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Autophagy

Autophagy. Autophagosome: A double membrane bound compartment that engulfs cytosol and degrades the cytoplasmic contents. Large: 400-1500 nm May originate from ER or from fusion of lipid-containing vesicles that form ‘sequestration crescent’. Autophagic pathways. Plants and animals

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Autophagy

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  1. Autophagy • Autophagosome: A double membrane bound compartment that engulfs cytosol and degrades the cytoplasmic contents. • Large: 400-1500 nm • May originate from ER or from fusion of lipid-containing vesicles that form ‘sequestration crescent’.

  2. Autophagic pathways • Plants and animals • Bacteria, viruses, parasites • Highly regulated processes • Stages: induction, execution, maturation

  3. Autophagy genes • Based on S. cerevisiae yeast studies: • ATG in yeast, homologs in other spp. • Involved in: • Tumor suppression • Starvation responses • Preventing premature cell senescence

  4. Role of autophagy • Does autophagy benefit the host as a defense mechanism? • Does autophagy benefit the microbe by facilitating survival and replication? • Or both?

  5. Role of autophagy • A host response to degrade intracellular pathogens? • Hepatic pathogen purging • 10 weeks: 75% of chimpanzee liver cells contained hepatitis B viral proteins. • 20 weeks: chimpanzees were virus free, with no evidence of extensive cell death!

  6. Role of autophagy • A microbe strategy for enhanced persistence and intracellular replication? • Murine hepatitis virus (MHV): • Cellular markers on viral membranes were consistent with autophagosomal markers. • ATG5 mutant had 1000-fold decrease in viral yield. • Plasmid expressing ATG5 restored viral yield.

  7. Induction • TOR (Target Of Rapamycin) kinase • Inhibits autophagy when phosphorylated. • Also regulates protein and amino acid synthesis. • Rapamycin and nutrient starvation dephosphorylate TOR, inducing autophagy. • Tamoxifen induces autophagy too (TOR?)

  8. Induction • Trimeric G proteins • High amino acids inactivate G proteins, so aa depletion may induce autophagy via G proteins. • PI3Ks (Phosphatidylinositol-3-kinases) • Essential for starvation induced autophagy. • 3-MA, wortmannin, LY294002 target PI3Ks, and result in inhibition of autophagy.

  9. Execution • Covalent linkage of Atg5 and Atg12 • Covalent lipidation of Atg8 • Enzymes Atg3, Atg7, and Atg10 are homologs of ubiquitylation enzymes but are used to modify pathway components instead of labeling them for degradation.

  10. Maturation • GTPases (Rab24) mediate vesicle fusion. • Intermediate autophagosomes • Fuse with endosomal vesicles. • Acquire LAMP, accumulate DAMP proteins. • Mature autolysosomes • Fuse with lysosomes. • Acquire cathepsins and acid phosphatases.

  11. Detection methods • Microscopy • Electron microscopy – ultrastructure, morphology, volumetrics, staining. • Biochemistry • Enzyme activity assays. • Radioactive degradation studies. • Marker studies • Autophagosomal or organelle markers. • Fluorescence or immunodetection.

  12. Bacterial susceptibility • Astragalus-Mesorhizobium: • Bacteria differentiate within membrane compartments until they can fix nitrogen and establish symbiosis. • Nutrient starvation: bacterial degradation observed. Autophagy? http://www.bioscience.drexel.edu/Homepage/immunology/presentations/group6/Aintro.htm

  13. Bacterial susceptibility • Rickettsiae conorii • Sensitive to NO produced by IFN, TNF-α. • Correlations between autophagosome-like structures and bacterial degradation. • Are autophagosomes destroying bacteria or just cleaning up after bacteria are killed?

  14. Bacterial susceptibility • Listeria monocytogenes • Enter host cells by phagocytosis, escape from phagosomes, multiply in cytoplasm. • ActA (actin) mutants are engulfed in autophagosome-like compartments. • Wortmannin reduces bacterial entry into autophagosomes. • Nutrient depletion increases bacterial entry into autophagosomes. http://www.rapidmicrobiology.com/news/603h48.php

  15. Bacterial subversion • Porphyromonas gingivalis • Infects human coronary artery endothelial cells. • Localizes to autophagosome-like compartments. • Wortmannin: compartments resembled lysosomes and acquired cathepsin earlier. Bacterial survival decreased. http://www.pgingivalis.org/ATCC33277(1).htm

  16. Bacterial subversion • Brucella abortus • Endosomal uptake, then autophagosomes. • Wortmannin reduced survival, cell starvation increased survival. • VirB mutants have Type IV secretion mutation that inhibits intracellular transport and growth. Mutants are localized to membrane compartments that acquire cathepsin earlier, resembling lysosomes.

  17. Bacterial subversion • Legionella pneumophila • Replicates in autophagosome-like compartments in macrophages. • Dot/icm mutants are defective in Type IV secretion involved with organelle trafficking or intracellular multiplication. Mutants localized to lysosomal-like vesicles, not autophagosomes. • ‘Pregnant pause’ model says that autophagosome maturation is delayed to allow for pathogen development.

  18. Bacterial subversion • Coxiella burnetti • Replicates within autophagosome-like acidic vesicles. • Rab7 mutants had altered size and numbers of vesicles containing bacteria. • Dot/icm homologs in Coxiella were able to return Dot/icm deficient Legionella to a wild phenotype.

  19. Viral susceptibility • Herpes-virus • PKR kinase – phosphorylates eukaryotic translation-initiation factor eIF2α to inhibit and deregulate cellular translation. • PKR can also induce autophagy, apoptosis, and activate NF-KB. • ICP34.5 – produced by herpes simplex virus 1 to antagonize PKR function by dephosphorylating eIF2α.

  20. Viral subversion • Positive-strand RNA viruses • Poliovirus, murine hepatitis virus (MHV), equine arterivirus (EAV), SARS human corona virus. • Require membranes for replication. • Autophagosomes are induced during infection, but are they part of the viral replication process or the host response to eliminate pathogens?

  21. TIP OF THE ICEBERG?

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