Apoptosis Mechanism Necrosis Pyroptosis Apoptosis: friend or

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Autophagy/xenophagy

• Autophagy eat onself
• Xenophagy eat foreign matter
• Highly conserved and regulated process that
  maintains cellular homeostasis and protects cells
  against starvation and microbe invasion
• 3 types
• macro-autophagy
• Microautophagy
• chaperone-mediated autophagy

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Manipulating host responses

• Autophagy
• Apoptosis

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Autophagic process

• Cytoplasmic organelles and portions of cytoplasm
  sequestered in double membrane-bound vacuoles
• Source of membrane multifactorial (ribosome-free
  ER?)
• Fuse with lysosomes

Nature reviews microbiology 2004 2301
Science 2004 306990
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3 Stages

• Initiation nutrient starvation, growth
  factor-mediated starvation
• Execution
• Maturation

Nature reviews microbiology 2004 2301
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InitiationSignaling pathways

• Trimeric G proteins
• Growth receptors
• PI3K (classes I III)
• protein phosphatases
• mTOR

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Execution

• Requires covalent-conjugation pathways
• Requires Atg3, Atg7, Atg10
• Homologs of ubiquitinylation proteins
• Modifies pathway components but does not target
  them for degradation

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Execution (cont)

• Production of PIP3 via hVPS34 (class III PI3K)
• Inhibited by wortmanin, 3-Methyladenine
• Promotes proteolysis-gtproduction of amino acids
• Complex formed between hVPS34, Atg14, Atg6
  (Beclin-1) which is necessary for downstream
  recruitment and localization of Atg5 Atg12
• Covalent linkage of Atg5 and Atg12 on
  pre-autophagosomal membrane
• Protease cleavage of Atg8 (MAP1 Light chain 3
  (LC3), followed by covalent lipidation to
  phosphatidylethanolamine and translocation to
  autophagosome membrane

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Fusion

• Autophagosomes fuse with endosomes
• Acquire characteristics of lysosomes
• LAMP1 2
• Accessible to DAMP
• Cathepsins, acid phosphatases
• Vesicle fusion mediated by Rabs (Rab24)

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Assays for autophagy

• EM
• Marker labeling
• Atg5 early autophagosomes
• Atg8/LCM3 early late
• Lysosomal markers (DAMP, lysosotracker)
• Monodansylcadaverine-fluorescent marker staining
• Genetically tractable organisms
• Knockout cells (Atg-5)
• Inhibitors (wortmannin, 3-MA)
• Inducers (amino acid starvation, rapamycin,
  IFN-gamma)

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Involved in many processes

• ATG6/Beclin 1 tumour suppressor gene
• Deleted in sporadic breast, ovarian, prostate CA
• Dauer formation in C. elegans
• ATG5 ATG7 Dictyostelium nitrogen starvation
• Prevent premature senescence
• Host defense against pathogen invasion

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Microbes autophagy

• Diverse strategies
• Host defense
• Bacterial manipulate it to survive intracellularly

Nature reviews microbiology 2004 2301
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Intracellular bacteria can by killed by autophagy

• Listeria (?ActA)

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Legionella

• pregnant pause hypothesis
• Initially enter into autophagosomes
• Require type IV effector to delay maturation into
  lysosomes

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Legionella

• Membranous vacuole surrounded by double membrane
• Contains ER protein (BiP)
• Contains LAMP1, cathepsin D
• Dot/ICM mutants enter late endosomes/lysosomes
• Controversial
• ER membrane
• Inhibited by DN SAR1 ARF1, involved in ER-golgi
  trafficking
• Normal intracellular development in dictyostelium
  Atg1, atg5, atg6, atg7, atg8 genes

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Streptococcus pyogenes aka Group A Strep (GAS)

• Sore throat
• Glomerulonephritis
• Rheumatic fever and valvular heart disease
• Toxin-mediated skin infections
• Impetigo
• Scarlet Fever
• Necrotizing fasciitis (flesh eating bacteria)
• Toxic shock-like syndrome

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GrpA strepAutophagy as host defense

• Binds to ECM produces toxins
• Invades non-phagocytic cells-gtvacuolar
  escape-gtdegraded by autophagy
• Initially enters early endosome (EEA1)
• Requires SLO to escape and enter autophagic
  pathway
• Vacuolar escape mutant (?SLO) avoids autophagy
• Atg5 deficient cells
• Vacuolar escape
• Avoids autophagic destruction
• Modest increase in intracellular bacterial titers
• Host cell ultimately undergo apoptosis

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M. Tb

• Induction of autophagy (AA starvation, rapamycin)
  augments phagolysome acidification
• Blocked by Wortmannin 3-MA
• Induction of autophagy promotes maturation of
  phagolysosome
• Acquisition of cathepsin D, Lamp-1, vacuolar
  ATPase, LBPA
• Induction of autophagy promotes co-localization
  of autophagic markers (LC3, beclin-1)
• Induction of autophagy inhibits mycobacterial
  survival
• Mimicked by IFN-gamma

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Shigellaa bug that disarms autophagyOgawa et al
Science 2005
Atg5
No autophagy
VirG
Atg5
?IcsBautophagy
IcsB
?VirG no autophagy
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There are many ways to die
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Apoptosis

• Programmed cell death
• Type I caspase mediated
• Type II autophagy-mediated
• Entire cell dismantled within membrane-enclosed
  vesicles
• Taken up by phagocytes, preventing release of
  intracellular components from dying cells
• Normal morphogenesis, removing genetically
  damaged cells, proper tissue homeostasis,
  invading microbes

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Mechanism

• Sequential activation of cysteine proteases
  (caspases)
• Caspase 1-related 1,4,5,13,14
• Cytokine processing and pro-inflammatory cell
  death
• Initiator caspases 2, 8, 9, 10
• Effector caspases 3,6,7
• Regulated process
• Extrinsic stimulation of Fas or TNFR surface
  receptors
• Intrinsic altered mitochondrial membrane
  integrity

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Necrosis

• Cell swelling and rupture
• Release in intracellular components
• Activation of inflammatory response
• Can be regulated
• Can occur in concert with or instead of apoptosis
  (e.g. if apoptosis is blocked)

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Pyroptosis

• Caspase-1-dependent cell death
• Convert IL-1B and IL-18 to active forms
• Induced by Shigella IpaB and Salmonella SipB

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Apoptosis friend or foe

• Host view
• Apoptosis is bad unless it takes away a
  privileged intracellular niche
• Pathogen view
• Inhibit apoptosis to provide for intracellular
  niche (Chlamydia)
• Promote apoptosis (Yersinia, Shigella)
• Dismantle host defense
• Promote microbe dissemination
• Many bacterial molecules can modulate apoptosis
• Toxins
• Type III secreted proteins

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Chlamydia species

• Obligate intracellular bacteria
• Interesting intracellular life cycle
• Chlamydia trachomatis
• Major cause of STDs in US and trachoma in
  developing countries
• Chlamydia pneumoniae
• Important cause of upper and lower respiratory
  infections
• Possible association with atherosclerotic heart
  disease

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• Diverges from endoctyic pathway immediately
• Acquires characteristics of golgli
• Sphingomyelin
• Cholesterol
• Rab1 (ER to golgi trafficking), Rab 11 (recycling
  endosomes, TGN, plasma membrane), Rab 6 (Golgi-ER
  trafficking, EE to TGN transport)

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Chlamydia modulates apoptosis

• Chlamydiae-infected cells are protected against
  mitochondria-dependent cell death but not
  caspase-3 mediated cell death
• Inhibits cytochrome C release
• Destroys pro-apoptotic BH3 domain containing
  proteins (Bim/Bod, Puma, Bad) which are upstream
  of Bax/Bak

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Chlamydia both induces and inhibits apoptosis

• Chlamydia protein associated with death domains,
  that interacts with death domains of TNF
  receptors to activate apoptotic caspases

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Salmonella-induced cell death is complicated

• Mutiple mechanisms
• Appears heterogeneous
• Apoptosis, necrosis, pyrotosis
• SPI-1-dependent caspase-1 dependent cell death
• Rapid
• Caspase-1 dependent
• Caspase-3 independent
• SipB-dependent
• SipB forms complex with Caspase-1

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• SPI-1 dependent caspase-1 independent cell death
• Slower
• SipB-dependent
• Expression of SipB in caspase-1 deficient cells
  lead to formation of lamellar structures..autophag
  osomes? Fusing to mitochondria
• SipB has fusogenic properties

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SPI-2-dependent macrophage cell death

• Prolonged incubation (24 hrs)
• Caspase-1-dependent
• Caspase-1-deficient mice are resistant to
  Salmonella infection

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