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Listeria monocytogenes phagocytic strategies.
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Listeria monocytogenes phagocytic strategies.

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  1. Listeria monocytogenes phagocytic strategies. Dr. Carmen Alvarez-Dominguez. Immunology Dpt. Hospital Santa Cruz de Liencres-IFIMAV/IES Zapatón. Cantabria. SPAIN.

  2. LISTERIA INNATE IMMUNITY **INNATEADAPTIVE (1.b.) IL-10 Th2 (1.a.) R2 (3.) Listeria Lysosome Ag presentation Y Phagosome IL-4 R1 Y T-Naive Th1 Co-stimulatory molecules Phagocyte (2.) IFN- IL-12, IL-18 (3.) • **INNATE • Cellular & bacterial proteins • Receptors • **Intracellular killing (Oxidative/Non-oxidative) • Cytosolic surveillance system (NOD-like receptors) • Cellular participants (MØs, PMNs, DC, other) • Additional participants (i.e., cytokines, Ag presentation) • STUDIES: • Cellular & bacterial proteins • Rab5, Ctsd, Limp-2/GAPDH, LLO • Cellular participants: MØs • Additional participants: cytokines

  3. Bacterium Phagosome Rab5?? Chlamydia Coxiella Helicobacter Rab4,11 Rab5 Rab7 Listeria Shigella Salmonella Mycobacterium Phago-lysosome Rab9 Rab9 modified- phagosome Rab6 Brucella Legionella GOLGI ER PHAGOCYTIC STRATEGIES • Stages: • Rabs involved: • *Rab5-> early timing • Rab4-> slow recycling • Rab11-> fast recycling • Rab7-> Le/Lyso transport - Phagosome formation (1) 1 - Modified phagosome (2) - Phago-Lysosome (3) • Secretory route (4) • Autophagosome (5) 2 - Cytosol (6) 3 4 6 5

  4. Cytosol Effector Membrane GAP GDI Rab-GDP GDI GEF Rab-GDP Rab-GTP Rab-GTP GDF Rab cycle Vps9 AQ AS Rab5a:Q79L(AQ) -> GTP always active Rab5a:S34N(AS) -> GDP blocked Vps9-> GDP/GTP exchange factor (GEF) GAP->GTP hydrolysis

  5. Listeria PHAGOCYTIC STRATEGY GAP Rab5a-GTP ENDOSOMA

  6. GAPDH Lmo 2459 Listeria PHAGOCYTIC STRATEGY Lmo 2459 (GAPDH-LM) - Blue sepharose-affinity column (binds proteins with NAD sites) - Rab5a* affinity column anti-GAPDH(N) antibody

  7. Listeria PHAGOCYTIC STRATEGY - GAPDH-LM is a Listeria virulence factor able to inactivate Rab5a: -binds to N-terminal -ADP-ribosylates Rab5a * *********************.**.** ******..***************** *** GAPDH-LM 1 MTVKVGINGFGRIGRLAFRRIQNVEGIEVVAINDLTDAKMLAHLLKYDTTQGRFDGEVEV 60 GAPDH-SP 1 mvvkvgingfgrigrlafrriqniegvevtrindltdpnmlahllkydttqgrfdgtvev 60 .* * ***. .** *.*.**.. *. **.**** ****. . ** *. . ******* GAPDH-LM 61 HDGFFKVNGKEVKVLANRNPEELPWGDLGVDIVLECTGFFTAQDKAELHIKA-GAKKVVI 119 GAPDH-SP 61 keggfevngnfikvsaerdpenidwatdgveivleatgffakkeaaekhlhtngakkvvi 120 .**. *.**.*.* **. *********************** * * **. ******** GAPDH-LM 120 SAPATGDMKTIVYNVNHETLDGTETVISGASCTTNCLAPMAKVLEDKFGVVEGLMTTIHA 179 GAPDH-SP 121 tapggndvktvvfntnhdildgtetvisgascttnclapmakalhdafgiqkglmttiha 180 ***** **.** ** *****.* **.**.******** *.* *.************** GAPDH-LM 180 YTGDQNTLDAPHPKGDFRRARAAAENIIPNTTGAAKAIGEVLPTLKGKLDGAAQRVPVPT 239 GAPDH-SP 181 ytgdqmildgphrggdlrraragaanivpnstgaakaiglvipelngkldgaaqrvpvpt 240 **.**** ***.*.***.**** ***. ..**** * .***** *...***** ***** GAPDH-LM 240 GSLTELVTVLDKKVTVDEVNAAMEAASDPETFGYTSDQVVSSDIKGMTFGSLFDETQTKV 299 GAPDH-SP 241 gsvtelvvtldknvsvdeinaamkaasn-dsfgytedpivssdivgvsygslfdatqtkv 299 . * **** *.************************ GAPDH-LM 300 LTVGDQQLVKTVAWYDNEMSYTAQLVRTLEYFAKIAK 336 GAPDH-SP 300 mevdgsqlvkvvswydnemsytaqlvrtleyfakiak 336

  8. Listeria PHAGOCYTIC STRATEGY - GAPDH-LM is a virulence factor that binds to Rab5a: -binding to N-terminal [Ab anti-GADPH(N)]

  9. Listeria PHAGOCYTIC STRATEGY • ADP-ribosylation of Rab5a by GAPDH-LM: • Rab5ADPr is retained in Phgs - GDI does not remove Rab5ADPr – Rab5ADPr GDP/GTP is lower Rab5ADPr by GAPDH-LM: -retained Rab5GDP on PhgsGDI cannot remove it due to low abinding - block GDP/GTP exchange by interfering with GEF binding.

  10. INTRACELLULAR KILLING: oxidative gp91 gp91 gp22 gp22 * rac2 rac2 p67 p67 p47 Rab5-GTP p47 O2- Active Inactive • MECHANISMS: • phox (Rab5a/Rac2) • iNOS IFN-g PHOX

  11. Oxidative listericidal mechanisms in phagosomes are regulated by Rab5GTP - phox activation - ROI production. Rab5 inhibition CFU: 16 32 1 4 Rab5-overexpression Rab5aGTP acts before Rac2GTP (Prada-Delgado et al., 2001, JBC 276:19059-67 ) CFU 12 1 30 9

  12. INTRACELLULAR KILLING: Non-oxidative • lysosomal proteins • pH • anti-microbial peptides • - Neutralizing antibodies

  13. INTRACELLULAR KILLING: Non-oxidative 1.- pH-> Not involved in Listeria degradation - Listeria phagosomes -> pH 5.0 2.- Are lysosomal proteases involved in Listeria killing? - activation of lysosomal proteases as Ctsd blocked Listeria growth . Ceramide production: ASMase-/- . IFN-g signalling -/- +/+ -/- +/+ (Prada-Delgado et al., 2001, JBC 276:19059-67 ) (Uttermöhlen et al.,2003, J. Immunol. 170: 2621-2628)

  14. Ctsd INTRACELLULAR KILLING: Non-oxidative 3.- are catepsins listericidal components? Restrictive cells: MØs Permisive cells: CHO . Ctsd activation Listeria killing . Inhibition of catepsin activity blocks Listeria killing (Prada-Delgado et al., 2005, Traffic 6:252-265)

  15. INTRACELLULAR KILLING: Non-oxidative - Ctsd is a relevant listericidal agent in MØs involved in innate immunity • Ctsd cleavage site in LLO is localized between WW of TACYs undecapeptide WEWWR (Del Cerro-Vadillo et al., 2006, J. Immunol. 176:1321-1325; Madrazo-Toca et al. , 2009, Mol Microbiol 72 :668 -682)

  16. %CFU(0): 31 100 98 50 %CFU(8):0,2 0,5 98 4,9 INTRACELLULAR KILLING: Non-oxidative • Ctsd enzymatic activity in TACYS • intracellular toxin: LLO (1-cleavage site) • extracellular toxin: PLY (2-cleavage sites) • blocks biological function: binding to membranes • PLY-->plasmatic membrane • LLO-->phagosomes • Immune features of LLO site: W491W492 • binding to Phgs + Ctsd cleavage site • W491: Phg binding, Ctsd sensitivity, immune advantage • W492: pore formation/cytotoxicity • W491W492: Phg sensor to produce >> or << LLO

  17. INTRACELLULAR KILLING: Non-oxidative

  18. ACKNOWLEDGEMENTS • Servicio de Inmunología. Hospital Santa Cruz de Liencres (HUMV)-IFIMAV/IES Zapatón. Cantabria. • CURRENT MEMBERS: • Lorena Fernadez-Prieto (Predoc-student) • Carlos Carranza-Cereceda (Predoc-student) • Estela Rodriguez-Del Rio (Student) • PREVIOUS MEMBERS: • Fidel Madrazo-Toca (Postdoctoral-fellow) • Elida Del Cerro-Vadillo (Student) • COLABORATORS: • *Eugenio Carrasco Marín (Santander. Spain) • M. Lopez-Fanárraga (Santander. Spain) • R. Tobes/E. Pareja (Granada. Spain) • J. Vandekerckhove (Ghent. Belgium) • J.R. de los Toyos (Asturias. Spain) • P. Saftig (Kiel. Germany) • D. Portnoy/J.A. Melton (CA. USA) • M. Roberts (Boston. USA)/H. Goldfine (PA. USA) • M. Mitsuyama (Japan) Bye! Bye!