Abstract
Elimination of fungal pathogens by phagocytes requires phagosome maturation, a process that involves the recruitment and fusion of intracellular proteins. The role of Dectin-1, a β-1,3-glucan receptor, critical for fungal recognition and triggering of Th17 responses, to phagosomal maturation has not been defined. We show that GFP-Dectin-1 translocates to the fungal phagosome, but its signal decays after 2 h. Inhibition of acidification results in retention of GFP-Dectin-1 to phagosome membranes highlighting the requirement for an acidic pH. Following β-1,3-glucan recognition, GFP-Dectin-1 undergoes tyrosine phosphorylation by Src kinases with subsequent Syk activation. Our results demonstrate that Syk is activated independently of intraphagosomal pH. Inhibition of Src or Syk results in prolonged retention of GFP-Dectin-1 to the phagosome signifying a link between Syk and intraphagosomal pH. β-1,3-glucan phagosomes expressing a signaling incompetent Dectin-1 failed to mature as demonstrated by prolonged Dectin-1 retention, presence of Rab5B, failure to acquire LAMP-1 and inability to acidify. Phagosomes containing Candida albicans also require Dectin-1-dependent Syk activation for phagosomal maturation. Taken together, these results support a model where Dectin-1 not only controls internalization of β-1,3-glucan containing cargo and triggers proinflammatory cytokines, but also acts as a master regulator for subsequent phagolysosomal maturation through Syk activation.
Highlights
Dectin-1 is able to recognize and phagocytose the fungal carbohydrate, -1,3-glucan, but its contribution to phagosomal maturation has not been explored
Through the use of chemical inhibitors or a signaling incompetent mutant of Dectin-1 incapable of activating spleen tyrosine kinase (Syk), we demonstrate that Dectin-1-dependent Syk activation is critical for acidification of -1,3glucan-containing phagosomes
GFP-Dectin-1 Retention to -Glucan-containing Phagosomes Is Dependent on Phagosomal pH—We first explored the kinetics of normal Dectin-1 translocation following capture and phagocytosis of -1,3-glucan beads
Summary
Dectin-1 is able to recognize and phagocytose the fungal carbohydrate, -1,3-glucan, but its contribution to phagosomal maturation has not been explored. The intracellular domain of Dectin-1 is phosphorylated by Src kinases resulting in activation of spleen tyrosine kinase (Syk) and an intracellular signaling cascade resulting in proinflammatory cytokine production capable of polarizing Th17 cells, essential for anti-fungal immunity [15,16,17,18,19,20,21,22]. Dectin-1 triggers phagocytosis, and in innate immune cells places cargo in membrane-delineated compartments termed phagosomes Maturation of these compartments occurs as a result of intracellular protein recruitment and vesicular fusion through protein chaperones such as Rab GTPase family members, or direct fusion with lysosomes. Recent observations from bacterial and fungal model systems demonstrate that specific signaling cascades as well as cross-presentation are
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