Haploid genetics and genome editing in immune responses to GAS (Group A Streptococci)

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 approaches, my specific project part will focus on the inflammatory response to the medically relevant pathogen Streptococcus pyogenes, and a selected set of PAMPs, such as cell wall extracts and bacterial nucleic acid preparations, known to signal threat to the host. 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.

Correct timing and balance of the immune response is key for fighting off infections yet preventing extensive damage to our own body. Thus, a constant challenge for our immune system is adjusting offense to achieve defense against pathogens. The lifespan of immune cells plays a central role in the adjustment of the immune response. One type of immune cells involved in response to infections are neutrophils, and incorrect regulation of those is associated with a number of human diseases. In our recent study published in the renowned Journal of Clinical Investigation and funded to a large part by this FWF grant we discovered how the lifespan of neutrophils is regulated during infection. Neutrophils are the most abundant type of white blood cells, and they have been known to have an extremely short lifespan that ranges from 1 4 days. Neutrophils are crucial for defense against bacterial infections: through chemotactic migration neutrophils rapidly accumulate in vast numbers at the site of infection where they deploy their anti-microbial armament. A failure in this process allows the pathogen to disseminate and take the host. However, an uncontrolled action of neutrophils may result in extensive tissue damage which can be as devastating to the body as the infection itself. A key mechanism that controls neutrophil activity is the programmed cell death called apoptosis. In our study we have discovered the pace maker of apoptosis of neutrophils that are actively engaged in fighting the pathogen. It turned out, that apoptosis of such pathogen- engaged neutrophils is regulated differently from that of the dormant ones which circulate in the bloodstream. We identified a specific protein, called tristetraprolin (TTP), as the main switch for turning off active neutrophils. TTP subdues factors which are anti-apoptotic. TTP only impinges on neutrophils after their encounter with the pathogen, whereas inactive circulating neutrophils remain unaffected and available for new fights. TTP does so by destabilizing the mRNA of the anti-apoptotic protein Mcl-1 thereby reducing the Mcl-1 protein levels. Intriguingly, we found out that deletion of TTP in neutrophils increased the number of active neutrophils at the site of infection substantially. This resulted in an augmented neutrophil response that blunted an otherwise lethal systemic spread of the pathogenic bacterium Streptococcus pyogenes in a model of invasive infection of the soft tissue. Infections are currently mainly being treated with antibiotics. A globally known problem is the growing antibiotic resistance, with the future outlook looking grim in light of impending resistances to even the last resort antibiotics. Thus, manipulation of the lifespan of activated neutrophils, possibly by pharmacological targeting of TTP, might represent a valid strategy for treatments of drug-resistant infections.