RESUMEN
All Aspergillus species form phialidic conidia (PC) when the mycelium is in contact with the air. These small, asexual spores are ideally suited for an airborne dissemination in the environment. Aspergillus terreus and a few closely related species from section Terrei can additionally generate accessory conidia (AC) that directly emerge from the hyphal surface. In this study, we have identified galactomannan as a major surface antigen on AC that is largely absent from the surface of PC. Galactomannan is homogeneously distributed over the entire surface of AC and even detectable on nascent AC present on the hyphal surface. In contrast, ß-glucans are only accessible in distinct structures that occur after separation of the conidia from the hyphal surface. During germination, AC show a very limited isotropic growth that has no detectable impact on the distribution of galactomannan. The AC of the strain used in this study germinate much faster than the corresponding PC, and they are more sensitive to desiccation than PC. During infection of murine J774 macrophages, AC are readily engulfed and trigger a strong tumor necrosis factor-alpha (TNFα) response. Both processes are not hampered by the presence of laminarin, which indicates that ß-glucans only play a minor role in these interactions. In the phagosome, we observed that galactomannan, but not ß-glucan, is released from the conidial surface and translocates to the host cell cytoplasm. AC persist in phagolysosomes, and many of them initiate germination within 24 h. In conclusion, we have identified galactomannan as a novel and major antigen on AC that clearly distinguishes them from PC. The role of this fungal-specific carbohydrate in the interactions with the immune system remains an open issue that needs to be addressed in future research.
RESUMEN
Carbachol-mediated activation of type M(3) muscarinic acetylcholine receptors induces the biosynthesis of the transcription factor Egr-1 in human SH-SY5Y neuroblastoma cells involving an activation of extracellular signal-regulated protein kinase. Carbachol triggered the phosphorylation of the ternary complex factor Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, and strikingly enhanced the transcriptional activation potential of Elk-1. Chromatin immunoprecipitation experiments revealed that Elk-1 binds in vivo to the 5'-upstream region of the Egr-1 gene in carbachol-stimulated neuroblastoma cells. Together, these data indicate that Elk-1 connects the intracellular signaling cascade elicited by activation of M(3) muscarinic acetylcholine receptors with the transcription of the Egr-1 gene. Lentiviral-mediated expression of either MAP kinase phosphatase-1 (MKP-1) or a constitutively active mutant of calcineurin A inhibited Egr-1 biosynthesis following carbachol stimulation, indicating that these phosphatases function as shut-off devices of muscarinic acetylcholine receptor signaling. Additionally, carbachol stimulation increased transcription of a chromatin-embedded collagenase promoter/reporter gene, showing that AP-1 activity is enhanced in carbachol-stimulated neuroblastoma. Expression experiments revealed that both MKP-1 and a constitutively active mutant of calcineurin A impaired carbachol-induced upregulation of AP-1 activity. The fact that carbachol stimulation of neuroblastoma cells activates the transcription factors Egr-1 and AP-1 suggests that changes in the gene expression pattern are an integral part of muscarinic acetylcholine receptor signaling.