Deciphering the molecular mechanisms underlying host cell exit of Orientia tsutsugamushi

• Applicant: Privatdozent Dr. Christian Keller
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens ; Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 531703706

Intracellular pathogens have evolved different strategies to exit from their infected host cell. Current knowledge on the molecular details about the exit events of intracellular bacteria has been derived from prototypic model organisms, such as Salmonella, Shigella, Listeria or Mycobacterium spp. However, there remain a wealth of nonmodel bacterial organisms that employ different exit strategies due to a fundamentally different biological life cycle. One example is the obligate intracellular human pathogen Orientia tsutsugamushi (Ot): Ot exits via cholesterol-rich lipid rafts in the plasma membrane as single bacteria that become enveloped by the host cell plasma membrane. At the host cell plasma membrane, a small budding neck is formed that eventually seals, thus allowing the release of encased bacteria. This exit mechanism, which resembles the budding of enveloped viruses, is unique among intracellular bacteria. However, the molecular details have not been elucidated to date. In the proposed project, we intend to apply omics approaches as well as super-resolution (SRM) and high-end electron microscopy, in collaboration with the SPP 2225 platforms 1 and 2, to resolve the ultrastructure of the budding process of Ot. In collaboration with the SPP 2225 imaging platform 2, we will localize the interacting host proteins and use SRM and 3D correlative light-electron microscopy to obtain detailed topographical information on the ultrastructure of budding Ot. Also, protein interactomes of bacterial surface proteins will be obtained by proximity ligation assays that involve the generation of biotin ligase fusion proteins (BioID2). In the selection for detailed characterization of interacting proteins, we will focus on candidates from the ESCRT machinery and ESCRT adapter proteins as well as the involvement of cytoskeletal proteins. The interaction of these proteins will be confirmed by classical immunoassays. Moreover, we will analyze the functional role of the newly identified host cell proteins in bacterial budding. To that end, we also aim to develop cell lines that allow the inducible expression of mutated proteins upon addition of specific ligands. As a complementary approach, hypothesizing that the budding process is not purely regulated by proteins, we will address the involvement and function of cellular long non-coding RNAs in the interaction with Ot surface proteins and in the budding process. By interacting with groups within the SPP 2225 framework that work on budding of Plasmodium merosomes or the host cell exit of Salmonella, we aim to define Orientia-specific or conserved features in host cell exit, thus shedding light on the different and common egress strategies employed by intracellular pathogens.

• DFG Programme: Priority Programmes
• Subproject of: SPP 2225: Exit strategies of intracellular pathogens