advances in understanding the molecular basis of pathogenesis …€¦ · isopol xvi savannah 2007...

Advances in understanding the molecular basisof pathogenesis of Listeria monocytogenes

Isopol XVI

Savannah 2007

Werner Goebel

Biocenter, University of Würzburg

WürzburgIn the old days today

…and some of her historic highlights

How it all began:

H. Seeliger (1921-1997) Professor of Hygiene and Microbiology at the University of Würzburg 1965-1989

Phase I (1980-86)

• J. Hacker C. Hughes M. Gilmore

- Uropathogenic E. coli – first virulencedeterminants (Hly and S- fimbriae genes)

- Chromosomal Pathogenicity (Hly)-Islands- Cytolysins of Gram-positive bacteria

Phase II 1986-1991

Start of Research in Listeria monocytogeneswith T. Chakraborty, S. Kathariou, J. Kreft

- LLO as virulence factor (S.K.)- PrfA and the PrfA-gene cluster (T.C) - ActA and ActA-induced actin polymerization (T.C)

- LLO, ILO, SLO (J.K)

H. Seeliger …..

……..at ISOPOL X, Pecs, Hungary, August 1988

Growth Cycle of L. monocytogenes insideMammalian Cells

Expression of most virulence genes depends on the transcriptional activator PrfA

inlA-inlB..

hpt (uhpT)

Steps 1-5 are determined by a number of chromosomal virulence genes

1

2

3

4

5

12+5 2+5 3+45 5all

according to PortnoyRecently some other genes, involved in virulence, like bsh, bilE, vip, aut havebeen reported as being PrfA-regulated

inlC…

P.Cossart

D.Portnoy, H.Goldfine

J.Wehland, T.Chakraborty

M. Kuhn:- iap/p60 - Host cell responsesR. Böckmann, C. Dickneite, S. Bohne- PrfA-dependent in vitro transcriptionF. Engelbrecht- Small internalins of Lm and Liv (inlC u.a.)A. Bubert/M. Goetz/B. Joseph/J. Slaghuis/J. Schaer- Metabolism and virulenceS. Altrock/Q. Luo/S. Mertins/R. Ecke- PrfA activity and carbohydrate metabolismS. Pilgrim/J. Stritzker/C. Schoen/Loeffler- Lm as carrier for DNA, RNA and protein

Later work:

Links Links betweenbetween metabolismmetabolism and and virulencevirulence-- CarbonCarbon ((C)C)--metabolismmetabolism underunder extraextra-- and and intracellularintracellular conditionsconditions

((PrfAPrfA activityactivity and and CC--metabolismmetabolism))

L. L. monocytogenesmonocytogenes as as carriercarrier systemsystem-- Transfer of Transfer of functionalfunctional DNA and RNA DNA and RNA intointo tumortumor cellscells

-- SpecificSpecific targetingtargeting of of L. L. monocytogenesmonocytogenes to (and to (and intointo) ) tumortumor cellscells

OVERVIEWOVERVIEW

Part I

Links between Metabolism and Virulence

in L. monocytogenes

The prfA mutant is highly virulence – attenuated

2

4

6

EGDe prfA prfA prfA

liver

spleen

5x103 5x103 5x105 5x107 bacteria i.v.

CFU

log

10 a

t day

3 po

st in

fect

ion

pycA pycA pycA

Example 1: A pycA mutant (defective in pyruvate carboxylase) isas avirulent as the prfA mutant

InlA

InlB

75 kD

75 kD

aroA

EG

De

InlA

InlA Protein

Stritzker et al., 2005

10

1

Invasion in Caco-2

aro

A

EG

De

relative rate of internalization

E-4-P + PEParoA

SH-7P

ChorismateMenmenF

Tyr, Phe Trp

Example 2: InlA production and internalization (into Caco-2 cells) of an aroA mutantis highly enhanced

Example 3: PrfA activity – and hence L. monocytogenesvirulence – is intimately linked to the metabolic conditions

-PrfA activity is inhibited in presence of PTS-sugars, like cellobiose, glucose ormannose

-PrfA mutants (PrfA*) were isolated (J.A. Vazquez-Boland and co-workers; N. Freitag and co-workers) that are constitutively active, i. e. PrfA* activity is no longer modulatedin presence of PTS sugars

The carbon (C) - metabolism of L. monocytogenes under extra- and

intracellular conditions was determined by:

- Transcriptome analysis and

- 13C-Glucose Flux Measurement (by NMR or MS) in defined minimal

media (with glucose or glycerol as carbon sources) and in J774

macrophages (in cooperation with the group of A. Bacher and W.

Eisenreich, TU Munich)

Listeria genus genome sequence performedin collaboration between Institut Pasteur and theGerman „Pathogenomics“ network (unpublished)

L. monocytogenes and L. innocua genome sequence(Glaser et al. 2002)

PEP

Glucose-6-P

Pyruvate

Acetyl-CoA

Oxaloacetate Citrate

2- Oxoglutarate

eno

pycA pdh

Glycolysis

Primary metabolism in presence of glucose

+ CO2/ATP

Gluconolactone

- CO2

Xylulose-5-P

Pentose-Phosphate-cycle

Erythrose-4-P

Tyr, Phe, TrpAsp, Thr, Lys, Cys, Met

Glu, Arg, Pro

Ala (Val, IIe, Leu)

Ser, Gly

HPr-His-P

HPr

HPr-Ser-P

hprK/P

CcpA

PrfA – low activity

Citrate cycle

Eisenreich et al. PNAS 2005;

Ecke et al., unpublished

-CO2

pyk

Succinyl-CoA

Active carboncataboliterepressioncontrol (CCR) CCR

IICIIC Enzyme IIEnzyme IIMembraneMembrane

IIAIIAGlcGlcPPPP

6 PEP:PTS 6 PEP:PTS forfor GluGlu8 PEP:PTS 8 PEP:PTS forfor CelCel4 PEP:PTS 4 PEP:PTS forfor ManMan

EIIB

EIIA

GlpF1 (lmo1539)

GlpF2 (lmo1167)

With C3-compounds (glycerol ordihydroxyacetone) as carbonsources Glycerol

Glycerol

G-3-P

Dha

DHAP GAP F-1,6-dP

PEP

Pyruvate

Ac-CoA+ CO2

Oxaloacetate

glpK(lmo1538; lmo1034) dhaK (lmo0347; lmo2695)

gspA (lmo1936)

eno

pyk

pdh

pycACO2/ ATP

glpD (lmo1293)

lmo0344 (?)lmo1737

PrfA – high activity

Joseph et al., unpublished

G-6-P

Dihydroxyacetone(Dha)

Growth in presenceof glycerol leads to (partial) CCR relieve

PrfA*

Hemolytic activity

G - in presence of glucose

C - in presence of cellobiose

Gly - in presence of glycerol

Even when the cellular concentration of PrfA protein is as high as PrfA* protein PrfAactivity is low in presence of glucose and cellobiose but high in presence of glycerol

Gly

Gly

Gly

Gly

InlCActA

LLO

IICIICGlcGlc Enzyme IIEnzyme II GlcGlcMembraneMembrane

IIAIIAGlcGlcPP

GlucoseGlucose--66--PPPP

PEP:PTSPEP:PTS

EIIB

EIIA

GlucoseGlucose

IICIICGlcGlc

IIAIIAGlcGlcPPPP

EIIB

EIIA

GlycerolGlycerol

GlpF

Glycerol

Inactive PrfA Active PrfA

(protein-protein-interactions measured by BIACORE )

Analysis of interactions of purified listerial proteins involvedin PTS/CCR control with PrfA

HPrHPr and and HPrHPr--SerSer--PPdo do notnot interactinteract withwith PrfAPrfA

A A purifiedpurified EIIAEIIAGlcGlc proteinproteininteractsinteracts weaklyweakly withwith PrfAPrfA

10uM CcpA

In vitro transcriptionInfluence of HPr, HPr-SerP and EIIA on PrfA-activity

• No inhibition of PrfA-activity by HPr or HPr-Ser46P

• Weak inhibition of PrfA-activity by EIIA

Phpt- PrfA+HisEIIA/ (32P-UTP)

0%

200%

400%

600%

800%

1000%

1200%

1400%

1600%

0 nM PrfA

8,5 nM

PrfA8,5

nM PrfA

10-4x E

IIA8,5

nM PrfA

10-3x E

IIA8,5

nM PrfA

10-2x E

IIA8,5

nM PrfA

10-1x E

IIA8,5

nM PrfA

1x EIIA

tran

scrip

tion

(% o

f tra

nscr

iptio

n w

ithou

t Prf

A)

In vitro transcription starting at Phpt in presenceof increasing amounts of EIIAGlc

Glycerol orother C3-C source

Glycerol-3-P GAP Dha-P

PEP

Pyruvate


Oxoglutarate

Acetyl-CoA

F1-6DP

Glu-6-P

Gluconolactone-6P

Alanine

Asparate

Glutamate

- CO2+ CO2 ,ATP

PPP

- CO2

DeducedDeduced intracellularintracellular CC--metabolismmetabolism of of L. L. monocytogenesmonocytogenes (in J774 (in J774 macrophagesmacrophages))

glpK glpD

pycA

pyk

pdh

High PrfA activity:

-expression of all PrfA-dependentgenes is induced

Eilert et al. unpublished

Valine

Serine, Glycine

Threonine

GlpF1 (lmo1539)

GlpF2 (lmo1167)

Glycerol

Glycerol

G-3-P

Dha

DHAP GAP Gluc-6-P

PEP

Pyruvate

Ac-CoA+ CO2

Oxaloacetate

glpK(lmo1538; lmo1034) dhaK (lmo0347; lmo2695)

gspA (lmo1936)

eno

pyk

pdh

pycA

glpD (lmo1293)

lmo0344 (?)lmo1737

Joseph et al., unpublished

Glucose-6-P

GAP DhaPGly-3-P

Dihydroxyacetone

Host cell -Glycolysis

A mutant unable to perform C3 metabolism, i.e. deficient in- the two glpK (glycerol-kinase) genes, - glpD (glycerol-3-P dehydrogenase), and-the two dhaK genes

show a strongly reduced intracellular replication efficiencyin macrophage- and epithelial cells under the in vitro culture conditions

1 3 5 7 9h pi

105

106

107

Intra

cellu

larb

acte

ria

WT strain

mutant

The pycA mutant is unable to grow intracellularlywt in BHIpycA in BHIprfA in BHI

2 4 6 8 10 14 h

8

9

7

Via

ble

bact

eria

lcou

nts

log1

0

time

pycA in MM+glucose

1 3 5 7 9

105

106

107

Intr

acel

lula

rbac

teria

h pitime of intracellular

replication in Caco-2 cells

TheThe pycApycA mutantmutant isis also also unableunable to to replicatereplicate in Cacoin Caco--2 2 cellscells

pycA mutant

wild-type strain

InlA

InlB

75 kD

75 kD

aroA

EG

De

InlA

Enhanced invasion and InlA production of the aroA mutant

aroA

EG

De

rela

tive

amou

ntof

inlA

trans

crip

t

50

100

InlA Protein inlA-RNA

Stritzker et al., 2005

10

1

Invasion

aroA

EG

De

relative rate of internalization

Specific properties of the Lm aroA mutant

-The aroA mutant is unable to produce aromatic amino acids but also

menaquinone which is the only quinone of Lm (required for aerobic respiration)

- Due to the inability to produce menaquinone the aroA mutant performs, like

a menF mutant, a predominantly anaerobic metabolism,

E-4-P + PEParoA

SH-7P

ChorismateMenaquinonemenF

Tyr, Phe Trp

-Due to the anaerobic metabolism intracellular replication of the aroA mutantis strongly impaired and hence the virulence of this mutant is highlyattenuated (LD50 > 107)

– this mutant is an excellent carrier for the introduction of protein, RNA and DNA into mammalian cells

The anaerobic metabolism leads to

- slight activation of PrfA and to

- strong activation of the translation of inlA(B) mRNA(possibly by a riboswitch mechanism of inlA(B) mRNA underanaerobic conditions)

Conclusion I

-L. monocytogenes can use various PTS carbohydrates as well as glycerol as carbon sources for extracellular growth

-Uptake of PTS carbohydrates (causing CCR) inhibits PrfA activity(cellobiose>glucose> mannose), whereas that of glycerol (partially relieving CCR) leads to high PrfA activity

- However, the two major components of the CCR control, CcpA and Hpr-Ser-P, are not direct modulators of PrfA activity

-C3 carbon sources seem to play also a major role for intracellular growth of L. monocytogenes (at least in in vitro cell cultures)

-Pyruvate carboxylase (PycA) is an essential enzyme for extra- and intracellularlisterial growth and hence a good metabolic target for the screening of antilisterialdrugs

-An aroA mutant performs anaerobic metabolism, is highly virulence attenuated butshows increased InlA-mediated invasiveness due to increased production of InlA(riboswitch of inlA(B) mRNA under anerobic conditions ?)

Part IIPart II

ListeriaListeria monocytogenesmonocytogenes as as carriercarrier forfor releaserelease of of functionalfunctionalDNADNA--, RNA, RNA-- and and proteinprotein intointo mammalianmammalian cellscells

Synthesis and Export of Proteins

via Signal -sequences

Secretion of theprotein

Anchoring of theprotein on the surfaceof the carrier bacteria

Lm as bacterial carrier withthe gene ( ) for an antigen

Three approaches for delivering protein or protein-encoding DNA orRNA into mammalian cells via L. monocytogenes:

a. By production and secretion of a protein-antigen by the carrier bacteria afteruptake by APC (DC and MP)

b. By delivery of DNA und RNA, encoding such antigens oder other functionalproteins into the cytosol of APC and other host cells which express the geneticinformation

Phage lysin-mediated

autolysis of thebacterial carrier

Plasmid-DNA

mRNA (with 5´-IRES-element)

DNA mRNA

Host cell

Host cell

Delivery of DNA and mRNA encoding EGFP by autolysing Listeriamonocytogenes works equally well in epithelial cells (with bactofectionrates up to 10%)

0,0

2,0

4,0

6,0

8,0

10,0

12,0

4 h

control (MOI10)

mRNA (MOI10)DNA (MOI10)

control (MOI100)mRNA (MOI100)

EG

FP-e

xpre

ssin

g ce

lls [%

]

t p.i.

DNA or mRNA

Pilgrim et al. 2003;

Schoen et al., 2005

nucleus

ProDrug-convertingenzyme is produced

Drug

tumor cell

Enzyme-encoding DNA

ProDrugtumor cell

Drug

Delivery of enzymes for conversion of non-toxic prodrugs into celltoxic drugs by using autolysing aro strains

Gene directed enzyme prodrug therapy (GDEPT)

autolysing Lm

Used Prodrug-Drug-Systems

• Conversion of 6-Methylpurine deoxyribose (MePdR) into the cell toxic drug 6-Methylpurine

(MeP) by PNP

MePdR MePPNP

6-methylpurine deoxyribose

methyl purine

deoD : encodes E. coli purine nucleoside phosphorylase (PNP)

ProDrug Drug

• Conversion of 5-Fluorocytosine (5-FC) into cell toxic drug 5-Fluorouridine- monophosphate (5-FUMP)

by CDA/UPT the gene product of fused gene fcuI

5-FC 5-FU 5-FUMPCDA UPT

5-Fluorocytosine 5-Fluorouracil 5-Fluorouridine-monophosphat

fcuI: gene fusion of yeast encoding cytosine deaminase (CDA) and uracilphosphoribosyltransferase (UPT)

ProDrug Drug Drug

Melanoma B16 cells are killed upon L. monocytogenes – mediated transfer of genes encoding prodrug-drug converting enzymes after treatment with the prodrugs

B16 melanoma cells treated with:( with and without DNA)

Prodrug MePdR(50mM)

Prodrug 5-FC (100 mM)

0

50

100

0 2 4 6

0

50

100

0 2 4 6

Perc

ento

f sur

vivi

ngB

16 c

ells

(trea

ted

/ unt

reat

ed)

Infected with Lm releasing DNA forPNP or CDA

Infected with Lm without DNA

days after treatment with prodrug

Luciferase-expressing Listeria monocytoenes as carriersof cell-toxic compounds into tumor tissue

day 1 (1 h) day 2

day 4 day 6

(Yu et al. (2004) Nat Biotechnol 22: 313)

http://www.biozentrum.uni-wuerzburg.de/

Tag 14 Tag 14

(Yu et al. (2004) Nat Biotechnol 22: 313)

Infected with Lm withoutDNA for Prodrug-Drugconverting Enzyme

Infected with Lm withDNA for Prodrug-Drugconverting enzyme

Rel

. enz

yme

activ

ityaf

ter1

4 da

ys

ca 105 bacteriawithin the tumor

30

60

TargetingTargeting of of L. L. monocytogenesmonocytogenes to to specificspecific tumortumor cellscells

InternalizationInternalization of of L. L. monocytogenesmonocytogenes intointo mammalianmammaliancellscells occursoccurs byby a a zipperzipper mechanismmechanism

MammalianMammalian cellcell withwith receptorreceptor R (R (EE--cadherincadherin oror Met )Met )

1.1. L. L. monocytogenesmonocytogenes isisequippedequipped withwith a a receptorreceptor--specificspecific ligandligand L (L (InlAInlA oror InlBInlB))

LL

RR

TargetingTargeting of of L. L. monocytogenesmonocytogenes to to specificspecific cellscells

MammalianMammalian cellcell withwith specificspecific receptorreceptor RR

2. L. 2. L. monocytogenesmonocytogenes equippedequippedwithwith receptorreceptor--specificspecific ligandligand L L bindsbinds to to receptorreceptor RR

RR

LL

TargetingTargeting of of L. L. monocytogenesmonocytogenes to to specificspecific cellscells

MammalianMammalian cellcell withwith specificspecific receptorreceptor RR

3. L. 3. L. monocytogenesmonocytogenes isis takentaken up up byby receptorreceptor –– ligandligand interactioninteraction

RR

LL

TargetingTargeting of of L. L. monocytogenesmonocytogenes to to specificspecific tumortumor cellcell

Tumor Tumor cellcell withwith overover--expressedexpressed specificspecificreceptorreceptor, , e.ge.g Her2/neuHer2/neu

1.1. L. L. monocytogenesmonocytogenes equippedequippedwithwith a a receptorreceptor--specificspecific ligandligand L L ((antibodyantibody againstagainst tumortumorreceptorreceptor))

RR

LL



2. L. 2. L. monocytogenesmonocytogenes equippedequippedwithwith antibodyantibody againstagainst tumortumorreceptorreceptor –– specificspecific recognitonrecogniton ??

RRLL



3.3. L. L. monocytogenesmonocytogenes equippedequippedantibodyantibody againstagainst tumortumor receptorreceptor-- specificspecific internalizationinternalization ????

RR

LL

Expression of Expression of S. S. aureusaureus proteinprotein A (SPA) on A (SPA) on thethe surfacesurface of of L. L. monocytogenesmonocytogenes ((LmLm--spaspa+) and +) and bindingbinding of of FITSFITS--labelledlabelled antibodiesantibodies to the to the listeriallisterial surfacesurface

S I S IS I S I

Specific invasion of Lm inlAB, spa+, loaded with Herceptin® (monoclonalantibody against Her2/neu), into Her2/neu - overexpressing cancer cells

SKBR-3 cells (Her2/neu +++) Caco-2 cells (Her2/neu -) HepG-2 (Her2/neu +)

50

100

Rel

ativ

e in

vasi

onra

te

EGDe

wt

EGDe

wt

EGDe

wt

EGDe

+ He

rcep

tin

EGDe

+ He

rcep

tin

EGDe

+ He

rcep

tin

inlAB

,spa+

inlA

B.sp

a+

inlAB

, spa

+in

lAB,

spa+

+Her

cept

in

inlAB

,spa+

Herc

eptin

inlA

B, s

pa+

+ He

rcep

tin

ThisThis newnew typetype of of antibodyantibody//receptorreceptor--mediatedmediated internalizationinternalization of of ListeriaListeriamonocytogenesmonocytogenes intointo tumortumor cellscells isis notnot observedobserved withwith unspecificunspecific antibodiesantibodies

1.01.0

2.02.0

0.10.1

HerceptinHerceptin®®/HER/HER--2/neu2/neu--mediated invasion of mediated invasion of LmLm--spaspa++ isisfollowedfollowed byby escapeescape of of LmLm--spaspa++ intointo thethe cytosolcytosol of of thethe tumortumorcellscells and and efficientefficient replicationreplication

a. Lm-PactA-gfp ((WTWT control)control)

c. Lm aroA,inlA/B-PactA-gfp

d. Lm aroA,inlA/B,spa+-PactA-gfp

withwith HerceptinHerceptin®®


withoutwithout

withoutwithout



b. Lm b. Lm inlA/B, spa+--PactAPactA--gfpgfpwithoutwithout

withoutwithoutSK-BR-3 cells

Conclusions II

- Efficient transfer of DNA and RNA encoding functional proteins intomammalian cells including primary phagocytes („bactofection“), can beachieved by the virulence- attenuated aroA L. monocytogenes mutant

- Release of DNA and RNA is greatly enhanced by phage lysin-mediatedintracellular destruction of the carrier bacteria

- RNA delivery results in a considerably earlier expression of the geneproduct in the bactofected cells (including APC) than DNA delivery and leads to a more efficient cellular immune response (mainly CTL)

- Specific targeting of L. monocytogenes to tumor cells can be achievedby receptor- antibody interaction and may improve the specific delivery of anti-cancerogenic agents into tumor cells

Who did the work:

Susanne Bauer Regine EckeAlexa Frentzen Monika Goetz Biju JosephKarin KnuthSonja MertinsSteffi Müller-Altrock Daniela LoefflerSabine PilgrimJenny SchaerChristoph SchoenJörg SlaghuisJochen Stritzker

Collaborations

Institut für Lebensmittelchemie, Würzburg:

Marcus Taupp

Peter Schreier

Institut für Medizinische Strahlenkunde und Zellforschung, Würzburg:

Joachim Fensterle

Ivaylo Gentschev

Ulf R. Rapp

Lehrstuhl für Physiologische Chemie II, Würzburg:

Ernst Conzelmann

Virchow Center, Würzburg

A.Szalay

Institut für Biochemie TU München

Adalbert Bacher/Wolfgang Eisenreich

Department of VeterinarianMedicine, University of Bristol

J.A. Vazquez-Boland

Thank you all !

……all the best for your research

on Listeria monocytogenes !

and

Collaborations

Institut für Lebensmittelchemie, Würzburg:

Marcus Taupp

Peter Schreier

Institut für Medizinische Strahlenkunde und Zellforschung, Würzburg:

Joachim Fensterle

Ivaylo Gentschev

Ulf R. Rapp

Lehrstuhl für Physiologische Chemie II, Würzburg:

Ernst Conzelmann

Virchow Center, Würzburg

A.Szalay

Institut für Biochemie TU München

Adalbert Bacher/Wolfgang Eisenreich

Department of VeterinarianMedicine, University of Bristol

J.A. Vazquez-Boland

PEP

Glucose-6-P

PTS-Gluc/Man

(Lmo0096-98;

lmo0781)

Other PTS

Pyruvate

Acetyl-CoA


2- Oxoglutarate

eno

pycA pdh

Glycolysis

+ CO2

Glucose-6 Phpt

Gluconolactone

- CO2

Xylulose-5-P

Pentose-Phospate-Cycle

Erythrose-4-P

Heptulose-7-P

Tyr, Phe, Trp

Asp, Thr, Lys, Cys, Met

Glu, Arg, Pro

Ala (Val, IIe, Leu)

Ser, Gly

HPr-His-P

HPr

HPr-Ser-P

hprK

CcpA

CCR

PrfA – low activity

Citrate cycle

Eisenreich et al. PNAS 2005;

Ecke et al., unpublished

-CO2

pyk

Succinyl-CoA

PrfA – high activity

P

P

Glucose-6-phosphate

Oral infection, i.v. or i.p.injection intothe vaccined organism

Transformation intothe carrier strain L.

monocytogenes ΔtrpS

origin of replication forL. monocytogenes

origin of replication forE. coli

cDNA of the antigen

polyadenylation signal

eukaryotic promoter

R

PlasmidpSP18

gene for phagelysin (ply118) under PactAcontrol

gene for trpS

DNA Delivery Plasmid pSP118

Delivery of plasmid DNA encoding EGFP to different cell lines – DNA expressed by the host cell after being released from the lysed bacteria

aroA Listeria monocytogenes autolysing aroA Listeria monocytogenes

<1% >7 %

HeLa cells

Problem: EGFP expression is slow and occurs only 24 – 48 post bactofection

Human DC

<1%

http://www.biozentrum.uni-wuerzburg.de/

ComponentsComponents of of listeriallisterial CCR CCR thatthat areare apparentlyapparently notnot involvedinvolved in in modulationmodulation of of PrfAPrfA activityactivity

crecre

GlucoseGlucose


IIBIIBGlcGlc

IIAIIAGlcGlcPP


CcpACcpA

S46S46--PP

HPrHPr HPr

HPr

FructoseFructose--1,61,6--PP22

PEP:PTSPEP:PTS

GlycolysisGlycolysis

S46S46--PP

HPrHPr--KinaseKinase

++

HPrHPr

H15H15--PP

HPrHPr

HPrHPr

S46S46--PP

EIEI

EIEI

PPPyruvatPyruvat

PEPEPP

ATATPP

ADPADP

CcpACcpA

HPrHPr

S46S46--PP

++

PPPPii

PPii

InvolvementInvolvement of of HprHpr--HisHis--PP

crecre

PTSPTSGlcGlc and and otherother PTSPTS


IIBIIBGlcGlc

IIAIIAGlcGlcPP


CcpACcpA

S46S46--PP

HPrHPr HPr

HPr


PEP:PTSPEP:PTS


S46S46--PP


++

HPrHPr

H15H15--PP

HPrHPr

HPrHPr

S46S46--PP

EIEI

EIEI

PPPyruvatPyruvat

PEPEPP

ATATPP

ADPADP

CcpACcpA

HPrHPr

S46S46--PP

++

PPPPii

PPiiL. monocytogenes has a large number of PTS genes (for 29 complete PTS and 8 incomplete ones): among those at least 6 are involved in glucose transport and at least 8 in cellobiosetransport

Hpr-His-PPRD-Regulators-P Glycerol-kinase-P

Dha-PDhaK-P

PEP

PrfA

Glucose and Glucose and otherother PTS PTS sugarssugars


IIBIIBGlcGlc

IIAIIAGlcGlcPPPP

CcpACcpA

S46S46--PP

HPrHPr HPr

HPr


PEP:PTSPEP:PTS


S46S46--PP


++

HPrHPr

H15H15--PP

HPrHPr

HPrHPr

S46S46--PP

EIEI

EIEI

PPPyruvatPyruvat

PEPEPP

ATATPP

ADPADP

CcpACcpA

HPrHPr

S46S46--PP

++

PPPPii

PPii

HPr-His-PPRD-Regulators-P Glycerol-kinase-P

Dha-PDha-K-P

HPr-His-P-meditated phosphorylation state and PrfA activity

in presence of cellobiose or glucose (PTS sugars)

PEP

PrfA

cellobiose, glucose, other PTS-sugars(under aerobic conditions)

different levels of Hpr-His-Pactive CCR, different levels of HPr-Ser-P

Low PrfA activity

P PTS - sugar

G G G G C C C C M M M M G G HPr HPr

HPr

HPr

HPr-His-PHPr-Ser-P

Ser-P

glucose cellobiose mannose

Active CCR leads to repression of:

P ?

ManyMany PTS PTS expressedexpressed

IICIIC Enzyme IIEnzyme IIMembraneMembrane

IIBIIBIIAIIA

PPPP

CcpACcpA

S46S46--PP

HPrHPr HPr

HPr


PEP:PTSPEP:PTS

S46S46--PP


++

HPrHPr

H15H15--PP

HPrHPr

HPrHPr

S46S46--PP

EIEI

EIEI

PPPyruvatPyruvat

PEPEPP

ATATPP

ADPADP

CcpACcpA

HPrHPr

S46S46--PP

++

PPPPii

PPii

HPr-His-PPRD-Regulators-P Glycerol-kinase-P

Dha-PDhaK-P

HPr-His-P-meditated phosphorylation state and PrfA activity in presence of glycerol

PEP

PrfA

High PrfA activity

Relieved CCR, low HPr-Ser-P

Relieved CCR resultsin expression of :

G G G G C C C C Y Y Y YOD : 0.6 1.0 0.6 1.0 0.6 1.0600nm

incubation (70°C): - + - + - + - + - + - +

HPr

WT WT WT

HPr

HPr-His-PHPr-Ser-P

HPr-Ser-His-P

G = glucose C = cellobiose Y = glycerol

glycerolHigh HPr-His-P

P ?

Passage of introduced DNA through the nucleus is slow: expression of antigen takes 24-48 h – during this time apoptosis of APC occurs

autolysing Lm

nucleus

antigen-presenting cell

antigen

CD8 T cell

CMI

MHC I

ER

Phage lysin(Ply118)-mediated

autolysisPlasmid-DNA

antigen-encoding DNA

apoptosisapoptosis

Solution:Delivery of mRNA by autolysing Listeria monocytogenes circumventspassage through the host cells nucleus

autolysing Lm

nucleus

antigen-presenting cell

antigen

CD8 T cell

CMI

MHC I

ER

Phage lysin(Ply118)-mediated

autolysismRNA

Loeffler et al (2006)

CD

44+

H-2

Kb:S

IINFE

KL te

tram

er/

CD

8+T

cells

[%]

41,0

0,0

2,0

4,0

6,0

8,0

10,0

40,0

42,011,3

1,3

OT-I transfer

mRNA-delivery

DNA-delivery

protein-delivery

Vα2+

Vß5+

/ C

D4+

T ce

lls [%

]0,0

0,5

1,0

9,5

10,0

10,5

9,4

0,40,6

OT-II transfer

ComparisonComparison of of DNADNA, , RNARNA and and proteinprotein delivery delivery efficienciesefficiencies in vivoin vivo

(CD8 response) (CD4 response)

L. L. monocytogenesmonocytogenes--mediatedmediated transfertransfer of of antigenantigen--encodingencoding DNA DNA oror RNA RNA resultsresultsmainlymainly in CD8 T in CD8 T cellcell responseresponse (Loeffler et al, 2006)(Loeffler et al, 2006)

advances in understanding the molecular basis of pathogenesis …€¦ · isopol xvi savannah 2007...

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