Identification of virulence factors mediating phagosomal escape of Staphylococcus aureus by transposon insertion site deep sequencing

金黄色葡萄球菌可以通过入侵宿主细胞来避开宿主免疫系统。据报道，细胞内金黄色葡萄球菌对宿主细胞具有细胞毒性。这种细胞毒性被认为依赖于病原体的吞噬体逃逸。我们之前已经证实，吞噬体逃逸可以通过膜活性肽与鞘磷脂酶β-毒素协同作用介导。然而，我们获得的数据表明，金黄色葡萄球菌可以使用替代毒素或其组合来诱导不同宿主背景下的吞噬体逃逸。为了通过遗传学方法确定与临床相关的金黄色葡萄球菌吞噬体逃逸有关的其他毒力因素，我们因此建议进行全基因组转座子突变筛选。为此，我们将生成一个转座子突变文库，用于感染宿主细胞系。无法逃离吞噬体的细菌将通过荧光激活的细胞分选分离出来。逃逸阳性和逃逸阴性金黄色葡萄球菌的DNA将通过多重新一代测序进行分析。通过这种所谓的TnSeq方法，我们将在每个获得的DNA文库样本中识别所有转座子插入位点。在靶向敲除方法中，我们将确认临床相关金黄色葡萄球菌菌株的最佳候选。

Staphylococcus aureus can avoid the host immune system by invasion of host cells. Intracellular S. aureus has been reported to be cytotoxic to the host cells. This cytotoxicity is thought to be dependent on a phagosomal escape of the pathogen. We previously have established that phagosomal escape can be mediated by membrane-active peptides in synergy with the sphingomyelinase β-toxin. However, we acquired data which suggest that alternative toxins or combinations thereof can be used by S. aureus to induce phagosomal escape in different host backgrounds. In order to identify additional virulence factors involved in phagosomal escape of clinically relevant S. aureus via a genetic approach, we therefore propose conducting a genome-wide transposon mutant screen. For that purpose we will generate a transposon mutant library which will be used to infect host cell lines. Bacteria unable to escape from the phagosome will be isolated by fluorescence activated cell sorting. DNA of escape-positive and escape-negative S. aureus will be analyzed by multiplexed next-generation sequencing. By this so-called TnSeq approach, we will identify all transposon insertions sites in each of the obtained DNA library samples. In targeted knock-out approaches we will confirm the best candidates in clinically relevant S. aureus strains.