Innate immune cells respond to microbial invaders using pattern recognition receptors that detect conserved microbial patterns. Among the cellular processes stimulated downstream of pattern recognition machinery is the initiation of autophagy, which plays protective roles against intracellular microbes. We have shown recently that Dictyostelium discoideum, which takes up bacteria for nutritive purposes, may employ pattern recognition machinery to respond to bacterial prey, as D. discoideum cells upregulate bactericidal activity upon stimulation by lipopolysaccharide (LPS). Here we extend these findings, showing that LPS treatment leads to induction of autophagosomal maturation in cells responding to the bacteria Staphylococcus aureus. Cells treated with the autophagy-inducing drug rapamycin clear internalized bacteria at an accelerated rate, while LPS-enhanced clearance of bacteria is reduced in cells deficient for the autophagy-related genes atg1 and atg9. These findings link microbial pattern recognition with autophagy in the social amoeba D. discoideum.

Cells of the innate immune system detect pathogen-associated molecular patterns (PAMPs) through highly conserved pattern-recognition machinery. The conservation of this machinery allows for its characterization in model organisms. The unique model organism Dictyostelium discoideum can exist as single-celled amoebae that phagocytize bacteria for nutritional uptake. However, it has not been determined if D. discoideum uses pattern-recognition machinery to detect PAMPs. Here we show that D. discoideum mounts a response against the bacterial cell wall PAMP, lipopolysaccharide (LPS). LPS treatment results in an increased clearance of phagoctyzed bacteria by D. discoideum. LPS-induced bactericidal activity appears to be dependent on MAPK and Tir domain-containing protein A (TirA)-mediated pathways. Recent results suggest that increased bactericidal activity upon treatment with LPS may involve the induction and maturation of autophagosomes. We show here, by following the delivery of the autophagosomal marker Atg8 to degradative compartments, that Staphylococcus aureus induces autophagosomal maturation in D. discoideum only upon addition of LPS. We are currently characterizing the signaling pathways underlying bacterial induction of autophagy in D. discoideum. Characterization of the cellular events induced upon microbial pattern recognition in D. discoideum should provide valuable insight into conversed cellular mechanisms underlying microbial detection and killing.

Pattern recognition machinery is used by cells of the innate immune system to detect conserved patterns on invading pathogens. Dictyostelieum discoideum is a model organism that phagocytizes bacteria for nutritional purposes, and we have shown recently that D. discoideum may use conserved pattern recognition machinery to detect and respond to their bacterial prey, since D. discoideum cells stimulated with the microbial pattern lipopolysaccharide (LPS) more efficiently clear phagocytized bacteria. Here we extend these findings, showing that LPS stimulates maturation of autophagosomes and transport of autophagosomal contents to lysosomes in D. discoideum cells exposed to Staphylococcus aureus. Induction of autophagy appears sufficient for increased bacterial killing in D. discoideum cells, as D. discoideum clears bacteria at in increased rate when cells are treated with the autophagy-inducing drug, rapamycin. Finally, we illustrate that LPS-enhanced transport of phagocytized S. aureus to the degradative lysosomes is significantly reduced in D. discoideum cells lacking the autophagy-related protein Atg1 or the toll/interleukin-1 receptor domain-containing protein TirA. Thus, for the first time, we link microbial pattern recognition and autophagy in this model organism, providing greater understanding of the highly conserved mechanisms underlying pathogen detection.