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

• Applicant: Privatdozent Dr. Martin J. Fraunholz
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens
• Term: from 2012 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 219106207

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.

• DFG Programme: Research Grants