Listeria monocytogenes

phagocytic strategies.

Dr. Carmen Alvarez-Dominguez.Immunology Dpt. Hospital Santa Cruz de Liencres-

IFIMAV/IES Zapatón. Cantabria. SPAIN.

Listeria

Phagocyte

Phagosome

Lysosome

R1

R2

IL-12, IL-18

Ag presentation

Y Y T-Naive

Th1

Th2

IFN-

IL-4

Co-stimulatorymolecules

IL-10

**INNATE ADAPTIVE(1.b.)

(3.)

(3.)(2.)

(1.a.)

**INNATE1. Cellular & bacterial proteins

a) Receptorsb) **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, LLOc)

2. Cellular participants: MØs3. Additional participants: cytokines

Bacterium

Rab5

Phagosome

Rab4,11

Rab5??

Rab7

Rab9Rab9

Phago-lysosome

modified-phagosome

GOLGI

ER

Rab6

1

234

5

6

Listeria

SalmonellaMycobacterium

BrucellaLegionella

CoxiellaHelicobacter

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

Rab-GDP

GDI

GDI

GDF Rab-GDP Rab-GTP

GEF

Rab-GTP

Effector

Cytosol

Membrane

GAP

Vps9

AS AQ

Rab5a:Q79L (AQ) -> GTP always activeRab5a:S34N (AS) -> GDP blocked Vps9 -> GDP/GTP exchange factor (GEF) GAP -> GTP hydrolysis

Rab cycle

Listeria PHAGOCYTIC STRATEGY

GAPENDOSOMA

Rab5a-GTP

Listeria PHAGOCYTIC STRATEGY

GAPDH

- Rab5a* affinity column

Lmo 2459Lmo 2459

(GAPDH-LM)

- Blue sepharose-affinity column(binds proteins with NAD sites)

anti-GAPDH(N) antibody

- 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 60GAPDH-SP 1 mvvkvgingfgrigrlafrriqniegvevtrindltdpnmlahllkydttqgrfdgtvev 60

.* * ***. .** *.*.**.. *. **.**** ****. . ** *. . *******GAPDH-LM 61 HDGFFKVNGKEVKVLANRNPEELPWGDLGVDIVLECTGFFTAQDKAELHIKA-GAKKVVI 119GAPDH-SP 61 keggfevngnfikvsaerdpenidwatdgveivleatgffakkeaaekhlhtngakkvvi 120

.**. *.**.*.* **. *********************** * * **. ********GAPDH-LM 120 SAPATGDMKTIVYNVNHETLDGTETVISGASCTTNCLAPMAKVLEDKFGVVEGLMTTIHA 179GAPDH-SP 121 tapggndvktvvfntnhdildgtetvisgascttnclapmakalhdafgiqkglmttiha 180

***** **.** ** *****.* **.**.******** *.* *.**************GAPDH-LM 180 YTGDQNTLDAPHPKGDFRRARAAAENIIPNTTGAAKAIGEVLPTLKGKLDGAAQRVPVPT 239GAPDH-SP 181 ytgdqmildgphrggdlrraragaanivpnstgaakaiglvipelngkldgaaqrvpvpt 240 **.**** ***.*.***.**** ***. ..**** * .***** *...***** *****GAPDH-LM 240 GSLTELVTVLDKKVTVDEVNAAMEAASDPETFGYTSDQVVSSDIKGMTFGSLFDETQTKV 299GAPDH-SP 241 gsvtelvvtldknvsvdeinaamkaasn-dsfgytedpivssdivgvsygslfdatqtkv 299

. * **** *.************************GAPDH-LM 300 LTVGDQQLVKTVAWYDNEMSYTAQLVRTLEYFAKIAK 336GAPDH-SP 300 mevdgsqlvkvvswydnemsytaqlvrtleyfakiak 336

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

Listeria PHAGOCYTIC STRATEGY

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 PhgsGDI cannot remove it due to low abinding- block GDP/GTP exchange by interfering with GEF binding.

INTRACELLULAR KILLING: oxidative

O2-

Active

gp22

rac2

p67p47

gp91gp22

rac2

p67p47

gp91

Rab5-GTP

Inactive

*

MECHANISMS:-phox (Rab5a/Rac2)-iNOS

IFN-

PHOX

Oxidative listericidal mechanisms in phagosomes are regulated by Rab5GTP

- phox activation - ROI production.

CFU: 16 32 1 4

Rab5aGTP acts before Rac2GTP(Prada-Delgado et al., 2001, JBC 276:19059-67 )

Rab5inhibition

Rab5-overexpression

CFU 12 1 30 9

INTRACELLULAR KILLING: Non-oxidative

- lysosomal proteins- pH- anti-microbial peptides- Neutralizing antibodies

-/- +/+ -/- +/+

(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

(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

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

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 membraneLLO-->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

INTRACELLULAR KILLING: Non-oxidative

Bye! 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)