Periodic Reporting for period 2 - PHAGENET (PHAGENET: PHAge GEnetic NETworking in the microbiome)
Período documentado: 2023-01-01 hasta 2024-06-30
The PHAGENET project seeks to elucidate the role of bacteriophages (phages) and phage-mediated horizontal gene transfer (HGT) in the functioning of the human gut microbiome. Complex microbial communities such as the human gut microbiome are believed to evolve and adapt primarily through HGT. HGT circumvents the barriers imposed by vertical evolution and allows individual strains to access the common distributed gene pool (the pangenome). The pangenome of any given microbial species, as well as the collective pangenome of the microbiome, act as “public libraries” of genetic material that can be re-shuffled and subjected to natural selection in the different genetic backgrounds of individual microbiome members.
The resulting dissemination of fitness-increasing “beneficial” traits is thought to be essential for the maintenance of functional diversity, resistance to adverse events and resilience in the microbiome. Likewise, “undesirable” (from a human perspective) traits such as antibiotic resistance genes (ARGs), toxins and virulence factors get a chance to be “sampled”, shared, and positively selected. Therefore, it should be expected that HGT plays a central role in the emergence and evolution of antibiotic-resistant pathogens, so called “superbugs”. These antibiotic resistant pathogens are one of the key unsolved problems faced by European and global healthcare systems, estimated to cause 25,000 deaths and economic losses of €1.5 billion annually in the EU alone.
The overarching aim of PHAGENET is to find out whether or not that phage-mediated HGT operates at high rate in the human gut microbiome, and whether or not it plays a major role in maintaining genetic diversity, resistance, and resilience. The central hypothesis underpinning this research project is that phages are the most abundant, the most sophisticated and most efficient gene-transfer pathway within the microbiome, acting to (a) increase the connectivity and accessibility of bacterial pangenomes in the microbiome; (b) create a repository or “filing” system of non-essential genes; (c) provide a channel for communication (genetic exchange, HGT) between individual human microbiome and the broader environment; and (d) help to rapidly spread specific fitness-promoting genes (including ARGs and virulence genes) and alleles required by bacterial populations.
The resulting dissemination of fitness-increasing “beneficial” traits is thought to be essential for the maintenance of functional diversity, resistance to adverse events and resilience in the microbiome. Likewise, “undesirable” (from a human perspective) traits such as antibiotic resistance genes (ARGs), toxins and virulence factors get a chance to be “sampled”, shared, and positively selected. Therefore, it should be expected that HGT plays a central role in the emergence and evolution of antibiotic-resistant pathogens, so called “superbugs”. These antibiotic resistant pathogens are one of the key unsolved problems faced by European and global healthcare systems, estimated to cause 25,000 deaths and economic losses of €1.5 billion annually in the EU alone.
The overarching aim of PHAGENET is to find out whether or not that phage-mediated HGT operates at high rate in the human gut microbiome, and whether or not it plays a major role in maintaining genetic diversity, resistance, and resilience. The central hypothesis underpinning this research project is that phages are the most abundant, the most sophisticated and most efficient gene-transfer pathway within the microbiome, acting to (a) increase the connectivity and accessibility of bacterial pangenomes in the microbiome; (b) create a repository or “filing” system of non-essential genes; (c) provide a channel for communication (genetic exchange, HGT) between individual human microbiome and the broader environment; and (d) help to rapidly spread specific fitness-promoting genes (including ARGs and virulence genes) and alleles required by bacterial populations.
1. Proof-of-principle demonstration of the use of long-read Nanopore sequencing on high-molecular weight encapsidated DNA from complex faecal phage communities to uncover different modes of phage transduction operating in the human gut microbiome (presented at the MENI meeting in Cork in 2023, and currently being prepared for publication).
2. Collaboration with University of York, resulting in the publication of the first high resolution virion structure from the most prevalent human gut phage (Bayfield, Shkoporov et al., Nature 2023, https://doi.org/10.1038/s41586-023-06019-2(se abrirá en una nueva ventana))
3. Additional studies of the human gut virome conducted and published under the auspices of PHAGENET: update of phage taxonomy by the International Committee on Taxonomy of Viruses (Turner, Shkoporov et al., Arch Virol 2023: https://doi.org/10.1007/s00705-022-05694-2)(se abrirá en una nueva ventana); a study of interpersonal variability of gut phageome/virome in IBD patients and healthy subjects (Stockdale, Shkoporov et al., Commun Biol 2023: https://doi.org/10.1038/s42003-023-04592-w)(se abrirá en una nueva ventana); and a study of gut phage transmission between mothers and their infants Garmaeva et al., bioRxiv 2023: https://doi.org/10.1101/2023.08.21.554108)(se abrirá en una nueva ventana); isolation and characterisation of novel Ruminococcus phages (Buttimer et al., Gut Microbes 2023: https://doi.org/10.1080/19490976.2023.2194794(se abrirá en una nueva ventana)).
4. Obtaining all necessary authorisations and approvals from national regulatory authorities and institutional committees (HPRA, EPA, CREC) to collect human biological samples and conduct in vitro and in vivo (mouse) experiments planned in this project. Commencement of the human sample collection and animal experiments.
5. Bioinformatic pipelines and SOPs developed for upcoming sequencing of human, animal and in vitro-generated microbiome/virome samples.
2. Collaboration with University of York, resulting in the publication of the first high resolution virion structure from the most prevalent human gut phage (Bayfield, Shkoporov et al., Nature 2023, https://doi.org/10.1038/s41586-023-06019-2(se abrirá en una nueva ventana))
3. Additional studies of the human gut virome conducted and published under the auspices of PHAGENET: update of phage taxonomy by the International Committee on Taxonomy of Viruses (Turner, Shkoporov et al., Arch Virol 2023: https://doi.org/10.1007/s00705-022-05694-2)(se abrirá en una nueva ventana); a study of interpersonal variability of gut phageome/virome in IBD patients and healthy subjects (Stockdale, Shkoporov et al., Commun Biol 2023: https://doi.org/10.1038/s42003-023-04592-w)(se abrirá en una nueva ventana); and a study of gut phage transmission between mothers and their infants Garmaeva et al., bioRxiv 2023: https://doi.org/10.1101/2023.08.21.554108)(se abrirá en una nueva ventana); isolation and characterisation of novel Ruminococcus phages (Buttimer et al., Gut Microbes 2023: https://doi.org/10.1080/19490976.2023.2194794(se abrirá en una nueva ventana)).
4. Obtaining all necessary authorisations and approvals from national regulatory authorities and institutional committees (HPRA, EPA, CREC) to collect human biological samples and conduct in vitro and in vivo (mouse) experiments planned in this project. Commencement of the human sample collection and animal experiments.
5. Bioinformatic pipelines and SOPs developed for upcoming sequencing of human, animal and in vitro-generated microbiome/virome samples.
Long read sequencing of intact DNA from phages and tranducing particles constitutes a novel approach to analysing transduction in single phage/bacteria co-cultures, as well as in complex microbiome, where it is capable of identifying characheristic patterns associated with different types of transduction. A pilot study using this approach has been conducted giving promising results and informing our future experiments. With the help of this technique and other approaches, the project is expected to gather unique data on transduction dynamics from a complex human gut microbial community, identify evolutionary patterns in the type and frequency of transduction events, extrapolate trends that will inform the fields of bacteriophage research and microbial evolution, as well as having implications for the treatment of human gut dysbiosis. In addition to answering the projects’ main research question, the action will deliver biotechnological resources, such as novel transposon libraries, a host-phage pair collection, and a faecal chemostat fermentation system optimally designed to investigate horizontal gene transfer in vitro.