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侵入宿主细胞来避开宿主免疫系统。胞内S.据报道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。我们先前已经确定，吞噬体逃逸可以通过与鞘磷脂酶β毒素协同作用的膜活性肽介导。然而，我们获得的数据表明，替代毒素或其组合可以使用的S。金黄色葡萄球菌诱导吞噬体逃逸在不同的宿主背景。为了确定参与临床相关的沙门氏菌吞噬体逃逸的其他毒力因子。因此，我们建议进行全基因组转座子突变体筛选。为此，我们将产生转座子突变体文库，其将用于感染宿主细胞系。不能从吞噬体逃逸的细菌将通过荧光激活细胞分选分离。逃逸阳性和逃逸阴性S.金黄色葡萄球菌将通过多重下一代测序进行分析。通过这种所谓的TnSeq方法，我们将鉴定每个获得的DNA文库样品中的所有转座子插入位点。在靶向敲除方法中，我们将在临床相关的S。金黄色葡萄球菌

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.