SFB796 - Associated project AP1

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AP1 : Didier Soulat
Functional characterization of phosphatases secreted by pathogens during host infection

 

Project summary

Infectious microorganisms have developed numerous strategies to drive the host-pathogen interaction for their own benefit. Among a large variety of strategy, the secretion of phosphatase by pathogen appears to be one of the most efficient to interfere with the host cell response. Indeed, the reaction of phosphorylation/dephosphorylation of protein and/or lipid is the most common modification used by cell to control their cellular machinery. Therefore, our research is aiming to understand how newly discovered phosphatases secreted by pathogens alter the host-pathogen interaction.

Our first infectious model is the intracellular Gram-positive bacteria Listeria monocytogenes, the causative agent of Listeriosis. This food-borne disease is rare but can cause clinically serious symptoms with a high case fatality rate in immuno-compromised individuals. After ingestion of contaminated food, L. monocytogenes crosses the epithelial barrier of the intestine and disseminates in deeper tissues via the lymphatics and the blood stream. At first, spleen and liver are infected. There, the bacterium replicates and uses these organs as reservoir for further spreading in the brain and the placenta. This pathogenicity relies on an elaborated arsenal of virulence factors which mainly support its intracellular life cycle. We recently identified a new virulence factor of L. monocytogenes, the secreted phosphatase LipA. This enzyme exhibits unusual features such as tyrosine as well as phosphoinositol-phosphate (PIP) phosphatase activity in vitro. Strikingly, bacteria lacking LipA are severely attenuated in virulence in vivo independently of the infection route, which goes in line with its expression pattern strictly dependent on the entry of the bacteria in the blood stream. Currently we are aiming (1) to determine the innate immune cells affected by LipA and (2) to identify the molecular nature of its molecular substrate taking advantage of catalytically inactive enzyme.

The Leishmania major parasite is the second infectious model studied in this project. This parasite is the causative agent of cutaneous leishmaniasis, a worldwide prevalent parasitic disease transmitted by sand fly bite. In its insect vector, the parasite resides in an extracellular, highly infective, promastigote form. Once in its mammal host, the parasite enters myeloid host cells with a high prevalence for macrophages. There, they transform into intracellular amastigotes, which facilitate the spread in the host organism. We recently identified two putative new virulence factors produced by Leishmania parasites. These two homologous tyrosine phosphatases, named Lp1 and Lp4, were shown to be partly secreted by Leishmania major via the exosome pathway. Interestingly, these phosphatases have a strong structural homology with the human phosphatase PRL-1 (Phosphatase of Regenerating Liver 1) that is involved in the regulation of numerous cellular features such as growth and cell motility. Based on this homology, we hypothesize that the Leishmania phosphatases could interfere with the PRL-1 function to modulate the host response during infection. To validate our hypothesis, we are currently characterizing biochemically the enzymatic activity of Lp1 and Lp4 to determine their range of substrate specificity in vitro. In addition, we are studying their subcellular localization during the infection of macrophage by Leishmania parasite. Finally, deletions of the phosphatase genes will be carried out to study their function in an in vivo model of cutaneous leishmaniasis.

All together, these two projects aim to identify and characterize new virulence factors of important human pathogens. The elucidation of their respective mode of action should help us to open new avenue in the development of better anti-microbial molecule.

 

Project relevant publications

Kastner, R., Dussurget, O., Archambaud, C., Kernbauer, E., Soulat, D., Cossart, P., and Decker, T.   (2011).   LipA, a tyrosine and lipid phosphatase involved in the virulence of Listeria monocytogenes.   Infection and immunity 79:2489-2498 .

Stockinger, S., Kastner, R., Kernbauer, E., Pilz, A., Westermayer, S., Reutterer, B., Soulat, D., Stengl, G., Vogl, C., Frenz, T., et al.   (2009).   Characterization of the interferon-producing cell in mice infected with Listeria monocytogenes.   PLoS pathogens 5:e1000355 .

Soulat, D., Burckstummer, T., Westermayer, S., Goncalves, A., Bauch, A., Stefanovic, A., Hantschel, O., Bennett, K.L., Decker, T., and Superti-Furga, G.   (2008).   The DEAD-box helicase DDX3X is a critical component of the TANK-binding kinase 1-dependent innate immune response.   The EMBO journal 27:2135-2146 .

Soulat, D., Bauch, A., Stockinger, S., Superti-Furga, G., and Decker, T.   (2006).   Cytoplasmic Listeria monocytogenes stimulates IFN-beta synthesis without requiring the adapter protein MAVS.   FEBS letters 580:2341-2346 .

Olivares-Illana, V., Meyer, P., Bechet, E., Gueguen-Chaignon, V., Soulat, D., Lazereg-Riquier, S., Mijakovic, I., Deutscher, J., Cozzone, A.J., Laprevote, O., et al.   (2008).   Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus.   PLoS biology 6:e143 .