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

耐药金黄色葡萄球菌(S.aureus)的出现和传播迫切需要开发更有效的预防和治疗措施。要做到这一点，需要更好地了解金黄色葡萄球菌感染的发病机制。虽然金黄色葡萄球菌通常被认为是一种胞外病原体，但越来越多的证据表明，金黄色葡萄球菌可以在非吞噬细胞内和专业吞噬细胞内生存和复制。吞噬细胞内的存活可能会促进细菌的持续存在和从局部感染部位传播，但金黄色葡萄球菌的增殖、细胞内生活方式和宿主反应之间的相互作用尚未得到很好的表征。本项目的目的是从吞噬细胞胞内隔间的成熟和细菌内化的摄取途径来表征金黄色葡萄球菌的增殖活性。此外，我们将确定金黄色葡萄球菌的增殖与体内吞噬细胞对病原体的接触之间的关系，以及金黄色葡萄球菌增殖的变化与吞噬细胞在感染过程中的转录反应之间的联系。为此，我们最近开发了一种方法，通过使用表达光转化荧光蛋白(MKikumeGR)的细菌来确定体内感染期间细胞内病原体的增殖状态。该系统允许通过光脉冲将绿色mKikumeGR蛋白转换为红色荧光。光转化后绿色荧光的恢复(由于绿色的从头产生和红色蛋白的稀释)与细菌的增殖率密切相关。因此，我们将能够表征含有金黄色葡萄球菌的液泡，同时读出寄生细菌的增殖状态。为了研究病原体增殖如何参与持续感染过程中荧光标记的中性粒细胞、单核细胞和树突状细胞的动态变化，我们将在活体双光子显微镜中使用金黄色葡萄球菌增殖报告株。此外，我们将分离感染高或低增殖细菌的吞噬细胞亚群，并通过双重RNA测序来确定不同吞噬细胞亚群及其细胞内金黄色葡萄球菌的转录反应。阐明促进细胞内允许生长的间隔室发育的途径，以及病原体增殖和吞噬细胞行为之间的联系，对于更好地理解金黄色葡萄球菌的致病机制至关重要。因此，通过表征细胞内生活方式所需的生态位，以及通过建立体内金黄色葡萄球菌增殖的测量，该项目的结果可以帮助设计新的干预措施，促进吞噬细胞清除细胞内金黄色葡萄球菌，并增强感染期间的免疫反应。

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.