Periodic Reporting for period 1 - AGAPE (Accessory Genome Accountability in Pseudomonas aeruginosa Persistent Infections)
Reporting period: 2021-07-01 to 2023-06-30
Antimicrobial resistance (AMR) is one of the leading causes of death in the world, with 1.27 million deaths attributable to these kind of infections. Emergence of resistance results from the adaptation of the pathogen to the host, in a complex process with many unknown players. In some cases, resistant bacterial pathogens can generate chronic infections, from the survival of a small subpopulation of bacteria after antibiotic treatment. Persistent infections critically decrease the life quality and expectancy of patients, in addition to posing a huge burden to the health sector. One of the pathogens generating chronic infections is Pseudomonas aeruginosa, a bacterium found in the airways of 70% of patients with Cystic Fibrosis (CF). This opportunistic pathogen takes advantage of the abnormal mucosity production in CF, quickly adapting its lifestyle to colonise the airways and utilise the available nutrients, while also escaping the immune system and antimicrobial treatment. Mostly, the success of P. aeruginosa relies on
its adaptability, which derives from its large genome, carrying genes for survival in a wide spectrum of environments. Most of these genes are not conserved among strains, composing the accessory genome (AG). Previous studies have shown the presence of virulence and resistance genes in the AG, yet a majority of accessory genes remain uncharacterised. Therefore, it has not been possible to assign a role to this variable portion of the genome in the establishment of persistent infections. A main objective of this project is to elucidate the relevance of the AG in CF infections, for which a genetic tool that allows for the systematic and high-throughput investigation of the AG components is required. In AGAPE, CRISPR interference (CRISPRi) libraries will be used to generate thousands of individual perturbations on accessory gene expression levels, in order to analyse their effect in bacterial fitness. CRISPRi will be carried out both in laboratory strains, in order to set up the technique, and in clinical isolates obtained from CF patients. AGAPE seeks to represent the full range of adaptability, by studying isolates that colonise patients from months to decades. In order to test the effect of the introduced perturbations in the AG, CRISPRi libraries will be used to infect lung organoids and air-liquid interfase cultures. These model systems will allow to identify relevant genetic players for the infection process. A second objective of this project is to characterise the dynamic conformation of the accessory genome of P. aeruginosa during host adaptation. Although previous studied have demonstrated that host adaptation results in genomic reduction, clinical isolates retain AG variability. Therefore, the project seeks to uncover the plasticity of the genome during infection, focusing on whether horizontal gene transfer of certain elements can be linked to infection persistence. For this, the sequences of P. aeruginosa clinical isolates obtained longitudinally from CF patients will be compared, in order to identify accessory genes conserved through infection. This information will be then cross-referenced with the CRISPRi functional analyses to obtain a detailed picture on the role of the accessory genome during P. aeruginosa persistent infections.
its adaptability, which derives from its large genome, carrying genes for survival in a wide spectrum of environments. Most of these genes are not conserved among strains, composing the accessory genome (AG). Previous studies have shown the presence of virulence and resistance genes in the AG, yet a majority of accessory genes remain uncharacterised. Therefore, it has not been possible to assign a role to this variable portion of the genome in the establishment of persistent infections. A main objective of this project is to elucidate the relevance of the AG in CF infections, for which a genetic tool that allows for the systematic and high-throughput investigation of the AG components is required. In AGAPE, CRISPR interference (CRISPRi) libraries will be used to generate thousands of individual perturbations on accessory gene expression levels, in order to analyse their effect in bacterial fitness. CRISPRi will be carried out both in laboratory strains, in order to set up the technique, and in clinical isolates obtained from CF patients. AGAPE seeks to represent the full range of adaptability, by studying isolates that colonise patients from months to decades. In order to test the effect of the introduced perturbations in the AG, CRISPRi libraries will be used to infect lung organoids and air-liquid interfase cultures. These model systems will allow to identify relevant genetic players for the infection process. A second objective of this project is to characterise the dynamic conformation of the accessory genome of P. aeruginosa during host adaptation. Although previous studied have demonstrated that host adaptation results in genomic reduction, clinical isolates retain AG variability. Therefore, the project seeks to uncover the plasticity of the genome during infection, focusing on whether horizontal gene transfer of certain elements can be linked to infection persistence. For this, the sequences of P. aeruginosa clinical isolates obtained longitudinally from CF patients will be compared, in order to identify accessory genes conserved through infection. This information will be then cross-referenced with the CRISPRi functional analyses to obtain a detailed picture on the role of the accessory genome during P. aeruginosa persistent infections.
To date, I have tested several CRISPRi systems in P. aeruginosa laboratory strains PAO1 and PA14, and a clinical isolate from the strain DK01. Having selected the best performing one on a fluorescence-decay experimental setup, I have designed and obtained an initial sgRNA library, which targets genes involved in transcriptional regulation, virulence, and pathogenesis, among others. This initial library will be used to test the limits of the CRISPRi high-throughput technique in P. aeruginosa. Once the ideal library size and CRISPRi protocol have been determined, the system will be transferred to clinical isolates of interest. After testing on these bacteria, new libraries targeting the accessory genome will be designed and put to the test both under planktonic and infection conditions. In parallel, the clinical isolates of interest have been selected from the available biobank, with the main selection criteria being the availability of longitudinal isolates spanning over 2 years of continuous infection (chronicity). The genomic sequences of these isolates are currently being compared, specifically between the earliest and the latest isolate of each strain, to determine the changes on the accessory genome and identify targets of interest for CRISPRi.
Beyond these results, I have engaged in science communication by participating in Skype a Scientist sessions and the Science is Wonderful event organized by the European Commission. I have also disseminated my project by giving an invited seminar at the Institute for Microbiology and Biotechnology, Rheinische Friedrich-Wilhelms-Universität Bonn and participating in the conference "Pseudomonas 2022" and the EMBO workshop "New Approaches to Combat Antibiotic-Resistant Bacteria 2022". I have expanded my network by organizing seminars and reaching out to possible collaborators within the Technical University of Denmark, in addition to expanding my social media presence on Twitter. Finally, I have engaged in teaching young researchers, through the supervision of a Master thesis and the planning and execution of the P. aeruginosa track of the PhD course "Advanced Experimental Synthetic Biology for Cell Factories" at the Technical University of Denmark.
Beyond these results, I have engaged in science communication by participating in Skype a Scientist sessions and the Science is Wonderful event organized by the European Commission. I have also disseminated my project by giving an invited seminar at the Institute for Microbiology and Biotechnology, Rheinische Friedrich-Wilhelms-Universität Bonn and participating in the conference "Pseudomonas 2022" and the EMBO workshop "New Approaches to Combat Antibiotic-Resistant Bacteria 2022". I have expanded my network by organizing seminars and reaching out to possible collaborators within the Technical University of Denmark, in addition to expanding my social media presence on Twitter. Finally, I have engaged in teaching young researchers, through the supervision of a Master thesis and the planning and execution of the P. aeruginosa track of the PhD course "Advanced Experimental Synthetic Biology for Cell Factories" at the Technical University of Denmark.
Through this project, I expect to identify among the accessory genes novel markers of persistent infection. In addition, I seek to highlight the genes that tolerate less the CRISPRi perturbations as potential targets for novel antibiotics. Overall, I wish to further the knowledge regarding bacterial chronic infections and the molecular mechanism behind them, with the bigger goal of helping patients that currently do not have a viable treatment. Furthermore, I am working towards training the next generation of scientists, and trying to encourage a younger audience to become scientists and be aware of the growing problem of antibiotic resistance.