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
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
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)
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