Autophagy/xenophagy

1. 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

2. Manipulating host responses • Autophagy • Apoptosis

3. 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 2:301 Science 2004 306:990

4. 3 Stages • Initiation: nutrient starvation, growth factor-mediated starvation • Execution • Maturation Nature reviews microbiology 2004 2:301

5. Initiation:Signaling pathways • Trimeric G proteins • Growth receptors • PI3K (classes I & III) • protein phosphatases • mTOR

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

7. Execution (cont) • Production of PIP3 via hVPS34 (class III PI3K) • Inhibited by wortmanin, 3-Methyladenine • Promotes proteolysis->production 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

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

9. 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)

10. 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

11. Microbes & autophagy • Diverse strategies • Host defense • Bacterial manipulate it to survive intracellularly Nature reviews microbiology 2004 2:301

12. Intracellular bacteria can by killed by autophagy • Listeria (∆ActA)

13. Legionella • “pregnant pause” hypothesis • Initially enter into autophagosomes • Require type IV effector to delay maturation into lysosomes

14. 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

15. 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

16. GrpA strepAutophagy as host defense • Binds to ECM & produces toxins • Invades non-phagocytic cells->vacuolar escape->degraded 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

18. 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

21. Atg5 IcsB VirG Shigellaa bug that disarms autophagyOgawa et al Science 2005 Atg5 No autophagy VirG Atg5 ∆IcsB:autophagy IcsB ∆VirG: no autophagy

22. There are many ways to die…

23. 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

24. 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

25. 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)

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

27. 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

28. 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

29. 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)

30. 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

31. Chlamydia both induces and inhibits apoptosis • Chlamydia protein associated with death domains, that interacts with death domains of TNF receptors to activate apoptotic caspases

32. 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

33. 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..autophagosomes? Fusing to mitochondria • SipB has fusogenic properties

34. SPI-2-dependent macrophage cell death • Prolonged incubation (24 hrs) • Caspase-1-dependent • Caspase-1-deficient mice are resistant to Salmonella infection