Autophagy

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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.

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Autophagic pathways

• Plants and animals
• Bacteria, viruses, parasites
• Highly regulated processes
• Stages induction, execution, maturation

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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

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Role of autophagy

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

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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!

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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.

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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?)

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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.

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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.

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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.

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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.

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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/immunolo
gy/presentations/group6/Aintro.htm
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Bacterial susceptibility

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

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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
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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
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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.

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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.

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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.

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Viral susceptibility

• Herpes-virus
• PKR kinase phosphorylates eukaryotic
  translation-initiation factor eIF2a 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
  eIF2a.

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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?

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TIP OF THE ICEBERG?