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Team projects

Projet 1:  Translocation control of the Dot/Icm effectors by c-di-GMP signaling

While investigating the function of the 22/23 c-di-GMP-metabolizing enzymes of the Lp1 Lens and Paris strains, we found that one diguanylate cyclase, DgcA (Lpl0780/Lpp809) directly contributes to their ability to infect both protozoan and mammalian cells, thanks to its role in a post-transcriptional control of the Dot/Icm effectors delivery. Our objective now is to elucidate the molecular mechanisms allowing this fine-tuned control by identifying the target of the DgcA-dependent c-di-GMP signaling pathway.  

Working model: DgcA localizes at the bacterial pole like the Dot/Icm T4SS. Identification of protein partners of DgcA and putative T4SS components able to bind the c-di-GMP is ongoing to gain new insights into the translocation control by the DgcA-dependent c-di-GMP signaling pathway.

 

Projet 2: Role of protein-kinase effectors LegK1-LegK4 in Lp1 infectious cycle

Among the 4 protein-kinases LegK1-LegK4, LegK1 was shown to induce the NF-kB pathway (Ge, PNAS 2009), and we have recently established that LegK2 targets ARP2/3 complex to inhibit actin polymerization at the surface on the LCV, and subsequently fusion of the LCV with late endosomes. We are now exploring the role of LegK3 and LegK4 by identifying the host cell targets of these effectors and their impact on the cell biology during infection.

Exclusive detection of LegK2 and actin on LCVs in D. discoideum. Bacteria in blue labeling, LegK2 on the LCVs in red and actin in green labeling.  Arrows point LegK2-positive LCVs (without actin labeling), and arrowheads highlight a LCV without LegK2 at its surface, and consequently stained for actin. Scale bar represents 10 µm.

 

 

 

 

Projet 3: Bacterial determinants of Lp1 associated with human cell infection - a global approach 

Although the Dot/Icm secretion plays a key role in the virulence of Lp1, it is not sufficient to explain the variable virulence observed between Lp1 strains. Indeed, the T4SS-encoding genes are very conserved whatever the virulence of the Lp1 strains; and the high level of variability of the effector repertoire could not be correlated to the variability of the Lp1 strains virulence. Thus, further research is needed now to investigate other bacterial determinants associated with LD severity. We are developing three complementary approaches to identify them: experimental evolution strategies, Tn-sequencing for infection of amoebae or human macrophages, and GWAS approaches by benefiting from the biological resources and clinical data collected by the NRCL.

 

Experimental evolution strategies to identify novel virulence determinants in Legionella

Initially isogenic lines of Legionella are subjected to serial passages in which a fraction of the population is used to re-inoculate fresh medium or eukaryotic cells. Bacterial adaptation over time is measured through fitness and comparative phenotypic analyses with the ancestral clone. Whole-genome sequencing, RNAseq and proteomic help identify what genes or functions have evolved.

 

Projet 4: Bacterial, host determinants and specific microbiota associated with severity of LD

 

We aim at studying the influence of both bacterial and host determinants on the severity of LD by investigating clinical data combined with analyses of patient specimens taken during infection. Regarding immunological aspects, we are evaluating in patients with opposite severity profiles the cells involved in the immune response by immunophenotyping and ex vivo immune functional assays. The impact of microbial environment on Lp1 virulence in lungs will be also explored using metagenomic approaches in order to identify specific microflora that could be associated to severity of infection and the changes in microbiota associated with disease progression.

 

 

 

 

 

Projet 5: Environmental determinants of human infection and severity of LD 

We are participating to a project about the impact of fine particulate matter and outdoor air pollution on human health (“Wanted” project funded by IDEX breakthrough program in 2018). The WHO estimates that 14% of outdoor air pollution-related premature deaths are due to chronic obstructive pulmonary disease or acute lower respiratory infections. Given that (1) Lp1 is an environmental bacterium, disseminated by man-made water systems, (2) a higher inflammation is associated with mortality for this pathogen, (3) exposure to fine particles causes pro-inflammatory responses, we will investigate the impact of fine particles exposure on the risk to develop a LD, and on the severity of the resulting disease.