Multidrug resistant bacterial infections are on the rise while antibiotic pipelines are drying out. This global threat urgently calls for novel antimicrobial therapies. Adjuvant immunotherapy represents a promising strategy to combat infections without fueling drug resistance. For this to work, protective immune responses must be scaled to the level of infectious threats (Blander and Sander Nat Rev Immunol 2012), thus allowing for efficient pathogen clearance while minimizing inflammation-induced tissue damage. However, the cellular machinery that accurately assesses these infectious threats in humans as well as the precise contribution of bacterial factors is not well understood. To fill this gap, we hereby propose to obtain an unbiased and comprehensive view on the bacterial recognition and human response network. To achieve this, we will exploit a unique approach and perform a genome-wide screen using haploid human immune cells (Carette et al. Science 2009) in which genes will be disrupted at saturating scale using gene trap mutagenesis (Carette et al. Nat Biotechnol 2011). By subjecting mutagenized cell pools to a selection scheme (e.g. survival, inflammatory response, internalization of PAMPs), mutants with desired phenotypes will be enriched and identified by deep sequencing. Following the identification of cellular targets, we will verify the functional relevance of these molecules by obtaining and testing cells from the human gene trap mutant collection of individual clones. This unique haploid clone collection contains conditional alleles, is DNA barcoded and will be provided by our external collaboration partner Haplogen and via the Research Center for Molecular Medicine (Ce-M-M-; Austria). Furthermore, we will explore CRISPR (RNA programmable Cas9)-mediated genome editing (discovered by E. Charpentier’s group) (Deltcheva et al. Nature 2011; Jinek et al. Science 2012) that has recently been expanded to mammalian cells, to inactivate genes of interest in relevant human immune cells (Cong et al. Science 2013; Mali et al. Science 2013). Finally, the biological in vivo function of selected target molecules will be investigated in relevant mouse models of infectious diseases. Using these screens we will focus on the inflammatory response to medically relevant pathogens like Streptococci (pneumoniae & pyogenes), and a selected set of PAMPs, such as cell wall extracts and bacterial nucleic acid preparations, known to signify bacterial viability (i.e. threat). The unique combination of expertise within our consortium, covering haploid genetic screens, RNA-programmable Cas9-mediated genome editing and proficiency in innate immune responses to bacterial pathogens and bacterial virulence strategies, provides a clear competitive advantage and the ideal setting to successfully apply this innovative and discovery driven strategy.

The project will develop a multipurpose, highly multiplexed, quantitative protein profiling (hmqPro) platform that can be adapted to a wide range of applications, particularly biomarker discovery. Biomarkers are a 43 Billion USD market spanning applications in diagnostics, drug discovery & development, personalized medicine, disease risk assessment. The method comprises an antibody-based profiling of a large number of proteins or protein posttranslational modifications against a large number of samples irrespective of source. Unlike competitor methods, hmqPro does not require platform-specific assay or detection equipment. Dual multiplexing implemented in a variant method, hmqPro-2D, will make the platform competitive against existing protein profiling methods in per-assay cost, flexibility and scalability. The hmqPro platform uniquely complements the technology portfolio of Epigenica AB and we will chart out two commercialization routes: first, hmqPro will be integrated into a service laboratory offering high-throughput sample-to-result epigenome and protein profiling services to academia, pharma and biotech industries. Second, we will package the method in a kit format distributed to R&D and clinical end users. In summary, the proposed project will deliver a new quantitative protein profiling platform that will have the capacity to impact the biomarker discovery market by combining unique flexibility with high-throughput.

This proposal describes the third core project of the Graphene Flagship. It forms the fourth phase of the FET flagship and is characterized by a continued transition towards higher technology readiness levels, without jeopardizing our strong commitment to fundamental research. Compared to the second core project, this phase includes a substantial increase in the market-motivated technological spearhead projects, which account for about 30% of the overall budget. The broader fundamental and applied research themes are pursued by 15 work packages and supported by four work packages on innovation, industrialization, dissemination and management. The consortium that is involved in this project includes over 150 academic and industrial partners in over 20 European countries.

My PhD experience in the field of immunology focused on deciphering vesicular pathways underlying T cell activation profoundly motivated my move to Dr. Bryceson’s lab, which offers a unique opportunity to explore cytotoxic lymphocyte (CL) exocytosis in the context of human disease. Cytotoxic lymphocyte exocytosis is critical for life, with mutations in genes required for cytotoxicity causative of severe, early-onset hyperinflammatory syndromes. Genetic studies have identified several cytosolic proteins required for CL exocytosis. However, understanding of how these proteins cooperate for exocytosis, their interaction partners and how their activities are regulated is still limited. I propose strategies to gain insight to the molecular regulation of human CL exocytosis. First, prompted by genetic studies, I aim to study the contribution of two distinct Munc13-4 isoforms to lymphocyte cytotoxicity by dissecting their role in cytotoxic granule (CG) exocytosis using advanced live-cell imaging as well as identifying their interaction partners using quantitative mass spectrometry (MS). Second, recycling endosome (RE) have recently been implicated in CG exocytosis, delivering syntaxin-11, an effector molecule required for CG fusion, to the plasma membrane. I plan to use a high-throughput MS approach to identify RE constituents and cargo in order to define new components that may be critical for CG exocytosis. Results will provide novel mechanistic insights to CL exocytosis, which will also be relevant for other exocytic systems. Providing detailed molecular understanding of lymphocyte cytotoxicity in both healthy and pathological conditions, insights promise to guide improved diagnosis and therapy of immune disorders associated with defects in lymphocyte cytotoxicity. Yearning to build a successful academic career, the excellence of the host laboratory will allow me to successfully purse this project and expand technical expertise and my scientific horizon.