Signaling in dendritic cells during Francisella tularensis invasion analyzed by phosphoproteomic approach

Tularemia disease is caused by Francisella tularensis, ahighly infectious Gram-negative bacterium. The delayed onset of the adaptive immunity followed by an excessive inflamation is characteristic for this infection disease. Francisella is capable of replication inside the primary phagocytes and avoiding their activation, which results in the imapired host response. The infected cells do not produce sufficient amount of pro-inflammatory stimuli and Francisella can use them as a niche for its multiplication. Dendritic cells (DCs) are important for the antigen presentation and these migratory cells can also seve as Francisella hosts.To understand the response of the cells infected by Francisella, a quntitative phosphoproteomic approach for the analysis of the intracellular signaling in DCs was used.The work was also focused on the beginning of the Francisella invasion, because these events play an important role in DCs activation and maturation. First, the primary bone marrow derived DCs (BMDCs) were labeled using SILAC and then infected either by virulent Francisella strain (FSC200) or by an attenuated mutant lacking dsbA gene. Infected BMDCs were lysed in different time points and further analysed using LC-MS after the enrichment for phosphosites differentally regulated during first 10 min after infection were related to the activation of PAKs, Akt-mTOR-p70S6K pathway and ERK signaling. No quantitative differences in BMDCs response were observed for both Francisella strains during the bacterial internalization. However, only the virulent Francisella strains induced a second phase of phosphorylation events in BMDCs 60 min after infection. This involved the activation of ERKs abd p38 kinases and their downstream transcription factors. In conclusion, the results show that the second phase of phosphorylation may represent the initial event of DCs activation that differs between the virulent and attenutaed Francisella strains.