Periodic Reporting for period 1 - Cell2Cell (What makes a successfull pathogen? Understanding the impact of cell-to-cell heterogeneity in chromatin structure on infection and adaptation) Reporting period: 2019-11-01 to 2021-10-31 Summary of the context and overall objectives of the project Infectious diseases have an enormous impact on worldwide mortality and society. They are a leading cause of death, particularly in low-income countries. In 2017, infectious diseases were responsible worldwide for 66% of deaths among children under the age of 5. In 2019, three infectious diseases -lower respiratory infections, malaria, and diarrheal diseases- were ranked in the top ten causes of death by the World Health Organization. The newly emerged infectious disease, COVID-19, became a main cause of death in 2020. While infections are most prevalent in developing countries, globalization and increasing migration make infectious diseases a challenge for the entire world. To improve current drug treatments and develop new vaccines, a better understanding of the pathogens and their specific requirements for establishing successful infections that cause the diseases is needed. Yet, little is known about what makes some individual pathogens within pathogen population more successful in establishing an infection than others.Cell-to-cell heterogeneity impacts the outcome of infection. It is a major driver in the adaptation of pathogens to ensure successful infections. Although single-cell technologies for mammalian cells are improving quickly, their development for unicellular pathogens has been lagging. Despite their impact on human health, relatively little is known about the factors controlling cellular heterogeneity in unicellular organisms. Thus, understanding how cell-to-cell heterogeneity benefits unicellular pathogens to ensure successful infections makes it a critical attempt to develop technologies to study unicellular pathogens at the single-cell level.To address these complex issues, Cell2Cell investigates how cell-to-cell chromatin heterogeneity enables pathogens to overcome challenges critical for persistent infection by focusing on the following main scientific objectives:1- Identifying factors and mechanisms that control cellular heterogeneity in chromatin in unicellular model systems and study how they contribute to the adaptation of pathogens to their hosts2- Developing tools to examine cell-to-cell variation in chromatin localization in yeast models and pathogens and study how heterogeneity in genome organization contributes to gene expression3- Elucidating how cell-to-cell heterogeneity contributes to the expansion of repressive chromatin structures (heterochromatin spreading) along chromosomes and across boundaries of chromatin regions Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far During the first two years of the project, work within work package 1 (WP1) was dedicated to the following key aspects: • developing data analysis pipelines to study transcriptional noise in single-cell• developing image processing strategies to follow gene expression switches and noise patterns over time• mapping the genomic pattern of histone exchange at high temporal resolution• implementing a genome-barcoding approach to map cell fate during hematopoiesis• isolating single Trypanosoma brucei cells using FACS (fluorescent activated cell sorting)• developing a pipeline for bulk RNA seq analysisAlso, joint efforts within WP1 between the groups of Cell2Cell members, Luisa Figueiredo and Nicolai Siegel, resulted in publishing a review on recent discoveries in cell-to-cell heterogeneity in Trypanosomes (Luzak et al, 2021). Within WP 2, the tasks focused on: • adapting an approach known as Genomic loci Positioning by Sequencing (GPSeq) assay to study radial gradients of chromatin-bound RNA in Saccharomyces cerevisiae model system• imaging RNA polymerase II (RNAPII) distribution in the nucleus and assessing its characteristics outside the nucleus• identifying stress-dependent genes at the genome-wide scale • developing an AI algorithm for single-cell classification in yeast cells and T. bruceiWithin WP 3, Cell2Cell has been: • setting up a pipeline for the analysis of single-cell multi-omic data• analyzing single-cell transcriptomics data of T. brucei during a switch in VSG expression• studying the spreading of heterochromatin at sub-telomeres in Saccharomyces pombe• characterizing heterochromatin boundaries in parasites• investigating cellular heterogeneity that leads to differentiation of male and female gametocytes essential for parasite transmissionWithin WP 4 “Training: Research & Transferable Skills”, Cell2Cell organized:• a virtual welcome event with modules in research integrity, open science, gender equality and gender dimension, and project and self-management• a lecture series on career path decisions as well as scientific topics in parasitology, chromatin biology, bioinformatics, and single-cell technologies • a virtual workshop in biostatistics • an in-person workshop in Stockholm (see the attached images) in imaging and DNA/RNA FISH data generation and analysisWithin WP 5 “Communication & Public Engagement”, the project covered activities that generate interest in the network’s research and communicate the importance of infectious diseases, chromatin, and cell-to-cell heterogeneity to the public. These activities included:• designing and publishing the webpage• publishing press releases, blog posts, flyers, and news on the Twitter account• participating in local events at the hosting institutionsWithin WP 6 “Dissemination & Exploitation”, Cell2Cell ensured that the results generated were reviewed for possible exploitation and disseminated according to the EU’s Open Science policy in the forms of scientific presentations, posters, publications, and dedicated news on the webpage.Within WP 7 “Project Management”, standard management actions were carried out, including organizing the interactions with the EC and with internal and external partners, ensuring the recruitment of all researchers, monitoring the overall progress of the project, organizing reports submitted to the EC, and assessing the risks and deciding on changes. Additionally, within WP 8 “Ethics Requirements”, Cell2Cell ensured compliance with the ethics requirements as described in the Grant Agreement. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The interdisciplinary and intersectoral nature of the projects within Cell2Cell has brought together international experts in parasite biology, yeast chromatin, single-cell methods, and bioinformatics from academia and industry. Thus, towards the end of the project, the consortium anticipates to generate: a) results that could be exploited through further research by scientists (inside and outside Cell2Cell) b) results with potential for commercial exploitation that will be protected and discussed with representatives from industry for commercialization or non-profit exploitation. Among the anticipated results are the development of technologies and pipelines focusing on the isolation and analysis of single cells and the generation of datasets containing information on the heterogeneity among different cells. These results will be of great socio-economic interest, particularly for the development of screens to better diagnose the causative agent of infection. In summary, the innovative scope of Cell2Cell as a platform to answer the key question “what makes a successful pathogen?” is of high relevance and will contribute to the ongoing global effort to better understand pathogens that cause diseases, improve current drug treatments, and develop new vaccines, in particular in light of the global impact of the COVID-19 pandemic during the last two years. Cell2Cell ESRs during the Imaging Workshop (21-26 Nov 2021, at KI in Stockholm, Sweden). A photo taken in Stockholm by the network manager, Lara Hassan. ESR4, Lara Lopez Escobar, during the Imaging Workshop (21-26 Nov 2021, at KI in Stockholm, Sweden).