Pseudomonas Phage MD8: Genetic Mosaicism and Challenges of Taxonomic Classification of Lambdoid Bacteriophages.
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ABSTRACT:
SUBMITTER: Evseev P
PROVIDER: S-EPMC8508860 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mosaicism denotes an individual who has at least two populations of cells with distinct genotypes that are derived from a single fertilized egg. Genetic variation among the cell lines can involve whole chromosomes, structural or copy-number variants, small or single-nucleotide variants, or epigenetic variants. The mutational events that underlie mosaic variants occur during mitotic cell divisions after fertilization and zygote formation. The initiating mutational event can occur in any types of cell at any time in development, leading to enormous variation in the distribution and phenotypic effect of mosaicism. A number of classification proposals have been put forward to classify genetic mosaicism into categories based on the location, pattern, and mechanisms of the disease. We here propose a new classification of genetic mosaicism that considers the affected tissue, the pattern and distribution of the mosaicism, the pathogenicity of the variant, the direction of the change (benign to pathogenic vs. pathogenic to benign), and the postzygotic mutational mechanism. The accurate and comprehensive categorization and subtyping of mosaicisms is important and has potential clinical utility to define the natural history of these disorders, tailor follow-up frequency and interventions, estimate recurrence risks, and guide therapeutic decisions.
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Project description:Shiga toxin is the major virulence factor of enterohemorrhagic Escherichia coli (EHEC), and the gene encoding it is carried within the genome of Shiga toxin-converting phages (Stx phages). Numerous Stx phages have been sequenced to gain a better understanding of their contribution to the virulence potential of EHEC. The Stx phages are classified into the lambdoid phage family based on similarities in lifestyle, gene arrangement, and nucleotide sequence to the lambda phages. This study explores the replication regions of non-lambdoid Stx phages that completely lack the O and P genes encoding the proteins involved in initiating replication in the lambdoid phage genome. Instead, they carry sequences encoding replication proteins that have not been described earlier, here referred to as eru genes (after EHEC phage replication unit genes). This study identified three different types of Eru-phages, where the Eru1-type is carried by the highly pathogenic EHEC strains that caused the Norwegian O103:H25 outbreak in 2006 and the O104:H4 strain that caused the large outbreak in Europe in 2011. We show that Eru1-phages exhibit a less stable lysogenic state than the classical lambdoid Stx phages. As production of phage particles is accompanied by production of Stx toxin, the Eru1-phage could be associated with a high-virulence phenotype of the host EHEC strain. This finding emphasizes the importance of classifying Stx phages according to their replication regions in addition to their Stx-type and could be used to develop a novel strategy to identify highly virulent EHEC strains for improved risk assessment and management.
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Project description:The Escherichia coli strain Nissle 1917 (EcN) is used as a probiotic for the treatment of certain gastrointestinal diseases in several European and non-European countries. In vitro studies showed EcN to efficiently inhibit the production of Shiga toxin (Stx) by Stx producing E. coli (STEC) such as Enterohemorrhagic E. coli (EHEC). The occurrence of the latest EHEC serotype (O104:H4) responsible for the great outbreak in 2011 in Germany was due to the infection of an enteroaggregative E. coli by a Stx 2-encoding lambdoid phage turning this E. coli into a lysogenic and subsequently into a Stx producing strain. Since EHEC infected persons are not recommended to be treated with antibiotics, EcN might be an alternative medication. However, because a harmless E. coli strain might be converted into a Stx-producer after becoming host to a stx encoding prophage, we tested EcN for stx-phage genome integration. Our experiments revealed the resistance of EcN towards not only stx-phages but also against the lambda phage. This resistance was not based on the lack of or by mutated phage receptors. Rather the expression of certain genes (superinfection exclusion B (sieB) and a phage repressor (pr) gene) of a defective prophage of EcN was involved in the complete resistance of EcN to infection by the stx- and lambda phage. Obviously, EcN cannot be turned into a Stx producer. Furthermore, we observed EcN to inactivate phages and thereby to protect E. coli K-12 strains against infection by stx- as well as lambda-phages. Inactivation of lambda-phages was due to binding of lambda-phages to LamB of EcN whereas inactivation of stx-phages was caused by a thermostable protein of EcN. These properties together with its ability to inhibit Stx production make EcN a good candidate for the prevention of illness caused by EHEC and probably for the treatment of already infected people.
2018-06-14 | GSE109932 | GEO
Project description:Shiga toxin (Stx) producing E. coli (STEC) such as Enterohemorrhagic E. coli (EHEC) are the major cause of foodborne illness in humans. In vitro studies showed the probiotic Escherichia coli strain Nissle 1917 (EcN) to efficiently inhibit the production of Stx. Life threatening EHEC strains as for example the serotype O104:H4, responsible for the great outbreak in 2011 in Germany, evolutionary developed from certain E. coli strains which got infected by stx2-encoding lambdoid phages turning the E. coli into lysogenic and subsequently Stx producing strains. Since antibiotics induce stx genes and Stx production, EHEC infected persons are not recommended to be treated with antibiotics. Therefore, EcN might be an alternative medication. However, because even commensal E. coli strains might be converted into Stx-producers after becoming host to a stx encoding prophage, we tested EcN for stx-phage genome integration. Our experiments revealed the resistance of EcN toward not only stx-phages but also against lambda-phages. This resistance was not based on the lack of or by mutated phage receptors. Rather it involved the expression of a phage repressor (pr) gene of a defective prophage in EcN which was able to partially protect E. coli K-12 strain MG1655 against stx and lambda phage infection. Furthermore, we observed EcN to inactivate phages and thereby to protect E. coli K-12 strains against infection by stx- as well as lambda-phages. Inactivation of lambda-phages was due to binding of lambda-phages to LamB of EcN whereas inactivation of stx-phages was caused by a thermostable protein of EcN. These properties together with its ability to inhibit Stx production make EcN a good candidate for the prevention of illness caused by EHEC and probably for the treatment of already infected people.
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Project description:21 is a temperate lambdoid coliphage, and the genes that encode the head proteins of lambda and 21 are descended from a common ancestral bacteriophage. The sequencing of terminase genes 1 and 2 of 21 was completed, along with that of a segment at the right end of 21 DNA that includes the R4 sequence. The R4 sequence, a site that is likely involved in termination of DNA packaging, was found to be very similar to the R4 sequences of lambda and phi 80, suggesting that R4 is a recognition site that is not phage specific. DNA packaging by 21 is dependent on a host protein, integration host factor. A series of mutations in gene 1 (her mutations), which allow integration host factor-independent DNA packaging by 21, were found to be missense changes that affect predicted alpha-helixes in gp1. gp2, the large terminase subunit, is predicted to contain an ATP-binding domain and, perhaps, a second domain important for the cos-cutting activity of terminase. orf1, an open reading frame analogous in position to FI, a lambda gene involved in DNA packaging, shares some sequence identity with FI. orf1 was inactivated with nonsense and insertion mutations; these mutations were found not to affect phage growth. 21 was also not able to complement a lambda FI mutant.
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Project description:Practical applications of bacteriophages in medicine and biotechnology induce a great need for technologies of phage purification. None of the popular methods offer solutions for separation of a phage from another similar phage. We used affinity chromatography combined with competitive phage display (i) to purify T4 bacteriophage from bacterial debris and (ii) to separate T4 from other contaminating bacteriophages. In 'competitive phage display' bacterial cells produced both wild types of the proteins (expression from the phage genome) and the protein fusions with affinity tags (expression from the expression vectors). Fusion proteins were competitively incorporated into the phage capsid. It allowed effective separation of T4 from a contaminating phage on standard affinity resins.
| S-EPMC3827602 | biostudies-other
Project description:Currently, the bacterial resistance, especially to most commonly used antibiotics has proved to be a severe therapeutic problem. Nosocomial and community-acquired infections are usually caused by multidrug resistant strains. Therefore, we are forced to develop an alternative or supportive treatment for successful cure of life-threatening infections. The idea of using natural bacterial pathogens such as bacteriophages is already well known. Many papers have been published proving the high antibacterial efficacy of lytic phages tested in animal models as well as in the clinic. Researchers have also investigated the application of non-lytic phages and temperate phages, with promising results. Moreover, the development of molecular biology and novel generation methods of sequencing has opened up new possibilities in the design of engineered phages and recombinant phage-derived proteins. Encouraging performances were noted especially for phage enzymes involved in the first step of viral infection responsible for bacterial envelope degradation, named depolymerases. There are at least five major groups of such enzymes - peptidoglycan hydrolases, endosialidases, endorhamnosidases, alginate lyases and hyaluronate lyases - that have application potential. There is also much interest in proteins encoded by lysis cassette genes (holins, endolysins, spanins) responsible for progeny release during the phage lytic cycle. In this review, we discuss several issues of phage and phage-derived protein application approaches in therapy, diagnostics and biotechnology in general.
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Project description:We report the genome sequences of two double-stranded DNA siphoviruses, POR1 infective for Pseudomonas oryzihabitans and PAE1 infective for Pseudomonas aeruginosa The phage POR1 genome showed no nucleotide sequence homology to any other DNA phage sequence in the GenBank database, while phage PAE1 displayed synteny to P. aeruginosa phages M6, MP1412, and YuA.
| S-EPMC4911491 | biostudies-literature
Project description:BackgroundCaM (calmodulin) mutations are associated with congenital arrhythmia susceptibility (calmodulinopathy) and are most often de novo. In this report, we sought to broaden the genotype-phenotype spectrum of calmodulinopathies with 2 novel calmodulin mutations and to investigate mosaicism in 2 affected families.MethodsCaM mutations were identified in 4 independent cases by DNA sequencing. Biochemical and electrophysiological studies were performed to determine functional consequences of each mutation.ResultsGenetic studies identified 2 novel CaM variants (CALM3-E141K in 2 cases; CALM1-E141V) and one previously reported CaM pathogenic variant (CALM3-D130G) among 4 probands with shared clinical features of prolonged QTc interval (range 505-725 ms) and documented ventricular arrhythmia. A fatal outcome occurred for 2 of the cases. The parents of all probands were asymptomatic with normal QTc duration. However, 2 of the families had multiple affected offspring or multiple occurrences of intrauterine fetal demise. The mother from the family with recurrent intrauterine fetal demise exhibited the CALM3-E141K mutant allele in 25% of next-generation sequencing reads indicating somatic mosaicism, whereas CALM3-D130G was present in 6% of captured molecules of the paternal DNA sample, also indicating mosaicism. Two novel mutations (E141K and E141V) impaired Ca2+ binding affinity to the C-domain of CaM. Human-induced pluripotent stem cell-derived cardiomyocytes overexpressing mutant or wild-type CaM showed that both mutants impaired Ca2+-dependent inactivation of L-type Ca2+ channels and prolonged action potential duration.ConclusionsWe report 2 families with somatic mosaicism associated with arrhythmogenic calmodulinopathy, and demonstrate dysregulation of L-type Ca2+ channels by 2 novel CaM mutations affecting the same residue. Parental mosaicism should be suspected in families with unexplained fetal arrhythmia or fetal demise combined with a documented CaM mutation.
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Project description:Despite their ubiquity, relatively few bacteriophages have been characterized. Here, we set out to explore Caulobacter bacteriophages (caulophages) in the rhizosphere and characterized Kronos, the first caulophage isolated from the rhizosphere. Kronos is a member of the Siphoviridae family since it has a long flexible tail. In addition, an analysis of the Kronos genome indicated that many of the predicted proteins were distantly related to those of bacteriophages in the lambdoid family. Consistent with this observation, we were able to demonstrate the presence of cos sites that are similar to those found at the ends of lambdoid phage genomes. Moreover, Kronos displayed a relatively rare head and tail morphology compared to other caulophages but was similar to that of the lambdoid phages. Taken together, these data indicate that Kronos is distantly related to lambdoid phages and may represent a new Siphoviridae genus.
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