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Autophagyxenophagy
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Intrinsic: altered mitochondrial membrane integrity. Necrosis. Cell swelling and rupture ... Possible association with atherosclerotic heart disease ... – PowerPoint PPT presentation
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Title: Autophagyxenophagy
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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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