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

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

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

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

5. Listeria PHAGOCYTIC STRATEGY GAP ENDOSOMA Rab5a-GTP

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

7. - GAPDH-LM is a Listeria virulence factor able to inactivate Rab5a: -binds to N-terminal -ADP-ribosylates Rab5a Listeria PHAGOCYTIC STRATEGY * *********************.**.** ******..***************** *** 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. - GAPDH-LM is a virulence factor that binds to Rab5a: -binding to N-terminal [Ab anti-GADPH(N)] Listeria PHAGOCYTIC STRATEGY

9. -ADP-ribosylation of Rab5a by GAPDH-LM: -Rab5 ADPr is retained in Phgs- GDI does not remove Rab5 ADPr – Rab5ADPr GDP/GTP is lower Rab5 ADPr by GAPDH-LM: -retained Rab5 GDP on Phgs  GDI cannot remove it due to low abinding - block GDP/GTP exchange by interfering with GEF binding.

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

11. Oxidative listericidal mechanisms in phagosomes are regulated by Rab5 GTP - phox activation - ROI production. CFU: 16 32 1 4 Rab5a GTP acts before Rac2 GTP (Prada-Delgado et al., 2001, JBC 276:19059-67 ) Rab5 inhibition Rab5- overexpression CFU 12 1 30 9

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

13. -/- +/+ -/- +/+ (Uttermöhlen et al.,2003, J. Immunol. 170: 2621-2628) (Prada-Delgado et al., 2001, JBC 276:19059-67 ) 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-  signalling

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

15. - 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) INTRACELLULAR KILLING: Non-oxidative

16. -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 %CFU(0): 31 100 98 50 %CFU(8): 0,2 0,5 98 4,9 -Immune features of LLO site: W 491 W 492 - binding to Phgs + Ctsd cleavage site - W 491 : Phg binding, Ctsd sensitivity, immune advantage - W 492 : pore formation/cytotoxicity - W 491 W 492 : Phg sensor to produce >> or << LLO

17. INTRACELLULAR KILLING: Non-oxidative

18. Bye! 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)