RESUMEN
Our observations of predatory fungi trapping rotifers in activated sludge and laboratory culture allowed us to discover a complicated trophic network that includes predatory fungi armed with bacteria and bacteriophages and the rotifers they prey on. Such a network seems to be common in various habitats, although it remains mostly unknown due to its microscopic size. In this study, we isolated and identified fungi and bacteria from activated sludge. We also noticed abundant, virus-like particles in the environment. The fungus developed absorptive hyphae within the prey. The bacteria showed the ability to enter and exit from the hyphae (e.g., from the traps into the caught prey). Our observations indicate that the bacteria and the fungus share nutrients obtained from the rotifer. To narrow the range of bacterial strains isolated from the mycelium, the effects of bacteria supernatants and lysed bacteria were studied. Bacteria isolated from the fungus were capable of immobilizing the rotifer. The strongest negative effect on rotifer mobility was shown by a mixture of Bacillus sp. and Stenotrophomonas maltophilia. The involvement of bacteriophages in rotifer hunting was demonstrated based on molecular analyses and was discussed. The described case seems to be an extraordinary quadruple microbiological puzzle that has not been described and is still far from being understood.
Asunto(s)
Fagos de Bacillus/fisiología , Hongos/patogenicidad , Rotíferos/microbiología , Animales , Bacillus/metabolismo , Fagos de Bacillus/genética , Bacterias , Quitinasas/metabolismo , Técnicas de Cocultivo , Consorcios Microbianos , Aguas del Alcantarillado/microbiología , Simbiosis , Eliminación de Residuos LíquidosRESUMEN
Nostocyclopeptides (Ncps) constitute a small class of nonribosomal peptides, exclusively produced by cyanobacteria of the genus Nostoc. The peptides inhibit the organic anion transporters, OATP1B3 and OATP1B1, and prevent the transport of the toxic microcystins and nodularin into hepatocytes. So far, only three structural analogues, Ncp-A1, Ncp-A2 and Ncp-M1, and their linear forms were identified in Nostoc strains as naturally produced cyanometabolites. In the current work, the whole genome sequence of the new Ncps producer, N. edaphicum CCNP1411 from the Baltic Sea, has been determined. The genome consists of the circular chromosome (7,733,505 bps) and five circular plasmids (from 44.5 kb to 264.8 kb). The nostocyclopeptide biosynthetic gene cluster (located between positions 7,609,981-7,643,289 bps of the chromosome) has been identified and characterized in silico. The LC-MS/MS analyzes of N. edaphicum CCNP1411 cell extracts prepared in aqueous methanol revealed several products of the genes. Besides the known peptides, Ncp-A1 and Ncp-A2, six other compounds putatively characterized as new noctocyclopeptide analogues were detected. This includes Ncp-E1 and E2 and their linear forms (Ncp-E1-L and E2-L), a cyclic Ncp-E3 and a linear Ncp-E4-L. Regardless of the extraction conditions, the cell contents of the linear nostocyclopeptides were found to be higher than the cyclic ones, suggesting a slow rate of the macrocyclization process.
Asunto(s)
Proteínas Bacterianas/metabolismo , Nostoc/metabolismo , Péptidos Cíclicos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Ciclización , Nostoc/genética , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Péptidos Cíclicos/genética , Péptidos Cíclicos/aislamiento & purificación , Conformación Proteica , Agua de Mar/microbiologíaRESUMEN
Two newly discovered bacteriophages, isolated from chicken feces and infecting Salmonella enterica strains, are described in this report. These phages have been named vB_Sen-TO17 and vB_Sen-E22, and we present their molecular and functional characterization. Both studied viruses are able to infect several S. enterica strains and develop lytically, but their specific host ranges differ significantly. Electron microscopic analyses of virions have been performed, and full genome sequences were determined and characterized, along with molecular phylogenetic studies. Genomes of vB_Sen-TO17 (ds DNA of 41,658 bp) and vB_Sen-E22 (dsDNA of 108,987 bp) are devoid of homologs of any known or putative gene coding for toxins or any other proteins potentially deleterious for eukaryotic cells. Both phages adsorbed efficiently (>95% adsorbed virions) within 10 min at 42 °C (resembling chicken body temperature) on cells of most tested host strains. Kinetics of lytic development of vB_Sen-TO17 and vB_Sen-E22, determined in one-step growth experiments, indicated that development is complete within 30-40 min at 42 °C, whereas burst sizes vary from 9 to 79 progeny phages per cell for vB_Sen-TO17 and from 18 to 64 for vB_Sen-E22, depending on the host strain. Virions of both phages were relatively stable (from several percent to almost 100% survivability) under various conditions, including acidic and alkaline pH values (from 3 to 12), temperatures from -80 °C to 60 °C, 70% ethanol, chloroform, and 10% DMSO. These characteristics of vB_Sen-TO17 and vB_Sen-E22 indicate that these phages might be considered in further studies on phage therapy, particularly in attempts to eliminate S. enterica from chicken intestine.
Asunto(s)
Bacteriófagos/aislamiento & purificación , Pollos/virología , Genoma Viral/genética , Salmonella enterica/genética , Animales , Bacteriófagos/clasificación , Bacteriófagos/genética , Bacteriófagos/ultraestructura , Pollos/genética , Heces/virología , Especificidad del Huésped/genética , Filogenia , Salmonella enterica/virología , Virión/genética , Virión/aislamiento & purificación , Virión/ultraestructuraRESUMEN
Molecular and functional characterization of a series of three bacteriophages, vB_SenM-1, vB_SenM-2, and vB_SenS-3, infecting various Salmonella enterica serovars and strains is presented. All these phages were able to develop lytically while not forming prophages. Moreover, they were able to survive at pH 3. The phages revealed different host ranges within serovars and strains of S. enterica, different adsorption rates on host cells, and different lytic growth kinetics at various temperatures (in the range of 25 to 42 °C). They efficiently reduced the number of cells in the bacterial biofilm and decreased the biofilm mass. Whole genome sequences of these phages have been determined and analyzed, including their phylogenetic relationships. In conclusion, we have demonstrated detailed characterization of a series of three bacteriophages, vB_SenM-1, vB_SenM-2, and vB_SenS-3, which reveal favorable features in light of their potential use in phage therapy of humans and animals, as well as for food protection purposes.
Asunto(s)
Bacteriófagos/clasificación , Salmonella enterica/clasificación , Salmonella enterica/virología , Bacteriófagos/genética , Bacteriófagos/fisiología , Genoma Viral , Especificidad del Huésped , Filogenia , Salmonella enterica/genética , Análisis de Secuencia de ADN , Temperatura , Secuenciación Completa del GenomaRESUMEN
A newly isolated bacteriophage infecting Enterococcus faecalis strains has been characterized, including determination of its molecular features. This phage, named vB_EfaS-271, has been classified as a Siphoviridae member, according to electron microscopy characterization of the virions, composed of a 50 nm-diameter head and a long, flexible, noncontractable tail (219 × 12.5 nm). Analysis of the whole dsDNA genome of this phage showed that it consists of 40,197 bp and functional modules containing genes coding for proteins that are involved in DNA replication (including DNA polymerase/primase), morphogenesis, packaging and cell lysis. Mass spectrometry analysis allowed us to identify several phage-encoded proteins. vB_EfaS-271 reveals a relatively narrow host range, as it is able to infect only a few E. faecalis strains. On the other hand, it is a virulent phage (unable to lysogenize host cells), effectively and quickly destroying cultures of sensitive host bacteria, with a latent period as short as 8 min and burst size of approximately 70 phages per cell at 37 °C. This phage was also able to destroy biofilms formed by E. faecalis. These results contribute to our understanding of the biodiversity of bacteriophages, confirming the high variability among these viruses and indicating specific genetic and functional features of vB_EfaS-271.
Asunto(s)
Bacteriófagos/crecimiento & desarrollo , Bacteriófagos/aislamiento & purificación , ADN Viral/análisis , Enterococcus faecalis/virología , Genoma Viral , Proteínas Virales/análisis , Virión/crecimiento & desarrollo , Bacteriófagos/genética , Bacteriófagos/ultraestructura , Especificidad del Huésped , Filogenia , Análisis de Secuencia de ADN , Aguas del Alcantarillado/microbiología , Proteínas Virales/metabolismo , Virión/genéticaRESUMEN
Genetic and immunological diseases, despite many attempts to develop effective treatments, still remain a great challenge for medicine. Current therapies of these diseases consist of pharmacological alleviation of symptoms, rehabilitation and psychological help which, although very important, are not sufficient. Therefore, searching for new therapeutics which could remove the major causes of these diseases is of particular importance for the society. Natural compounds reveal many biological activities which makes them candidates for drugs in such diseases. One of them is genistein, a compound from the group of flavonoids. As it affects multiple processes, genistein has become in the center of interest of many scientists working on diseases of various etiology, course and inheritance. It was used in experimental therapies of some genetic diseases (Huntington's disease, amyotrophic lateral sclerosis Parkinson disease, cystic fibrosis), as well as autoimmunological diseases and allergies. Clinical trials with the use of genistein in treatment of patients suffering from Alzheimer's diseases and mucopolysaccharidosis type III are ongoing. The employment of differential properties of genistein in attempts to treat each of these diseases is of special interest. In this review, detailed molecular mechanisms of genistein action are summarized in the light of therapies of the above mentioned genetic and immunological diseases, including description of therapeutic potentials of each activity of this isoflavone, efficiency of its action, and its potential use as a drug in the future.
Asunto(s)
Genisteína/farmacología , Genisteína/uso terapéutico , Enfermedades del Sistema Inmune/tratamiento farmacológico , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/genética , Humanos , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/genética , Mucopolisacaridosis III/tratamiento farmacológico , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/genéticaRESUMEN
One of the hopes for overcoming the antibiotic resistance crisis is the use of bacteriophages to combat bacterial infections, the so-called phage therapy. This therapeutic approach is generally believed to be safe for humans and animals as phages should infect only prokaryotic cells. Nevertheless, recent studies suggested that bacteriophages might be recognized by eukaryotic cells, inducing specific cellular responses. Here we show that in chickens infected with Salmonella enterica and treated with a phage cocktail, bacteriophages are initially recognized by animal cells as viruses, however, the cGAS-STING pathway (one of two major pathways of the innate antiviral response) is blocked at the stage of the IRF3 transcription factor phosphorylation. This inhibition is due to the inability of RNA polymerase III to recognize phage DNA and to produce dsRNA molecules which are necessary to stimulate a large protein complex indispensable for IRF3 phosphorylation, indicating the mechanism of the antiviral response impairment.
Asunto(s)
Bacteriófagos , Terapia de Fagos , Humanos , Animales , Bacteriófagos/fisiología , Pollos , Inmunidad , AntiviralesRESUMEN
Cyanobacteria of the Nostoc genus belong to the most prolific sources of bioactive metabolites. In our previous study on Nostoc edaphicum strain CCNP1411, the occurrence of cyanopeptolins and nostocyclopeptides was documented. In the current work, the production of anabaenopeptins (APs) by the strain was studied using genetic and chemical methods. Compatibility between the analysis of the apt gene cluster and the structure of the identified APs was found. Three of the APs, including two new variants, were isolated as pure compounds and tested against four serine proteases and carboxypeptidase A (CPA). The in vitro enzymatic assays showed a typical activity of this class of cyanopeptides, i.e., the most pronounced effects were observed in the case of CPA. The activity of the detected compounds against important metabolic enzymes confirms the pharmaceutical potential of anabaenopeptins.
Asunto(s)
Nostoc , Péptidos Cíclicos , Carboxipeptidasas A/metabolismo , Nostoc/genética , Nostoc/metabolismo , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Serina Proteasas/metabolismoRESUMEN
The characterization of a recently isolated bacteriophage, vB_Eco4M-7, which effectively infects many, though not all, Escherichia coli O157 strains, is presented. The genome of this phage comprises double-stranded DNA, 68,084 bp in length, with a GC content of 46.2%. It contains 96 putative open reading frames (ORFs). Among them, the putative functions of only 35 ORFs were predicted (36.5%), whereas 61 ORFs (63.5%) were classified as hypothetical proteins. The genome of phage vB_Eco4M-7 does not contain genes coding for integrase, recombinase, repressors or excisionase, which are the main markers of temperate viruses. Therefore, we conclude that phage vB_Eco4M-7 should be considered a lytic virus. This was confirmed by monitoring phage lytic development by a one-step growth experiment. Moreover, the phage forms relatively small uniform plaques (1 mm diameter) with no properties of lysogenization. Electron microscopic analyses indicated that vB_Eco4M-7 belongs to the Myoviridae family. Based on mass spectrometric analyses, including the fragmentation pattern of unique peptides, 33 phage vB_Eco4M-7 proteins were assigned to annotated open reading frames. Importantly, genome analysis suggested that this E. coli phage is free of toxins and other virulence factors. In addition, a similar, previously reported but uncharacterized bacteriophage, ECML-117, was also investigated, and this phage exhibited properties similar to vB_Eco4M-7. Our results indicate that both studied phages are potential candidates for phage therapy and/or food protection against Shiga toxin-producing E. coli, as the majority of these strains belong to the O157 serotype.