Analysis of determinants driving bacterial persistence in water – elucidation of the ClpS interactome of persisting Legionella pneumophila.

• Applicant: Dr. Philipp Aurass
• Subject Area: Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology ; Microbial Ecology and Applied Microbiology
• Term: from 2019 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 420596046

Legionella pneumophila is the causative agent of Legionnaires` disease, a severe pneumonia. It inhabits natural freshwater environments and man-made water systems. Human infection occurs by inhalation of contaminated aerosols, often generated by technical installations. Although it is known that L. pneumophila efficiently persists for long periods in water, the molecular adaptation program of the pathogen allowing survival is underexplored. Therefore in a recent project, the transcriptome of L. pneumophila during stationary growth phase compared to persistence in water was analysed by RNA sequencing. We identified specific transcripts that were significantly induced during persistence. Among those were transcripts of clpS encoding the adapter protein ClpS of the ClpAP complex, a member of the caseinolytic proteases family (Clp). Clp have been correlated with stress tolerance and virulence in various pathogens. ClpS as an adapter protein functions in selective detection, sequestration, and delivery of substrates containing a recognition signature to ClpAP for subsequent degradation. Despite the need to save maintenance costs during long-term nutrient limitation, clpS transcript quantity was significantly induced, suggesting its pivotal role in adaptation and survival. Thus, the aim of the proposed project is to characterize the processes supporting survival of Legionella during persistence in water. Specifically, it is of interest to identify proteins selectively targeted and controlled by ClpS under persistence conditions. In detail, 1) L. pneumophila clpS, clpA, and clpP mutants will be generated, microbiologically characterized, and their capability to persist in water will be analyzed, 2) ClpS will be recombinantly expressed and employed to trap and to identify the persistency-specific ClpS-targeted subproteome of Legionella, 3) the identified ClpS targets will be validated with respect to their implication in persistence by genetic modification of their ClpS recognition residues. We expect to clarify the following important research questions: 1) Does ClpS together with ClpAP support persistence and virulence of L. pneumophila? 2) Does ClpS serve as an adapter protein for specific, regulatory proteolysis during persistence? 3) Which proteins are proteolytically controlled by ClpS during persistence and in which functional pathways are they involved? 4) Does genetic modification of ClpS recognition residues result in substrate stabilization and persistency defects?The expected findings of the project will complement our understanding of what makes Legionella a professional survivor of water stress over the long term, a feature contributing to its success as a pathogen.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Dr. Ralph Isberg