Role Of C-di-GMP in the Kinetics of legionella Effector Translocation – ROCKET

Secretion system (SS)-mediated delivery of effector proteins into host cells plays a key role in bacterial virulence. Despite recent progress in the structure-function studies of various SS (e.g. T3SS, T4SS, T6SS, etc.), the signals that activate effector transfer have largely remained a closed book, especially when secretion machineries deal with the export of several, and at times numerous, effectors. Recently, we discovered that the secretion of more than 300 effectors by the Dot/Icm T4SS of Legionella pneumophila is both spatially and temporally controlled by the intracellular second messenger c-di-GMP. In particular, we showed that a specific c-di-GMP synthesizing enzyme – the diguanylate cyclase (DGC) Lpl0780/Lpp0809 – co-localizes with and significantly contributes to accurate triggering of effector secretion by the T4SS machinery at a specific stage of the bacterial life cycle (Allombert, Jaboulay et al., J. Mol. Biol., 2021). In this project we will harness complementary expertise in microbiology, host-pathogen interactions, bacterial genetics, dinucleotide signaling, and structural biology to decipher the structural and functional bases of c-di-GMP-dependent control of T4SS-mediated effector secretion.
On the basis on robust and original data, we hypothesize that (i) the DGC Lpl0780/Lpl0809 manipulates the local c-di-GMP concentration at the pole, (ii) the c-di-GMP synthesized by the DGC Lpl0780/Lpp0809 could target the proximal secretion machine either by limited diffusion or direct dinucleotide signal relay, and (iii) the interactions between the coupling protein complex, the chaperones and/or others unknown partners can thus been modified to finely orchestrate loading of the ~ 300 effectors on the Icm/Dot T4SS in coherence with their role during the infection. We propose to address this hypothesis through the realization of 4 work packages, that aim at: (1) Deciphering the molecular basis of c-di-GMP delivery by Lpl0780/Lpp0809 at the bacterial pole; (2) Characterizing the impact of locally increased c-di-GMP concentration in proximity to the Icm/Dot T4SS; (3) Establishing the structural basis of the Icm/Dot machinery control by c-di-GMP; (4) Validating the structure-based molecular mechanisms of c-di-GMP-dependent Icm/Dot control and investigating its impact on Legionella virulence.
This ambitious project will expand our knowledge on the as yet enigmatic structure and mechanisms of a prevalent type of bacterial secretion macroassemblies, the Type 4 Secretion System, and will contribute an in-depth investigation of a novel target of the extraordinary second messenger c-di-GMP. The two teams have a long-standing and complementary expertise in Legionella cell biology, virulence and infectious biology, c-di-GMP receptor identification and intracellular signaling mechanisms, structural biology and bacterial secretion (Krasteva et al., Science 2010; Levet-Paulo et al., J. Biol Chem. 2011; Goyal, Krasteva et al. Nature 2014; Allombert et al. Infect. Immun. 2014; Michard et al., mBio 2015; Matsuyama*, Krasteva* et al. PNAS 2016; Pecastaing et al. Biofouling 2016; Krasteva et al. Nature Communications 2017; Zouhir et al. mBio 2020; Abidi et al. Science Advances 2021; Allombert*, Jaboulay* et al. J. Mol. Biol. 2021). Importantly, the proposed research targets a new paradigm for the control of efficient orchestration of multi-effector secretion by secretion systems in general and opens important perspectives to further characterize the host-pathogen interactions of a clinically important infectious agent. Finally, it will provide insights for the development of novel prevention and treatment strategies targeting c-di-GMP signaling pathways as sustainable alternatives to classical antibiotics targets for the development of efficient and specific virulence modulators.