Periodic Reporting for period 2 - Cell2Cell (What makes a successfull pathogen? Understanding the impact of cell-to-cell heterogeneity in chromatin structure on infection and adaptation)
Reporting period: 2021-11-01 to 2024-04-30
Infectious diseases have a devastating impact on global mortality and society. The WHO has previously listed lower respiratory infections, malaria, and diarrheal diseases among the top ten causes of death worldwide. COVID-19 has further highlighted this thread, becoming a major cause of death in 2020. While infectious diseases are more prevalent in developing countries, globalization makes them a challenge for the entire world. To improve treatments scientists need a deeper understanding of how pathogens establish successful infections and what factors within a pathogen population make some individuals more successful than others.
Cell-to-cell heterogeneity is a key driver for adaptation, allowing pathogens to overcome host defenses – yet little is known about the factors that control cellular heterogeneity. While single-cell technologies for studying mammalian cells have advanced rapidly, the development of similar tools for unicellular pathogens has lagged behind. Thus, understanding how cell-to-cell heterogeneity benefits unicellular pathogens makes it crucial to develop technologies that allow the study of unicellular pathogens at the single-cell level.
The PhD network Cell2Cell addressed these challenges by focusing on cellular variations in chromatin structure and gene expression that enable pathogens to adapt to new host environments, evade immune responses, and differentiate into states that allow new infections. Specifically, Cell2Cell aimed to:
(1) Identify factors and mechanisms that control cellular heterogeneity in chromatin in unicellular pathogens and yeast model systems and study how they contribute to adaptation.
(2) Develop tools to examine cell-to-cell variation in chromatin localization and how heterogeneity in genome organization contributes to altered gene expression in pathogens and yeast models.
(3) Elucidate how cell-to-cell heterogeneity contributes to the expansion of transcriptionally repressed chromatin structures along chromosomes and across boundaries separating active from inactive chromatin regions.
Cell-to-cell heterogeneity is a key driver for adaptation, allowing pathogens to overcome host defenses – yet little is known about the factors that control cellular heterogeneity. While single-cell technologies for studying mammalian cells have advanced rapidly, the development of similar tools for unicellular pathogens has lagged behind. Thus, understanding how cell-to-cell heterogeneity benefits unicellular pathogens makes it crucial to develop technologies that allow the study of unicellular pathogens at the single-cell level.
The PhD network Cell2Cell addressed these challenges by focusing on cellular variations in chromatin structure and gene expression that enable pathogens to adapt to new host environments, evade immune responses, and differentiate into states that allow new infections. Specifically, Cell2Cell aimed to:
(1) Identify factors and mechanisms that control cellular heterogeneity in chromatin in unicellular pathogens and yeast model systems and study how they contribute to adaptation.
(2) Develop tools to examine cell-to-cell variation in chromatin localization and how heterogeneity in genome organization contributes to altered gene expression in pathogens and yeast models.
(3) Elucidate how cell-to-cell heterogeneity contributes to the expansion of transcriptionally repressed chromatin structures along chromosomes and across boundaries separating active from inactive chromatin regions.
Bringing together experts in infection and chromatin biology, single-cell methodologies, imaging techniques and bioinformatics, the network provided the young scientists with a broad training in experimental and computational skills, complemented by various dedicated workshops in transferrable skills. The scientific work packages 1-3 investigated how cellular heterogeneity in chromatin structures can arise and contribute to adaptation and successful infection, focusing on different organizational levels of chromatin and their consequences for gene expression.
In Work Package 1, we explored factors and molecular mechanisms that promote cell-to-cell heterogeneity at the local chromatin level, using yeast and the unicellular pathogen Trypanosome brucei as model systems. Together, these studies demonstrated that metabolism and tissue environment are key determinants of altered chromatin conformations and phenotypic variation. The studies further revealed that antigen switching, a common mechanism to evade elimination by the host immune response, involves genomic rearrangements following DNA double-strand breaks, the repair of which depends on the availability of homologous repair templates.
In Work Package 2, we investigated how cellular diversity in genome organization contributes to adaptation and the ability of pathogens to evade the host’s immune system. Key achievements have been advancing existing methods to overcome challenges, as for example posed by the many repeat regions in pathogen genomes and the small size of nuclei in unicellular yeast models. These efforts include the adaptation of the Genomic loci Positioning by Sequencing (GPSeq) approach and the 10x Genomics protocol for single-cell RNA-sequencing for yeast cells entering and exciting quiescence.
In Work Package 3, we investigated the cellular heterogeneity of chromatin structures that separate actively transcribed euchromatin from transcriptionally repressed heterochromatin and promote differentiation into infection-competent states. The use of reporter genes to monitor heterochromatin expansion revealed locus-specific factors that control heterochromatin spreading in fission yeast and similar techniques have been applied in the malaria parasite P. falciparum. Heterochromatin-associated factors were further identified by proximity-labeling assays. Together, this WP resulted in new single-cell analysis tools and data integration pipelines, providing valuable resources for future research on how pathogens exploit cell-to-cell variation for transmission.
In Work Package 4, we focused on the research training and transferable skills component of the network. Despite all the COVID-19 adversities, the Cell2Cell network managed to organize three in-person workshops on imaging and DNA/RNA FISH data generation and analysis (Stockholm, November 2021), next generation sequencing (Budapest, July 2022), and bioinformatics and single-cell data analysis (Istanbul, January 2023). These workshops also included various soft skills modules ranging from entrepreneurship and scientific thinking to career path decisions.
As part of Work Package 5 “Communication & Public Engagement”, the network initiated activities to communicate research results and the importance of infectious diseases and cell-to-cell heterogeneity to the general public. We had a strong online presence on our Cell2Cell website (https://cell2cell.eu/(opens in new window)) and the @Cell2_Cell X account (formerly known as Twitter), where the network regularly posted news to increase the visibility for the general public.
As part of Work Package 6 “Dissemination & Exploitation”, we ensured that our research results were reviewed for possible exploitation and dissemination according to the EU Open Science policy, presented at conferences and published in peer-reviewed journals. In addition, we organized two large events open to the scientific community: The Summer School "Chromatin and Infection Biology at the Single-Cell Level" in Sesimbra in September 2023 and the Final Symposium "Dynamics in chromatin organization and RNA regulation: adaptation, infection - and beyond" in Giessen in April 2024. This conference was a joint event with the local PhD network RTG2235 and combined with a career day event for ESRs prior to the symposium.
In Work Package 1, we explored factors and molecular mechanisms that promote cell-to-cell heterogeneity at the local chromatin level, using yeast and the unicellular pathogen Trypanosome brucei as model systems. Together, these studies demonstrated that metabolism and tissue environment are key determinants of altered chromatin conformations and phenotypic variation. The studies further revealed that antigen switching, a common mechanism to evade elimination by the host immune response, involves genomic rearrangements following DNA double-strand breaks, the repair of which depends on the availability of homologous repair templates.
In Work Package 2, we investigated how cellular diversity in genome organization contributes to adaptation and the ability of pathogens to evade the host’s immune system. Key achievements have been advancing existing methods to overcome challenges, as for example posed by the many repeat regions in pathogen genomes and the small size of nuclei in unicellular yeast models. These efforts include the adaptation of the Genomic loci Positioning by Sequencing (GPSeq) approach and the 10x Genomics protocol for single-cell RNA-sequencing for yeast cells entering and exciting quiescence.
In Work Package 3, we investigated the cellular heterogeneity of chromatin structures that separate actively transcribed euchromatin from transcriptionally repressed heterochromatin and promote differentiation into infection-competent states. The use of reporter genes to monitor heterochromatin expansion revealed locus-specific factors that control heterochromatin spreading in fission yeast and similar techniques have been applied in the malaria parasite P. falciparum. Heterochromatin-associated factors were further identified by proximity-labeling assays. Together, this WP resulted in new single-cell analysis tools and data integration pipelines, providing valuable resources for future research on how pathogens exploit cell-to-cell variation for transmission.
In Work Package 4, we focused on the research training and transferable skills component of the network. Despite all the COVID-19 adversities, the Cell2Cell network managed to organize three in-person workshops on imaging and DNA/RNA FISH data generation and analysis (Stockholm, November 2021), next generation sequencing (Budapest, July 2022), and bioinformatics and single-cell data analysis (Istanbul, January 2023). These workshops also included various soft skills modules ranging from entrepreneurship and scientific thinking to career path decisions.
As part of Work Package 5 “Communication & Public Engagement”, the network initiated activities to communicate research results and the importance of infectious diseases and cell-to-cell heterogeneity to the general public. We had a strong online presence on our Cell2Cell website (https://cell2cell.eu/(opens in new window)) and the @Cell2_Cell X account (formerly known as Twitter), where the network regularly posted news to increase the visibility for the general public.
As part of Work Package 6 “Dissemination & Exploitation”, we ensured that our research results were reviewed for possible exploitation and dissemination according to the EU Open Science policy, presented at conferences and published in peer-reviewed journals. In addition, we organized two large events open to the scientific community: The Summer School "Chromatin and Infection Biology at the Single-Cell Level" in Sesimbra in September 2023 and the Final Symposium "Dynamics in chromatin organization and RNA regulation: adaptation, infection - and beyond" in Giessen in April 2024. This conference was a joint event with the local PhD network RTG2235 and combined with a career day event for ESRs prior to the symposium.
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. As a network, we have generated important results that can be exploited through further research by scientists (within and outside Cell2Cell) and have also potential for commercial exploitation. These include the development of technologies and pipelines focused on the isolation and analysis of single cells and the generation of datasets containing information on 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 outcome of the Cell2Cell consortium addressing the key question “What makes a successful pathogen?” will make a significant impact on the ongoing global effort to better understand pathogens, improve current drug treatments, and develop new vaccines.