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Revealing the mechanism of host membrane rupture by invasive pathogens and its role in triggering the immune response

Final Report Summary - RUPTEFFECTS (Revealing the mechanism of host membrane rupture by invasive pathogens and its role in triggering the immune response)

Pathogens have been evolving with their hosts for millions of years adopting tailored strategies to propagate within the host body and evading the host immune response. It is crucial to understand the underlying strategies employed by pathogens on the cellular and molecular level in order to develop novel weapons against them. On the cellular level, host-pathogen interactions are generally very dynamic and tightly regulated. Therefore, it has been difficult to establish methodologies that can capture this cross-talk. To overcome these hurdles, we have been developing novel approaches to monitor the dynamic host-pathogen interactions. Particularly, we have taken advantage of methodologies based on the principle of fluorescence, which we use and/or develop to obtain information on molecular processes in single living cells. We have addressed the following key questions in the field of cellular microbiology; (i) how do entero-pathogenic bacteria invade cells and (ii) how do the latter sense the incoming pathogens.

Using Shigella invasion of epithelial cells as our initial model, we have investigated how this pathogen hijacks host cellular machineries and ruptures the vacuolar membranes upon cellular uptake to escape into the host cytoplasm. These studies have deciphered novel underlying principles of its infection strategy, and they revealed novel paradigms in fundamental membrane cell biology, namely concerning endomembrane biogenesis and repair. We have clarified the role of a special host compartment, the macropinosomes, for the intracellular niche formation of the bacterial pathogens. Comparative studies with different endomembrane rupturing pathogens, especially Salmonella, Mycobacteria and Listeria have defined the common molecular principles of the process of vacuolar escape and the involvement of the host in the process. Further, we have exploited our approaches to investigate endomembrane integrity/rupture following the cellular trafficking/digestion of antigens during cross-presentation.

Then, we have addressed how membrane damage and the change of intracellular localization of invasive pathogens impact on the induced host immune response. For this, we have established two technologies to study gene expression at the single cell level. First, a cell sorting approach coupled with transcriptional gene profiling that yields information on subsets of genes differentially regulated dependent on the pathogen localization. Secondly, the role of these genes can be further studied in individual cells with an assay to track the host gene expression response quantitatively during pathogen invasion with a timely resolution of seconds to minutes. These methods have revealed that Shigella induces a localization-dependent host immune response that changes even from an intravacuolar localization to a cytosolic one.

Together, we have been deciphering the molecular mechanisms how invasive bacteria interfere with eukaryotic cellular trafficking and signal transduction pathways to alter host immune responses. Further, we have developed a number of innovative single-cell based technologies that gives insights at both, the host and the pathogen, during their intimate contacts during infection. Thus, beyond providing novel targets for antimicrobial therapy, our study paves the way to understand fundamental cellular processes.