Biology subjects

Cole, R.

Publications and source records attributed to Cole, R..

3 recordsLinked to original sources

bioRxiv2019

K. pneumoniae ST258 genomic variability and bacteriophage susceptibility

Multidrug resistant carbapenemase-producing Klebsiella pneumoniae capable of causing severe disease in humans is classified as an urgent threat by health agencies worldwide. Bacteriophages are being actively explored as potential therapeutics against these multidrug resistant pathogens. We are currently developing bacteriophage therapy against carbepenem-resistant K. pneumoniae belonging to the genetically diverse, globally disseminated clonal group CG258. In an effort to define a robust experimental approach for effective selection of lytic viruses for therapy, we have fully characterized the bacterial genomes of 18 target strains, tested them against novel lytic bacteriophages, and generated phage-susceptibility profiles. The genomes of K. pneumoniae isolates carrying blaNDM and blaKPC were sequenced and isolates belonging to CG258 were selected for susceptibility testing using a panel of lytic bacteriophages (n=65). The local K. pneumoniae CG258 population was dominated by isolates belonging to sequence type ST258 clade 1 (86%). The primary differences between ST258 genomes were variations in the capsular locus (cps) and in prophage content. We showed that CG258-specific lytic phages primarily target the capsule, and that successful infection is blocked in many, post-adsorption, by immunity conferred by existing prophages. Five bacteriophages specifically active against K. pneumoniae ST258 clade 1 (n=5) belonging to the Caudovirales order were selected for further characterization. Our findings show that effective control of K. pneumoniae CG258 with phage will require mixes of diverse lytic viruses targeting all relevant cps variants and allowing for variable prophage content. These insights will facilitate identification and selection of therapeutic phage candidates against this serious pathogen.\n\nImportanceBacteriophages are natural agents that exclusively and selectively kill bacteria and have the potential to be useful in the treatment of multidrug resistant infections. K. pneumoniae CG258 is a main agent of life-threatening sepsis that is often resistant to last-line antibiotics. Our work highlights some key requirements for developing bacteriophage preparations targeting this pathogen. By defining the genomic profile of our clinical K. pneumoniae CG258 population and matching it with bacteriophage susceptibility patterns, we found that bacteriophage ability to lyse each strain correlates well with K. pneumoniae CG258 structural subtypes (capsule variants). This indicates that preparation of bacteriophage therapeutics targeting this pathogen should aim at including phages against each bacterial capsular subtype. This necessitates a detailed understanding of the diversity of circulating isolates in different geographical areas in order to make rational therapeutic choices.

microbiology↗

bioRxiv2019

Deleting a UBE3A substrate rescues impaired hippocampal physiology and learning in Angelman syndrome mice

In humans, loss-of-function mutations in the UBE3A gene lead to the neurodevelopmental disorder Angelman syndrome (AS). AS patients have severe impairments in speech, learning and memory, and motor coordination, for which there is currently no treatment. In addition, UBE3A is duplicated in >1-2% of patients with autism spectrum disorders - a further indication of the significant role it plays in brain development. Altered expression of UBE3A, an E3 ubiquitin ligase, is hypothesized to lead to impaired levels of its target proteins, but identifying the contribution of individual UBE3A targets to UBE3A-dependent deficits remains of critical importance. Ephexin5 is a putative UBE3A substrate that has restricted expression early in development, regulates synapse formation during hippocampal development, and is abnormally elevated in AS mice, modeled by maternally-derived Ube3a gene deletion. Here, we report that Ephexin5 is a direct substrate of UBE3A ubiquitin ligase activity. Furthermore, removing Ephexin5 from AS mice specifically rescued hippocampus-dependent behaviors, CA1 physiology, and deficits in dendritic spine number. Our findings identify Ephexin5 as a key driver of hippocampal dysfunction and related behavioral deficits in AS mouse models. These results demonstrate the exciting potential of targeting Ephexin5, and possibly other UBE3A substrates, to improve symptoms of AS and other UBE3A-related developmental disorders.

neuroscience↗

bioRxiv2018

Proteogenomic annotation of the Chinese hamster reveals extensive novel translation events and endogenous retroviral elements

A high quality genome annotation greatly facilitates successful cell line engineering. Standard draft genome annotation pipelines are based largely on de novo gene prediction, homology, and RNA-Seq data. However, draft annotations can suffer from incorrectly predictions of translated sequence, incorrect splice isoforms and missing genes. Here we generated a draft annotation for the newly assembled Chinese hamster genome and used RNA-Seq, proteomics, and Ribo-Seq to experimentally annotate the genome. We identified 4,333 new proteins compared to the hamster RefSeq protein annotation and 2,503 novel translational events (e.g., alternative splices, mutations, novel splices). Finally, we used this pipeline to identify the source of translated retroviruses contaminating recombinant products from Chinese hamster ovary (CHO) cell lines, including 131 type-C retroviruses, thus enabling future efforts to eliminate retroviruses by reducing the costs incurred with retroviral particle clearance. In summary, the improved annotation provides a more accurate platform for guiding CHO cell line engineering, including facilitating the interpretation of omics data, defining of cellular pathways, and engineering of complex phenotypes.

bioinformatics↗