Listeria monocytogenes phagocytic strategies.

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

• 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
PHAGOCYTIC STRATEGIES

• Stages
• Rabs involved
• Rab5-gt early timing
• Rab4-gt slow recycling
• Rab11-gt fast recycling
• Rab7-gt Le/Lyso transport

4
Rab cycle
Vps9
AQ
AS
Rab5aQ79L (AQ) -gt GTP always active Rab5aS34N
(AS) -gt GDP blocked Vps9 -gt
GDP/GTP exchange factor (GEF)
GAP -gt GTP hydrolysis
5
Listeria PHAGOCYTIC STRATEGY
6
Listeria PHAGOCYTIC STRATEGY
- Blue sepharose-affinity column (binds proteins
with NAD sites)
anti-GAPDH(N) antibody
7
Listeria PHAGOCYTIC STRATEGY
- GAPDH-LM is a Listeria virulence factor able to
inactivate Rab5a -binds to N-terminal -ADP-ribo
sylates Rab5a
..
.. GAPDH-LM 1
MTVKVGINGFGRIGRLAFRRIQNVEGIEVVAINDLTDAKMLAHLLKYDTT
QGRFDGEVEV 60 GAPDH-SP 1 mvvkvgingfgrigrlafrriqn
iegvevtrindltdpnmlahllkydttqgrfdgtvev 60
. . . .... . . . .
. . GAPDH-LM 61 HDGFFKVNGKEVKVLANRNPE
ELPWGDLGVDIVLECTGFFTAQDKAELHIKA-GAKKVVI
119 GAPDH-SP 61 keggfevngnfikvsaerdpenidwatdgveiv
leatgffakkeaaekhlhtngakkvvi 120
.. ... .
. GAPDH-LM 120 SAPATGDMKTIVYNVNHETLDGT
ETVISGASCTTNCLAPMAKVLEDKFGVVEGLMTTIHA
179 GAPDH-SP 121 tapggndvktvvfntnhdildgtetvisgasct
tnclapmakalhdafgiqkglmttiha 180
. . .. .
. GAPDH-LM 180 YTGDQNTLDAPHPKGDFRRA
RAAAENIIPNTTGAAKAIGEVLPTLKGKLDGAAQRVPVPT
239 GAPDH-SP 181 ytgdqmildgphrggdlrraragaanivpnstg
aakaiglvipelngkldgaaqrvpvpt 240
. ... . .. .
... GAPDH-LM 240 GSLTELVTVLDKKVTVDEVNA
AMEAASDPETFGYTSDQVVSSDIKGMTFGSLFDETQTKV
299 GAPDH-SP 241 gsvtelvvtldknvsvdeinaamkaasn-dsfg
ytedpivssdivgvsygslfdatqtkv 299 .
. GAPDH-LM 300
LTVGDQQLVKTVAWYDNEMSYTAQLVRTLEYFAKIAK
336 GAPDH-SP 300 mevdgsqlvkvvswydnemsytaqlvrtleyfa
kiak 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

• MECHANISMS
• phox (Rab5a/Rac2)
• iNOS

IFN-g
PHOX
11
Oxidative listericidal mechanisms in phagosomes
are regulated by Rab5GTP
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-gt Not involved in Listeria degradation -
Listeria phagosomes -gt 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 27619059-67 )
(Uttermöhlen et al.,2003, J. Immunol. 170
2621-2628)
14
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 6252-265)
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.
1761321-1325 Madrazo-Toca et al. , 2009, Mol
Microbiol 72 668 -682)
16
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--gtplasmatic membrane
• LLO--gtphagosomes

• 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 gtgt or ltlt LLO

17
(No Transcript)
18
(No Transcript)