Convergent Met and voltage-gated Ca²⁺channel signaling on Ras-Erk MAPK drives migratory activation of dendritic cells parasitized byToxoplasma gondii

Abstract

24Ras-Erk MAPK signaling controls many of the principal pathways involved in 25 metazoan cell motility, drives metastasis of multiple cancer types and is targeted in 26chemotherapy. Yet, its putative roles in immune cell functions or in infections have 27 remained elusive. Here, using primary dendritic cells (DCs) in an infection model with 28 the protozoan Toxoplasma gondii, we show that two pathways activated by infection 29 converge on Ras-Erk MAPK signaling to promote migration of parasitized DCs. We 30 identify signaling through the receptor tyrosine kinase Met (also known as HGFR) as a 31 driver of T. gondii-induced DC hypermotility. Further, we show that voltage-gated Ca 2+ 32 channel (VGCC, subtype CaV1.3) signaling impacts the migratory activation of DCs 33 via calmodulin-calmodulin kinase II. We report that VGCC and Met signaling converge 34 on Ras GTPase to drive Erk1/2 phosphorylation and migratory activation of T. gondii-35 infected DCs. The data provide a molecular basis for the hypermigratory mesenchymal-36 to-amoeboid transition (MAT) of parasitized DCs. The emerging concept suggests that 37 parasitized DCs acquire metastasis-like migratory properties to promote infection-38 related dissemination. 39 40 understood (Ebert et al., 2016; Scheele et al., 2007). Further, in primary dendritic cells 65 (DCs), Ras-Erk signaling remains largely unexplored (Riegel et al., 2019). 66Owing to host-pathogen coevolution with reciprocal selection, the study of host-67 pathogen interactions has emerged as a powerful approach to gain insight into basic cell 68 biology. The protozoan Toxoplasma gondii is a model obligate intracellular pathogen 69 due to its wide host range among warm-blooded vertebrates and ability to actively 70 invade nucleated cells (Sibley, 2004). 71

The unicellular eukaryotic parasite Toxoplasma gondii hijacks the migration machinery of mononuclear phagocytes to promote its dissemination

“…Ras activation ultimately leads to Erk1/2 phosphorylation via Raf-Mek. Further, Ras-Erk signalling was activated downstream of the receptor tyrosine kinase (RTK) MET, situating Ras as a central signalling node and point of convergence for VGCC and MET signalling (Olafsson et al, 2020). These data are in line with paradigms in neuronal cells, where VGCC-dependent CaM activity regulates cytoskeleton organisation and cell migration via Ras-Erk signalling (Rosen et al, 1994).…”

“…As laid out above, autocrine GABAergic signalling is activated in parasitised DCs (Fuks et al, 2012), which in turn triggers VGCC-mediated Ca 2+ influx (Kanatani et al, 2017). More recent data revealed that Ca 2+ influx via the VGCC subtype Ca V 1.3 mediates activation of the small GTPase Ras via calmodulin (CaM) and CaM kinase II (CaMkII) signalling (Olafsson et al, 2020) (Figure 2). Ras activation ultimately leads to Erk1/2 phosphorylation via Raf-Mek.…”

“…The RTK MET, also known as c-MET, scatter factor receptor or hepatocyte growth factor receptor, has been associated with cancer metastasis but also more recently with DC/Langerhans cell migration in skin immunity (Baek et al, 2012). It was recently reported that MET is activated in DCs upon T. gondii infection with an impact on DC hypermotility (Olafsson et al, 2020). Interestingly, secretion of METs only known ligand hepatocyte growth factor (Hgf) was not significantly elevated upon T. gondii infection, while inhibition of integrin (ITGB1)-linked tyrosine kinases (FAK, PYK2) led to reduced Erk activation.…”

“…Within minutes of invasion, T. gondii induces a hypermigratory phenotype in host phagocytes, which is characterised by cytoskeletal reorganization and migratory activation (Weidner et al, 2013). (2) T. gondii infection triggers GABAergic signalling in DCs and in monocytes (Fuks et al, 2012), which leads to influx of Ca 2+ via VGCCs (mainly Ca V 1.3) (Kanatani et al, 2017) and downstream activation of Ras GTPase via CaM-CaMkII signalling (Olafsson et al, 2020). 'P' indicates protein phosphorylation.…”

“…(4) Secreted TIMP-1 activates CD63-ITGB1-FAK-Src/Pi3k signalling leading to cytoskeletal rearrangements and shifts the cell into a hypermotile state that facilitates dissemination (Olafsson et al, 2019). 5The receptor tyrosine kinase (RTK) Met is rapidly activated upon T. gondii infection, partly through cytoplasmic transactivation via FAK and Pyk2 (Olafsson et al, 2020). 6The VGCC-CaM-CaMkII and Met signalling pathways converge on the activation of Ras GTPase (Olafsson et al, 2020).…”