Haploid genetics, genome editing and pneumococcal recognition

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 my specific project part will focus on the inflammatory response to the medically relevant pathogen Streptococcus pneumoniae, 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.

Bacterial infections are a leading cause of morbidity and mortality worldwide. In this project (which was part of a collaborative consortium within the ERA-Infect framework), we attempted to tackle the problem of multidrug resistant bacterial infections by exploiting novel strategies like adjuvant immunotherapy to improve host defense mechanisms during bacterial infections. To allow for efficient pathogen clearance while minimizing inflammation-induced tissue damage, we investigated the cellular response machinery that accurately assesses infectious threats in the host as well as the precise contribution of bacterial factors, as a pre-requisite to understand potential targets for immunotherapy.We hereby discovered essential regulatory factors that importantly shape the inflammatory response to bacteria. As such, we identified the protective role of type I interferons in the lungs, as these molecules importantly help maintain tissue barriers and thereby prevent systemic spread of bacteria like Streptococcus pneumoniae. In another approach we were able to mechanistically explain the cause for high numbers of bacterial infections in people suffering from hemolytic disorders. We discovered that heme, the molecule released from red blood cells, strongly inhibits the uptake and elimination of bacteria. As a result, elevated heme levels disable the host to clear bacteria, which results in high lethality rates during sepsis. Importantly so, we managed to identify a drug that can prevent these effects and can restore the host defense mechanisms in vivo. This type of immunoadjuvant therapy might serve as a model to prevent high rates of infection in patients with hemolysis in the future.