Elucidating the mechanism of membrane effector secretion through the Legionella Dot/Icm type IV b secretion system

• Applicant: Professor Samuel Wagner, Ph.D.
• Subject Area: Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 541310473

Legionellae employ type 4 b secretion systems (T4bSS) to establish a parasitic lifestyle within amoeba. To this end, these systems facilitate the translocation of a cocktail of more than 300 different bacterial effector proteins into the eukaryotic target cells. In the context of infection, the same effectors are translocated into alveolar macrophages by pathogenic Legionella spp. like L. pneumophila, leading to Legionnaires’ disease. About 20% of all effectors of T4bSS are transmembrane domain (TMD)-containing effectors that have their final destination in one of the membranes of the eukaryotic host. Transmembrane segments within effectors pose a potential targeting conflict as two sequential secretion signals for two different pathways are concatenated in the same protein: the C-terminal secretion signal for transport through the T4bSS on the one hand and hydrophobic segments that signal inner membrane targeting on the other hand. We previously showed that a subset of TMD-effectors of T4bSS of L. pneumophila possess TMDs of reduced hydrophobicity and by this avoid inner membrane targeting and facilitate direct delivery of these effectors to the T4bSS from the cytoplasmic side. However, T4bSS are also able to secrete TMD-effectors of higher hydrophobicity that could be targeted by the signal recognition particle and integrate into the bacterial inner membrane. Indeed, we obtained preliminary evidence that highly hydrophobic TMD-effectors follow a two-step secretion pathway with an inner membrane intermediate. For secretion, these TMD-effectors seem to require a C-terminal secretion signal that is located on the cytoplasmic side of the membrane. We hypothesize that they are extracted from the bacterial membrane towards the cytoplasm and subsequently handed over to the T4bSS core complex for secretion. We herein propose to investigate the mechanistic details of how the T4bSS machinery copes with the secretion of these TMD-effectors. Using a combination of bioinformatics, genetic dissection and mutant analysis, protein-protein interaction analysis, secretion analysis and selective ribosome profiling, we expect that the results obtained by the proposed work - Will provide a clear biochemical classification of TMD-effectors according to the path they take for their T4bSS-dependent secretion; - Will show how secretion of T4bSS effector proteins in general and TMD-effectors in particular is integrated into the overall context of protein biogenesis and maintenance inside the bacterial cell; - Will proof that strongly hydrophobic TMD-effectors are secreted through an inner membrane intermediate; - Will provide a detailed picture of the necessary and allowed traits of strongly hydrophobic TMD-effectors that are secreted through an inner membrane intermediate. Over all, the work proposed herein has the potential to yield seminal insights into the intricate molecular machines facilitating the interplay of bacteria with their eukaryotic hosts.

• DFG Programme: Research Grants