Listeria monocytogenes phagocytic strategies Dr Carmen AlvarezDominguez Immunology

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

LISTERIA INNATE IMMUNITY **INNATE 2. 3. Cellular & bacterial proteins a) Receptors b) **Intracellular killing (Oxidative/Non-oxidative) c) Cytosolic surveillance system (NOD-like receptors) Cellular participants (MØs, PMNs, DC, other) Additional participants (i. e. , cytokines, Ag presentation) 1. 2. 3. Cellular & bacterial proteins a) b) Rab 5, Ctsd, Limp-2/GAPDH, LLO c) Cellular participants: MØs Additional participants: cytokines **INNATE (1. b. ) (1. a. ) R 2 Listeria R 1 Lysosome Phagosome (2. ) Phagocyte ADAPTIVE IL-10 Ag presentation YY 1. STUDIES: (3. ) Th 2 IL-4 T-Naive Co-stimulatory molecules IL-12, IL-18 (3. ) Th 1 IFN-

PHAGOCYTIC STRATEGIES • Stages: - - • Phagosome formation (1) Modified phagosome (2) Phago-Lysosome (3) Secretory route (4) Autophagosome (5) Cytosol (6) Rabs involved: – *Rab 5 -> early timing – Rab 4 -> slow recycling – Rab 11 -> fast recycling – Rab 7 -> Le/Lyso transport Bacterium Chlamydia Phagosome 1 Rab 5? ? Coxiella Helicobacter Listeria 4 Shigella 3 Salmonella Mycobacterium 6 Rab 9 Rab 6 2 Rab 4, 11 Rab 5 Rab 7 modifiedphagosome GOLGI ER Phago-lysosome Rab 9 5 Brucella Legionella

Rab cycle Cytosol GDI Rab-GDP GDI Vps 9 GEF GDF Effector AQ AS Rab-GDP Rab-GTP Membrane GAP Rab 5 a: Q 79 L (AQ) -> GTP always active Rab 5 a: S 34 N (AS) -> GDP blocked Vps 9 -> GDP/GTP exchange factor (GEF) GAP -> GTP hydrolysis

Listeria PHAGOCYTIC STRATEGY GAP Rab 5 a-GTP ENDOSOMA

Listeria PHAGOCYTIC STRATEGY Lmo 2459 (GAPDH-LM) Lmo 2459 GAPDH - Rab 5 a* affinity column anti-GAPDH(N) antibody - Blue sepharose-affinity column (binds proteins with NAD sites)

Listeria PHAGOCYTIC STRATEGY - GAPDH-LM is a Listeria virulence factor able to inactivate Rab 5 a: -binds to N-terminal -ADP-ribosylates Rab 5 a GAPDH-LM 1 GAPDH-SP 1 * ***********. ** ******. . ********* *** MTVKVGINGFGRIGRLAFRRIQNVEGIEVVAINDLTDAKMLAHLLKYDTTQGRFDGEVEV 60 mvvkvgingfgrigrlafrriqniegvevtrindltdpnmlahllkydttqgrfdgtvev 60 GAPDH-LM 61 GAPDH-SP 61 . * * ***. . ** *. *. **. ****. . ** *. . ******* HDGFFKVNGKEVKVLANRNPEELPWGDLGVDIVLECTGFFTAQDKAELHIKA-GAKKVVI 119 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 LTVGDQQLVKTVAWY DNEMSYTAQLVRTLEYFAKIAK 336 GAPDH-SP 300 mevdgsqlvkvvswydnemsytaqlvrtleyfakiak 336

Listeria PHAGOCYTIC STRATEGY - GAPDH-LM is a virulence factor that binds to Rab 5 a: -binding to N-terminal [Ab anti-GADPH(N)]

Listeria PHAGOCYTIC STRATEGY -ADP-ribosylation of Rab 5 a by GAPDH-LM: - Rab 5 ADPr by GAPDH-LM: -retained Rab 5 GDP on Phgs GDI cannot remove it due to low abinding - block GDP/GTP exchange by interfering with GEF binding.

INTRACELLULAR KILLING: oxidative MECHANISMS: -phox (Rab 5 a/Rac 2) -i. NOS gp 91 gp 22 * rac 2 IFN- rac 2 p 67 p 47 Inactive Active PHOX Rab 5 -GTP O 2 -

Oxidative listericidal mechanisms in phagosomes are regulated by Rab 5 GTP - phox activation - ROI production. Rab 5 inhibition CFU: 16 32 1 Rab 5 overexpression CFU (Prada-Delgado et al. , 2001, JBC 276: 19059 -67 ) 12 1 30 9 Rab 5 a. GTP acts before Rac 2 GTP 4

INTRACELLULAR KILLING: Non-oxidative - lysosomal proteins - p. H - anti-microbial peptides - Neutralizing antibodies

INTRACELLULAR KILLING: Non-oxidative 1. - p. H-> Not involved in Listeria degradation - Listeria phagosomes -> p. H 5. 0 2. - Are lysosomal proteases involved in Listeria killing? - activation of lysosomal proteases as Ctsd blocked Listeria growth. Ceramide production: ASMase-/- +/+ -/- . IFN- signalling +/+ (Uttermöhlen et al. , 2003, J. Immunol. 170: 2621 -2628) (Prada-Delgado et al. , 2001, JBC 276: 19059 -67 )

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

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)

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 üPLY-->plasmatic membrane üLLO-->phagosomes -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

INTRACELLULAR KILLING: Non-oxidative

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!