Metagenomic analysis of therapeutic PYO phage cocktails from 1997 to 2014

Phage therapy has regained interest in recent years due to the alarming spread of antibiotic resistance. Whilst phage cocktails are commonly sold in pharmacies in countries such as Georgia and Russia, this is not the case in western countries due to western regulatory agencies requiring a thorough characterization of the drug. Here, DNA sequencing of constituent biological entities constitutes a first step. The pyophage (PYO) cocktail is one of the main commercial products of the Georgian Eliava Institute of Bacteriophage, Microbiology and Virology and is used to cure skin infections. Since its first production in the 1930s, the composition of the cocktail has been periodically modified to add phages effective against emerging pathogenic strains. In this paper, we compared the composition of three PYO cocktails from 1997 (PYO97), 2000 (PYO2000) and 2014 (PYO2014). Based on next generation sequencing, de novo assembly and binning of contigs into draft genomes based on tetranucleotide distance, thirty and twenty-nine phage draft genomes were predicted in PYO97 and PYO2014, respectively. Of these, thirteen and fifteen shared high similarity to known phages. Eleven draft genomes were found to be common in the two cocktails. One of these showed no similarity to publicly available phage genomes. Representatives of phages targeting E. faecalis, E. faecium, E. coli, Proteus, P. aeruginosa and S. aureus were found in both cocktails. Finally, we estimated larger overlap of the PYO2000 cocktail to PYO97 compared to PYO2014. Using next generation sequencing and metagenomics analysis, we were able to characterize and compare the content of PYO cocktails separated by 17 years in time. Even though the cocktail composition is upgraded every six months, we found it to remain relatively stable over the years.Fil: Villarroel, Julia. Technical University of Denmark; DinamarcaFil: Larsen, Mette Voldby. GoSeqIt ApS; DinamarcaFil: Kilstrup, Mogens. Technical University of Denmark; DinamarcaFil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

The in vitro redundant enzymes PurN and PurT are both essential for systemic infection of mice in Salmonella enterica serovar Typhimurium

Metabolic enzymes show a high degree of redundancy, and for that reason they are generally ignored in searches for novel targets for anti-infective substances. The enzymes PurN and PurT are redundant in vitro in Salmonella enterica serovar Typhimurium, in which they perform the third step of purine synthesis. Surprisingly, the results of the current study demonstrated that single-gene deletions of each of the genes encoding these enzymes caused attenuation (competitive infection indexes [CI] of <0.03) in mouse infections. While the ΔpurT mutant multiplied as fast as the wild-type strain in cultured J774A.1 macrophages, net multiplication of the ΔpurN mutant was reduced approximately 50% in 20 h. The attenuation of the ΔpurT mutant was abolished by simultaneous removal of the enzyme PurU, responsible for the formation of formate, indicating that the attenuation was related to formate accumulation or wasteful consumption of formyl tetrahydrofolate by PurU. In the process of further characterization, we disclosed that the glycine cleavage system (GCV) was the most important for formation of C(1) units in vivo (CI = 0.03 ± 0.03). In contrast, GlyA was the only important enzyme for the formation of C(1) units in vitro. The results with the ΔgcvT mutant further revealed that formation of serine by SerA and further conversion of serine into C(1) units and glycine by GlyA were not sufficient to ensure C(1) formation in S. Typhimurium in vivo. The results of the present study call for reinvestigations of the concept of metabolic redundancy in S. Typhimurium in vivo

The Transcriptional and Gene Regulatory Network of Lactococcus lactis MG1363 during Growth in Milk

In the present study we examine the changes in the expression of genes of Lactococcus lactis subspecies cremoris MG1363 during growth in milk. To reveal which specific classes of genes (pathways, operons, regulons, COGs) are important, we performed a transcriptome time series experiment. Global analysis of gene expression over time showed that L. lactis adapted quickly to the environmental changes. Using upstream sequences of genes with correlated gene expression profiles, we uncovered a substantial number of putative DNA binding motifs that may be relevant for L. lactis fermentative growth in milk. All available novel and literature-derived data were integrated into network reconstruction building blocks, which were used to reconstruct and visualize the L. lactis gene regulatory network. This network enables easy mining in the chrono-transcriptomics data. A freely available website at http://milkts.molgenrug.nl gives full access to all transcriptome data, to the reconstructed network and to the individual network building blocks

CDKL5 expression is modulated during neuronal development and its subcellular distribution is tightly regulated by the C-terminal tail

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with Rett syndrome (RTT), West syndrome, and X-linked infantile spasms, sharing the common feature of mental retardation and early seizures. CDKL5 is a rather uncharacterized kinase, but its involvement in RTT seems to be explained by the fact that it works upstream of MeCP2, the main cause of Rett syndrome. To understand the role of this kinase for nervous system functions and to address if molecular mechanisms are involved in regulating its distribution and activity, we studied the ontogeny of CDKL5 expression in developing mouse brains by immunostaining and Western blotting. The expression profile of CDKL5 was compared with that of MeCP2. The two proteins share a general expression profile in the adult mouse brain, but CDKL5 levels appear to be highly modulated at the regional level. Its expression is strongly induced in early postnatal stages, and in the adult brain CDKL5 is present in mature neurons, but not in astroglia. Interestingly, the presence of CDKL5 in the cell nucleus varies at the regional level of the adult brain and is developmentally regulated. CDKL5 shuttles between the cytoplasm and the nucleus and the C-terminal tail is involved in localizing the protein to the cytoplasm in a mechanism depending on active nuclear export. Accordingly, Rett derivatives containing disease-causing truncations of the C terminus are constitutively nuclear, suggesting that they might act as gain of function mutations in this cellular compartment

Functional consequences of mutations in CDKL5, an X-linked gene involved in infantile spasms and mental retardation

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with Rett syndrome, West syndrome, and X-linked infantile spasms sharing the common features of generally intractable early seizures and mental retardation. Disease-causing mutations are distributed in both the catalytic domain and in the large COOH terminus. In this report, we examine the functional consequences of some Rett mutations of CDKL5 together with some synthetically designed derivatives useful to underline the functional domains of the protein. The mutated CDKL5 derivatives have been subjected to in vitro kinase assays and analyzed for phosphorylation of the TEY (Thr-Glu-Tyr) motif within the activation loop, their subcellular localization, and the capacity of CDKL5 to interact with itself. Whereas wild-type CDKL5 autophosphorylates and mediates the phosphorylation of the methyl-CpG-binding protein 2 (MeCP2) in vitro, Rett-mutated proteins show both impaired and increased catalytic activity suggesting that a tight regulation of CDKL5 is required for correct brain functions. Furthermore, we show that CDKL5 can self-associate and mediate the phosphorylation of its own TEY (Thr-Glu-Tyr) motif. Eventually, we show that the COOH terminus regulates CDKL5 properties; in particular, it negatively influences the catalytic activity and is required for its proper sub-nuclear localization. We propose a model in which CDKL5 phosphorylation is required for its entrance into the nucleus whereas a portion of the COOH-terminal domain is responsible for a stable residency in this cellular compartment probably through protein-protein interactions