) on the growth of Salmonella were modeled together with temperature using the response surface methodology. Six serovars of Salmonella enterica were inoculated (ca. 10(3) cells/ml) in brain-heart infusion broth with different levels of the studied factors and then incubated at different temperatures. The results showed that while Salmonella growth was affected by all the factors, temperature was the most important factor influencing the time to detection of the pathogen. All factors, including temperature, showed significant two-way interactions. The presence of enterocins A and B, lactate, and EDTA had an inhibitory effect that was enhanced at suboptimal temperatures for growth, thus delaying the time to detection. Moderate-low concentrations of lactate and EDTA increased the inhibitory effect of enterocins A and B. The effectiveness of these bacteriocins was not further enhanced by high concentrations of lactate (>3.6%) or EDTA (>200 mg/l). The mathematical model obtained from these analyses provides a useful tool to assess the effects of natural antimicrobials and their interactions with other growth-related factors on the growth response of Salmonella. The results can be applied to determine the most effective combination of hurdles to be used in the preservation of food products (AU)

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Antibioticoterapia con probióticos / No disponible

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A halocin acting on Na+/H+ exchanger of Haloarchaea as a new type of inhibitor in NHE of mammals

The capability of halocin H6 (a bacteriocin-like protein produced by haloarchaeaHaloferax gibbonsii) to inhibit Na+/H+ exchange (NHE) in mammalian cells and its cardio-protective efficacy on the ischemic and reperfused myocardium were evaluated in the present study. H6 inhibits NHE activity (measured by a flow cytometry method) in a dose-dependent form of cell lines of mammalian origin (HEK293, NIH3T3, Jurkat and HL-1) as well as in primary cell culture from human skeletal muscle (myocytes and fibroblasts).In vivo, an ischemia-reperfusion model in dogs by coronary arterial occlusion was used (two hours of regional ischemia and three hours of reperfusion). In animals treated with halocin H6 there was a significant reduction of premature ventricular ectopic beats and infarct size, whereas blood pressure and heart rate remained unchanged. Up to date, halocin H6 is the only described biological molecule that exerts a, specific inhibitory activity in NHE of eukaryotic cells (AU)

En el presente trabajo se evalúa la capacidad de la halocina H6 (una proteína tipo bacteriocina producida por la haloarchaeaHaloferax gibbonsii) para inhibir el intercambiador Na+/H+ (NHE) de céludas de mamífero y su posible eficacia cardioprotectora frente a los daños causados por isquemia-reperfusión del miocardio. En experimentosin vitro H6 inhibe la actividad de NHE (determinada por citometría de flujo) de forma dosis-dependiente tanto en líneas celulares de mamíferos (HEK293, NIH3T3, Jurkat y HL-1) como en cultivos primarios de miocitos y fibroblastos aislados de músculo esquelético humano. En experimentosin vivo se utilizó un modelo de isquemia-reperfusión en perros por oclusión de la arteria coronaria (dos horas de isquemia y tres de reperfusión). En animales tratados con halocina H6 se produjo una disminución significativa a nivel estadístico, tanto del número de latidos ectópicos ventriculares como del tamaño del infarto, mientras que no se produjeron cambios tanto en la presión sanguínea como en el ritmo cardíaco. Hasta la fecha la halocina H6 es la única molécula biológica descrita que ejerce una actividad inhibidora específica sobre el NHE de células eucariotas (AU)

Characterization of a bacteriocin-like substance produced by Bacillus amyloliquefaciens isolated from the Brazilian Atlantic forest / Caracterización de una substancia del tipo bacteriocina producida por Bacillus amyloliquefaciens aislado del bosque atlántico brasileño

A Bacillus strain producing a bacteriocin-like substance was characterized by biochemical profiling and 16S rDNA sequencing. Phylogenetic analysis indicated that the strain has high sequence similarity with Bacillus amyloliquefaciens. The antimicrobial substance was inhibitory to pathogenic and food-spoilage bacteria, such as Listeria monocytogenes, Bacillus cereus, Serratia marcescens, and Pasteurella haemolytica. It was stable over a wide temperature range, but lost activity when the temperature reached 121 degrees C/15 min. Maximum activity was observed at acidic and neutral pH values, but not at alkaline pH. The antimicrobial substance was sensitive to the proteolytic action of trypsin, papain, proteinase K, and pronase E. Except for iturins, other antimicrobial peptides have not been described for B. amyloliquefaciens. The identification of a bacteriocin-like inhibitory substance active against L. monocytogenes addresses an important aspect of food protection (AU)

Se ha caracterizado una cepa de Bacillus productora de una substancia del tipo bacteriocina mediante perfiles bioquímicos y secuenciación del rDNA 16S. El análisis filogenético indicó que la cepa presentaba una gran similitud de secuencia con Bacillus amylolique faciens. La sustancia antimicrobiana era inhibitoria para algunas bacterias patógenas y causantes del deterioro de los alimentos como Listeria monocytogenes, Bacillus cereus, Serrati amarcescens y Pasteurella haemolytica. La sustancia era estable en una amplia gama de temperaturas, pero perdía la actividad cuando la temperatura alcanzaba 121°C/15 min. La actividad máxima fue observada a valores de pH ácidos y neutros, pero no a pH alcalinos. La sustancia antimicrobiana era sensible a la acción proteolítica de la tripsina, la papaína, la proteinasa K y la pronasa E. Excepto las iturinaos, no se han descrito otros péptidos antimicrobianos de B. amyloliquefaciens. La identificación de una sustancia tipo bacteriocina activa contra L. monocytogenes cubre un aspecto importante en la protección de los alimentos (AU)

Antibacterial activity of cerein 8A, a bacteriocin-like peptide produced by Bacillus cereus / Actividad antibacteriana de la cereína 8A, un péptido de tipo bacteriocina producido por Bacillus cereus

The mode of action of cerein 8A, a bacteriocin produced by the soil bacterium Bacillus cereus 8A, was investigated. The effect of cerein 8A was tested against Listeria monocytogenes and a bactericidal effect at 400 arbitrary units (AU)/ml was observed. In addition, cerein 8A was bactericidal against Bacillus cereus at 200 AU/ml, and inhibited the growth of Escherichia coli and Salmonella Enteritidis. Stronger inhibition of these gram-negative bacteria was achieved when the chelating agent EDTA was added together with bacteriocin. The effect of cerein 8A on B. cereus and L. monocytogenes was also investigated by Fourier transform infrared spectroscopy (FTIR). Treated cells had an important frequency increase at 2920 cm-1 and a decrease at 1400 cm-1, corresponding to assignments of fatty acids. Transmission electron microscopy showed damaged cell walls and loss of protoplasmic material. These results suggest that the mode of action of cerein 8A is to interfere with cell membranes and the cell wall (AU)

Se investigó el modo de acción de la cereína 8A, una bacteriocina producida por la bacteria del suelo Bacillus cereus 8A. El efecto de la cereína 8A fue probado contra Listeria monocytogenes, obteniendo un efecto bactericida a concentraciones de 400 unidades arbitrarias (AU)/ml. La cereína 8A también tuvo un efecto bactericida contra Bacillus cereus a una concentración de 200 AU/ml. La bacteriocina inhibió el crecimiento de Escherichia coli y Salmonella Enteritidis. Mayor inhibición contra estas bacterias gram-negativas se consiguió cuando a la bacteriocina se le añadió el agente quelante EDTA. El efecto de la cereína 8A sobre B. cereus y L. monocytogenes también fue investigado por espectroscopía de infrarrojos de transformación de Fourier (FTIR). Las células tratadas mostraron un importante crecimiento en frecuencia de 2920 cm-1 y un decrecimiento de 1400 cm-1 de banda, correspondiéndose con la asignación de los ácidos grasos. La microscopía electrónica de transmisión mostró que las células habían padecido daños en la pared celular, con pérdida de material protoplásmico. Los resultados sugieren que el modo de acción de la cereína 8A se produce mediante su intervención en las membranas celulares y en la pared celular (AU)

Colicin S8 export: extracellular and cytoplasmic colicin are different

The properties of colicin S8 are different for the cytoplasmic, periplasmic and extracellular protein. Interactions with its specific receptors reflect this. Active cell extracts separate into a non-anionic along with an anionic fraction by DEAE-Sephacell chromatography. Previously, we have purified cell-associated colicin S8 as an aggregation of highly related polypeptides; cytoplasmic colicin S8 seems to be post-translationally processed into an aggregation of polypeptides of molecular mass ranging from 45,000 Da to 60,000 Da. We suggest that a conformational change to colicin S8 may occur related to the export process (AU)

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Producción de baccteriocina por aislamientos clínicos de estafilococos / Bacteriocin production by clinical Staphylococcus isolates

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Bacterial membrane injuries induced by lactacin F and nisin / Lesiones en la membrana bacteriana inducidas por lactacina F y nisina

The combined action of nisin and lactacin F, two bacteriocins produced by lactic acid bacteria, is additive. In this report, the basis of this effect is examined. Channels formed by lactacin F were studied by experiments using planar lipid bilayers, and bactericidal effects were analyzed by flow cytometry. Lactacin F produced pores with a conductance of 1 ns in black lipid bilayers in 1 mM KCI at 10 mV at 20 degrees C. Pore formation was strongly dependent on voltage. Although lactacin F formed pores at very low potential (10 mV), the dependence was exponential above 40 mV. The injuries induced by nisin and lactacin F in the membranes of Lactobacillus helveticus produced different flow cytometric profiles. Probably, when both bacteriocins are present, each acts separately; their cooperation may be due to an increase in the number of single membrane injuries (AU)

La acción combinada de la nisina y la lactacina F (dos bacteriocinas producidas por las bacterias del ácido láctico) es aditiva. En este artículo se examina la base de este fenómeno. Los canales que forma la lactacina F fueron estudiados mediante experimentos en bicapas lipídicas, mientras que los efectos bactericidas fueron analizados mediante citometría de flujo. La lactacina F produjo poros con una conductancia de 1 nS en bicapas lipídicas negras en KCl 1 mM a 10 mV y 20ºC. La formación del poro es altamente dependiente del voltaje. Aunque la lactacina F forma poros a potenciales muy bajos (10 mV), la dependencia a partir de los 40 mV es exponencial. Las lesiones inducidas por la nisina y la lactacina F en las membranas de Lactobacillus helveticus producen perfiles de flujo citométrico distintos. Seguramente, cuando actúan juntas, cada molécula actúa por separado; su cooperación puede deberse a un aumento en el número de lesiones unimembranales. (AU)

Purification and biological characterization of halocin H1 from Haloferax mediterranei M2a

The production of halocins, bacteriocin-like proteins of ecological significance, is a frequent characteristic of species from the family Halobacteriaceae. Halocin H1, produced by Haloferax mediterranei strain M2a, is a single 31-kDa polypeptide. Its purification was achieved by combining two chromatographic systems: Sepharose 4B linked to bacitracin followed by hydroxylapatite Bio-gel HTP. Halocin H1 required concentrations of NaCl higher than 1.5 M to maintain its activity. Haoarchaeal strains showed a differential degree of sensitivity to the action of this halocin (AU)

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Classification and mode of action of membrane-active bacteriocins produced by gram-positive bacteria

Bacteriocins are ribosomally synthesized antimicrobial peptides produced by microorganisms belonging to different eubacterial taxonomic branches. Most of them are small cationic membrane-active compounds that form pores in the target cells, disrupting membrane potentials and causing cell death. The production of small cationic peptides with antibacterial activity is a defense strategy found not only in bacteria, but also in plants and animals. Bacteriocins are classified according to different criteria by different authors; in this review, we will summarize the principal bacteriocin classifications, highlight their main physical and chemical characteristics, and describe the mechanism of some selected bacteriocins that act at the membrane level (AU)

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