L17

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1. What alternative role does the Toll signaling pathway have in adult flies? What experiment showed this, and what were the results of this experiment?

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In adult flies, the toll-signaling pathway is used for antimicrobial peptide production in the Gram+ and fungal infections. The first experiment to indicate that toll had an immune function was one that studied % survival of different mutants exposed to bacterial and fungal infections, the strains with toll pathway mutations had like a 3% survival.

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2. What are antimicrobial peptides and what do they do? Where are they made in Drosophila and which antimicrobial peptides act against which pathogens?

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Microbial peptides aid in the destruction of parasitic microbes. We know specifically that defensins (punch holes in membranes) are highly conserved in plants, insects, and animals. Drosophila transcribe antimicrobial peptides in the “fat body” and all work by disrupting the PM of invaders using cysteine residues that allow them to integrate into membrane. 7 inducible in drosophila.
Fungi: Drosomycin, Metchnikowin, Cecropin
Gram(+)Bacteria: Defensin, Metchnikowin
Gram(-) bacteria: Attacin, Cecropin, Diptericin, Drosocin

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Gram(-) bacteria antimicrobial peptides

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Attacin, Cecropin, Diptericin, Drosocin

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Gram(+)Bacteria antimicrobial peptides

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Defensin, Metchnikowin

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Fungi antimicrobial peptides

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Drosomycin, Metchnikowin, Cecropin

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3. What are the two types of host immune responses? Compare and contrast these two types of immunity. What are the three responses of Drosophila’s innate immunity?

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Innate: Immediate response. First and second lines of defense.
1st line = physical barriers (skin and stuff)
2nd line = macrophages (cellular) and systematic recognition of “invariant patterns” that are not present within the organism, such as viral RNA. Several proteolytic cascades.
3 innate immune responses in drosophila:
1. Humoral response using antimicrobial peptides (7 inducible)
2. Plasmatocytes/hematocytes doing phagocytosis
3. Melanization of wounds produces toxic oxygen species that recruit to site of wound to bind, encapsulate, and degrade pathogens at the wound.
Drosophila have some level of viral immunity through dicer and Argonaut dicing viral RNA.
Adaptive: Delayed response. Passive and Active immunity. Only a few vertebrates have this.
Tailored to maximally eliminate specific antigens. Accelerated somatic mutations. Irreversible genetic recombinations of antigen receptor gene segments. Accelerated clonal expansion, etc., etc., etc.

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4. What is the significance of NFkB in innate immunity? Which receptor/signaling pathway is involved in the activation of NFkB and NFkB-like transcription factors in mammals and Drosophila, respectively?

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Drosophila share intracellular signaling pathways involved in innate immunity with vertebrates. These pathways lead to the activation of transcription factor NFkB, which is homologous to Dorsal and Dif. This suggests that activation of NFkB and NFkB-like transcription factors could be the original and central signaling pathway in innate immunity. Partly because if antimicrobial peptides are the earliest form of defense, then the receptors that activate them are likely the earliest receptors dedicated to host defense.

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5. What advantage does having 10 TLR’s in humans gain, as opposed to the fruit fly having only one Toll receptor? What common goal do all of these TLRs have with regard to innate immunity?

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More toll receptors = more pathogen-common molecules that can activate the innate response. The common goal of all these TLR’s is to activate NFkB or NFkB-like dependant pathway. This will result in the release of cytokines(inflammation) and antimicrobial proteins.

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6. What family of proteins does the Toll signaling pathway activate? What do these proteins do? What are their names in Drosophila?

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The toll signaling pathway leads to activation of Rel family proteins, which are all NFkB homologues(Dif, Dorsal, Relish) or NFkB themselves and therefore transcription factors. DiF = Dorsal related immunity factor. Dorsal = embryogenesis, DiF = immunity.

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7. Does the Toll receptor directly bind pathogens? What is suggested to be involved in pathogen recognition in the immune response?

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Toll does not directly bind to pathogens. Instead, pathogens bind to cleaved, activated Spatzle. Basically, you have a pathogen recognition molecule binding to spatzle and interacting with the serine protease that cleaves it.

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8. How is Spatzle distributed in the perivitelline space of the egg? How is Spatzle activated only in the ventral third of the egg? What are two “cues” that can activate Toll via Spatzle activation in the fly?

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Prepro-spatzle is processed in the ER, and then prospatzle is secreted into of perivitrelline space. Easter, which is activated in ventral third of perivitrelline space, cleaves spatzle in the ventral third of the perivitrelline space. Two cues: microbes and embryogenesis.

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9. What form is Spatzle synthesized in the egg?

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Prepro-protein synthesized and processed into a proprotein in the ER. The pro-protein is then secreted into the perivitelline space.

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10. What processing does Spatzle protein undergo before it is secreted into the perivitelline space and where is it processed? In what form is Spatzle secreted into the perivitelline space?

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The signaling peptide is removed in the ER before it is secreted. Spatzle is secreted into the perivitelline space as a pro-protein. A serine protease (easter) then cleaves spatzle’s pro-domain, which normally masks a very hydrophobic region which, once exposed, drives spatzle to the PM. This results in a conformational change that produces an active spatzle ligand.

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11. What serine protease is responsible for activating Spatzle? How is this serine protease itself regulated to be active only in the ventral side of the embryo (two ways)?

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Easter is responsible for cleaving the spatzle. Spatzle is 382 residues long. The N-terminus is the regulatory domain whereas the C-terminus is the actual signaling domain. Easter cleaves off the entire N-terminus, leaving only the 106 residue C-terminal signaling domain.

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12. What is the function of Spatzle’s prodomain? How long (in amino acids) is the active ligand form?

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Spatzle prodomain hides the hydrophobic region that drives active spatzle to the PM. Active spatzle is 106 residues long.

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13. Which end of the Spatzle active protein binds to the Toll receptor as a dimer? How does Spatzle binding affect the curvature of the Toll protein?

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The C-terminus of spatzle is the signaling domain, and therefore binds to the toll receptor as a dimer. The spatzle dimer cross-links two toll proteins together and increases the curvature (curve becomes tighter). The ability of toll to bind and recognize spatzle is attributed to its leucine-rich repeat scaffolding.

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14. What is the effect of injecting Spatzle into the hemolymph of adult flies?

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Injecting spatzle into the hemolymph of flies induces a toll-dependent immune response. It is believed that there are specific pathogen recognition molecules that are activating in concert with spatzle to initiate the cleavage of its pro-domain.

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15. Which pathway is involved in recognition of gram (+) bacteria? Fungal pathogens? Gram (-) bacteria?

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Gram(+): Toll pathway with GNBP-1 and PGRP-SA (semmelweis gene)
Fungi: Toll pathway GNBP-1 helps
Gram(-): Imd (ImMunoDeficiency) pathway. PGRP-LE = secreted PGRP-LC = Transmembrane.

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16. Which pathway does Imd resemble in mammals? What does activation of this pathway lead to?

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Imd resembles the TNF (Tumor necrosis factor) pathway in mammals. Activation of this pathway leads to activation of Relish, a Rel family protein with NFkL homology.

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17. Compare and contrast activation of Toll receptor in embryo and adult flies.

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In the drosophila embryo, activation of the Toll receptor leads to the activation of cactus kinase which releases cactus, dorsal’s chaperone, and therefore the translocation of the dorsal TF into nuclei.
In adults, Toll receptor activation is triggered in response to gram(+) and fungal infections, and leads to the activation of Rel family proteins, which are homologous to NFkL and are therefore transcription factors. These TFs trigger immune responses, specifically the production of antimicrobial peptides. DiF is the NFkB-like transcription factor that is activated by toll in immune response in adult flies.