Convergent Met and voltage-gated Ca2+ channel signaling drives hypermigration of Toxoplasma-infected dendritic cells.

Ras-Erk MAPK signaling controls many of the principal pathways involved in metazoan cell motility, drives metastasis of multiple cancer types and is targeted in chemotherapy. However, its putative roles in immune cell functions or in infections have remained elusive. Here, using primary dendritic cells (DCs) in an infection model with the protozoan Toxoplasma gondii, we show that two pathways activated by infection converge on Ras-Erk MAPK signaling to promote migration of parasitized DCs. We report that signaling through the receptor tyrosine kinase Met (also known as HGF receptor) contributes to T. gondii-induced DC hypermotility. Furthermore, voltage-gated Ca2+ channel (VGCC, subtype CaV1.3) signaling impacted the migratory activation of DCs via calmodulin-calmodulin kinase II. We show that convergent VGCC signaling and Met signaling activate the GTPase Ras to drive Erk1 and Erk2 (also known as MAPK3 and MAPK1, respectively) phosphorylation and hypermotility of T. gondii-infected DCs. The data provide a molecular basis for the hypermigratory mesenchymal-to-amoeboid transition (MAT) of parasitized DCs. This emerging concept suggests that parasitized DCs acquire metastasis-like migratory properties that promote infection-related dissemination.

Voltage-dependent calcium channel signaling mediates GABAA receptor-induced migratory activation of dendritic cells infected by Toxoplasma gondii.

The obligate intracellular parasite Toxoplasma gondii exploits cells of the immune system to disseminate. Upon T. gondii-infection, γ-aminobutyric acid (GABA)/GABAA receptor signaling triggers a hypermigratory phenotype in dendritic cells (DCs) by unknown signal transduction pathways. Here, we demonstrate that calcium (Ca2+) signaling in DCs is indispensable for T. gondii-induced DC hypermotility and transmigration in vitro. We report that activation of GABAA receptors by GABA induces transient Ca2+ entry in DCs. Murine bone marrow-derived DCs preferentially expressed the L-type voltage-dependent Ca2+ channel (VDCC) subtype Cav1.3. Silencing of Cav1.3 by short hairpin RNA or selective pharmacological antagonism of VDCCs abolished the Toxoplasma-induced hypermigratory phenotype. In a mouse model of toxoplasmosis, VDCC inhibition of adoptively transferred Toxoplasma-infected DCs delayed the appearance of cell-associated parasites in the blood circulation and reduced parasite dissemination to target organs. The present data establish that T. gondii-induced hypermigration of DCs requires signaling via VDCCs and that Ca2+ acts as a second messenger to GABAergic signaling via the VDCC Cav1.3. The findings define a novel motility-related signaling axis in DCs and unveil that interneurons and DCs share common GABAergic motogenic pathways. T. gondii employs GABAergic non-canonical pathways to induce host cell migration and facilitate dissemination.

Yersinia pseudotuberculosis efficiently escapes polymorphonuclear neutrophils during early infection.

The human-pathogenic species of the Gram-negative genus Yersinia preferentially target and inactivate cells of the innate immune defense, suggesting that this is a critical step by which these bacteria avoid elimination and cause disease. In this study, bacterial interactions with dendritic cells, macrophages, and polymorphonuclear neutrophils (PMNs) in intestinal lymphoid tissues during early Yersinia pseudotuberculosis infection were analyzed. Wild-type bacteria were shown to interact mainly with dendritic cells, but not with PMNs, on day 1 postinfection, while avirulent yopH and yopE mutants interacted with PMNs as well as with dendritic cells. To unravel the role of PMNs during the early phase of infection, we depleted mice of PMNs by using an anti-Ly6G antibody, after which we could see more-efficient initial colonization by the wild-type strain as well as by yopH, yopE, and yopK mutants on day 1 postinfection. Dissemination of yopH, yopE, and yopK mutants from the intestinal compartments to mesenteric lymph nodes was faster in PMN-depleted mice than in undepleted mice, emphasizing the importance of effective targeting of PMNs by these Yersinia outer proteins (Yops). In conclusion, escape from interaction with PMNs due to the action of YopH, YopE, and YopK is a key feature of pathogenic Yersinia species that allows colonization and effective dissemination.

Colonization of cecum is important for development of persistent infection by Yersinia pseudotuberculosis.

Yersiniosis is a human disease caused by the bacterium Yersinia pseudotuberculosis or Yersinia enterocolitica. The infection is usually resolved but can lead to postinfectious sequelae, including reactive arthritis and erythema nodosum. The commonly used Yersinia mouse infection model mimics acute infection in humans to some extent but leads to systemic infection and eventual death. Here, we analyzed sublethal infection doses of Y. pseudotuberculosis in mice in real time using bioluminescent imaging and found that infections using these lower doses result in extended periods of asymptomatic infections in a fraction of mice. In a search for the site for bacterial persistence, we found that the cecum was the primary colonization site and was the site where the organism resided during a 115-day infection period. Persistent infection was accompanied by sustained fecal shedding of cultivable bacteria. Cecal patches were identified as the primary site for cecal colonization during persistence. Y. pseudotuberculosis bacteria were present in inflammatory lesions, in localized foci, or as single cells and also in neutrophil exudates in the cecal lumen. The chronically colonized cecum may serve as a reservoir for dissemination of infection to extraintestinal sites, and a chronic inflammatory state may trigger the onset of postinfectious sequelae. This novel mouse model for bacterial persistence in cecum has potential as an investigative tool to unveil a deeper understanding of bacterial adaptation and host immune defense mechanisms during persistent infection.

Immune response to diphtheria toxin-mediated depletion complicates the use of the CD11c-DTR(tg) model for studies of bacterial gastrointestinal infections.

Dendritic cells play an important role in the immune response against pathogens, as they are responsible for the activation and control of both innate and adaptive immune responses. The CD11c-DTR(tg) model, which allows transient elimination of dendritic cells by diphtheria toxin-treatment (DTx), has been extensively used to study the importance of this immune cell during steady-state and infection conditions in mice. Infecting dendritic cell-depleted mice orally with Yersinia pseudotuberculosis results in a markedly reduced level of infection compared with infection of non-depleted mice. We show here that it is not the lack of dendritic cells per se that is responsible for the reduced infection efficiency, instead it is an immune response induced by the DTx-treatment that prevents the bacteria from establishing colonization in Peyer's patches. The DTx-induced depletion initiates an immune response, with elevated serum levels of keratinocyte-derived cytokine (KC) and recruitment of polymorphonuclear neutrophils to dendritic cell-containing organs, such as Peyer's patches. Since the window for having an animal depleted of dendritic cells is limited in time for this model, the DTx-mediated effect on the immune system complicates the use of this model in studies of early events during bacterial infections.

Cell type-specific effects of Yersinia pseudotuberculosis virulence effectors.

One important feature of Yersinia pseudotuberculosis that enables resistance against the host immune defence is delivery of the antiphagocytic effectors YopH and YopE into phagocytic cells. The tyrosine phosphatase YopH influences integrin signalling, and YopE impairs cytoskeletal dynamics by inactivating Rho GTPases. Here, we report the impact of these effectors on internalization by dendritic cells (DCs), which internalize antigens to orchestrate host immune responses. We found that this pathogen resists internalization by DCs via YopE. YopH that is important for blocking phagocytosis by macrophages and neutrophils and which is also present inside the DCs does not contribute to the resistance. However, the YopH targets Fyb and p130Cas show higher expression levels in macrophages than in DCs. Furthermore, live cell microscopy revealed that the cells internalize Y. pseudotuberculosis in different ways: the macrophages utilize a locally restricted receptor-mediated zipper mechanism, whereas DCs utilize macropinocytosis involving constitutive ruffling that randomly catches bacteria into membrane folds. We conclude that YopH impacts early phagocytic signalling from the integrin receptor to which the bacterium binds and that this tight receptor-mediated stimulation is absent in DC macropinocytosis. Inactivation of cytoskeletal dynamics by YopE affects ruffling activity and hence also internalization. The different modes of internalization can be coupled to the major functions of these respective cell types: elimination by phagocytosis and antigen sampling.

Pathogenic Yersinia Promotes Its Survival by Creating an Acidic Fluid-Accessible Compartment on the Macrophage Surface.

Microbial pathogens and host immune cells each initiate events following their interaction in an attempt to drive the outcome to their respective advantage. Here we show that the bacterial pathogen Yersinia pseudotuberculosis sustains itself on the surface of a macrophage by forming acidic fluid-accessible compartments that are partially bounded by the host cell plasma membrane. These Yersinia-containing acidic compartments (YACs) are bereft of the early endosomal marker EEA1 and the lysosomal antigen LAMP1 and readily form on primary macrophages as well as macrophage-like cell lines. YAC formation requires the presence of the Yersinia virulence plasmid which encodes a type III secretion system. Unexpectedly, we found that the initial formation of YACs did not require translocation of the type III effectors into the host cell cytosol; however, the duration of YACs was markedly greater in infections using translocation-competent Y. pseudotuberculosis strains as well as strains expressing the effector YopJ. Furthermore, it was in this translocation- and YopJ-dependent phase of infection that the acidic environment was critical for Y. pseudotuberculosis survival during its interaction with macrophages. Our findings indicate that during its extracellular phase of infection Y. pseudotuberculosis initiates and then, by a separate mechanism, stabilizes the formation of a highly intricate structure on the surface of the macrophage that is disengaged from the endocytic pathway.