Investigation of Staphylococcus aureus intracellular survival strategies using a new genetically encoded proliferation reporter system

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The emergence and spread of antibiotic-resistant Staphylococcus aureus (S. aureus) demands an urgent development of more efficient prevention and treatment efforts. To achieve this, a better understanding of the pathogenesis of S. aureus infections is required. Although S. aureus is generally considered an extracellular pathogen, a growing body of evidence indicates that S. aureus can survive and replicate within non-phagocytic as well as within professional phagocytic cells. Survival within phagocytes might foster bacterial persistence and dissemination from the local site of infection, but the interplay between S. aureus proliferation, intracellular lifestyle and the host response is not well characterized. The aim of this project is to characterize the proliferative activity of S. aureus with respect to both maturation of intracellular compartments of phagocytic cells and the uptake pathways by which the bacteria are internalized. Furthermore, we will determine the relationship between S. aureus proliferation and pathogen engagement by phagocytes in vivo, and the link between variations in S. aureus proliferation and the transcriptional response of the phagocytes during infection. To this end, we have recently developed a method for determining the proliferative status of intracellular pathogens during in vivo infection by using photoconvertible fluorescence protein (mKikumeGR)-expressing bacteria. This system permits switching the green mKikumeGR protein to red fluorescence by a light pulse. The recovery of green fluorescence after photoconversion (due to de novo production of the green and dilution of the red protein) closely correlates with the bacterial proliferation rate. Therefore, we will be able to characterize the S. aureus-containing vacuole and simultaneously read out the proliferative status of the inhabiting bacteria. In order to investigate how pathogen proliferation is involved in the dynamics of fluorescence-labelled neutrophils, monocytes and dendritic cells in the ongoing infection, we will use the S. aureus proliferation reporter strain in intravital 2-Photon microscopy. Furthermore, we will isolate the phagocytes subpopulations infected by either high or low proliferating bacteria, and determine the transcriptional response of the different phagocyte subpopulations and their intracellular S. aureus by dual RNA-sequencing. Elucidation of pathways that promote development of an intracellular compartment permissive for growth, and of the link between pathogen proliferation and phagocyte behaviour, is critical for gaining a better understanding of pathogenic mechanisms of S. aureus. Therefore, by characterizing the niche required for the intracellular lifestyle, and by establishing measurement of S. aureus proliferation in vivo, the results of this project can help to design new interventions to promote clearance of intracellular S. aureus by phagocytic cells, and to boost the immune response during infection.