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A Ride with A Ride with Listeria monocytogenesListeria monocytogenes: : The Trojan HorseThe Trojan Horse
Presented By:Presented By:Josh HaasJosh Haas
Krista KusinskiKrista KusinskiShruti PoreShruti Pore
Solmaz ShadmanSolmaz ShadmanMithaq VahediMithaq Vahedi
Everything you’d want know Everything you’d want know about Listeriosisabout Listeriosis
BackgroundBackgroundEntryEntryEscape With LLOEscape With LLOEscape With PLCEscape With PLCActin Base MotilityActin Base MotilityTreatmentsTreatmentsPreventionPrevention
(Portnoy et al., 2002)
History and EpidemiologyHistory and Epidemiology
First discovered in 1924 in England by E.G.D. First discovered in 1924 in England by E.G.D. Murray, R.A. Webb, and M.B.R. Swann.Murray, R.A. Webb, and M.B.R. Swann.
First reported disease in humans, in Denmark First reported disease in humans, in Denmark in 1929.in 1929.
Outbreak of Listeriosis in California, in 1985, Outbreak of Listeriosis in California, in 1985, killed 18 people and 30 fetuses.killed 18 people and 30 fetuses.
LM used as a model organism to study LM used as a model organism to study immune response to intracellular pathogens.immune response to intracellular pathogens.
Some FactsSome Facts
ListeriosisListeriosis is an bacterial infection caused is an bacterial infection caused by a gram-positive bacterium, by a gram-positive bacterium, Listeria Listeria monocytogenes (LM)monocytogenes (LM)
EffectsEffects epithelial cells, hepatocytes, and epithelial cells, hepatocytes, and neurons neurons
http://encyclopedia.laborlawtalk.com/wiki/images/thumb/6/66/250px-Listeria.jpg
Who Is most susceptible?Who Is most susceptible?
Pregnant women, and fetus Pregnant women, and fetus 20 times more than healthy people20 times more than healthy people
People with immune deficiencies People with immune deficiencies People with AIDS People with AIDS
300 times more at risk than healthy people300 times more at risk than healthy peopleThe elderlyThe elderly
Listeriosis: the way to the Listeriosis: the way to the porcelain throneporcelain throne
Flu-like symptoms : fever, muscle aches, nausea, and diarrhea
Pregnant women treatment can prevent a spontaneous abortion
fever and chills (stillbirth)
Stiff neck, loss of balance, and seizures can occur if the infection spreads to the CNS
Meningitis
Clip Art
The Workings of the Trojan The Workings of the Trojan HorseHorse
The main route of acquisition of The main route of acquisition of ListeriaListeria is is through the ingestion of contaminated food through the ingestion of contaminated food products products
Isolated from raw meat, dairy products, Isolated from raw meat, dairy products, vegetables, and seafood vegetables, and seafood
Soft cheeses and unpasteurized milk/dairy Soft cheeses and unpasteurized milk/dairy products should be products should be AVOIDEDAVOIDED
Google images
How the Trojan Horse got in: How the Trojan Horse got in: LM ENTRY MECHANISMSLM ENTRY MECHANISMS
Entry
(Portnoy et al., 2002)
InlA
E-cadherin
α & β catenin
Actin
Internalin (InlA) is involved in LM Internalin (InlA) is involved in LM entryentry
Yes, LM entry depends on Internalin (InlA)
Lecuit et al., 1997
Entry
InlA InlAClip Art
Clip Art
E-Cadherin is the receptor for InlA E-Cadherin is the receptor for InlA that is involved in entry of LMthat is involved in entry of LM
Entry
The E-cadherin ectodomain is important in cell adhesion, and cytoplasmic E-cadherin is required for LM entry
Lecuit et al., 2000
Clip Art
Clip Art
αα and and ββ-catenins are important in -catenins are important in LM uptakeLM uptake
Yes, E-cadherin binds β-catenin which binds α-catenin
Lecuit et al., 2000
Entry
Actin is important in LM uptakeActin is important in LM uptake
Actin is recruited during LM entry
Lecuit et al., 2000
Entry
Myosin VIIa is recruited at the site Myosin VIIa is recruited at the site of entry of LMof entry of LM
Yes!
Entry
PUTTING IT ALL TOGETHER…PUTTING IT ALL TOGETHER…
Reproduced with modifications from Sousa et al., 2004
Entry
(Portnoy et al., 2002)
InlA
E-cadherin
α & β caterin
Actin
LLO
PFO
SLO
HISLLO
Entry Escape LLO
Listeriolysin O (LLO)Listeriolysin O (LLO)
Pore-forming toxin, Pore-forming toxin, Coded by Coded by hlyhly gene geneMember of thiol-activated Member of thiol-activated
toxins toxins *Perfringolysin O (PFO), Streptolysin O *Perfringolysin O (PFO), Streptolysin O (SLO), etc.(SLO), etc.
Works with Works with Phospholipases to lyse Phospholipases to lyse vacuolesvacuoles (Vazquez et al., 2001)
LLO acting on a sheep erythrocyte (bar=100 nanometers)
Escape LLO
Is LLO unique in its ability to Is LLO unique in its ability to lyse the phagolysosome?lyse the phagolysosome?
Control bacteria after 3 and 5 hours
B. Subtilis expressing LLO after 3 and 5 hours
B. Subtilis expressing PFO after 3 and 5 hours
Portnoy et al., 1992
Escape LLO
Is LLO more active at a lower Is LLO more active at a lower pH?pH?
Purified Protein injected in cuvette Purified Protein injected in cuvette containing erythrocytescontaining erythrocytes
Hemolysis measured as decrease in right-Hemolysis measured as decrease in right-angle scatterangle scatter
Portnoy et al., 1992
Dashed lines represent activity at pH 5.5, while solid lines represent activity at pH 7.0
Escape LLO
Can PFO confer virulence?Can PFO confer virulence?
Expressed PFO in Expressed PFO in Listeria monocytogenesListeria monocytogenesPFO mediated escape from vacuolePFO mediated escape from vacuoleDisrupted the plasma membraneDisrupted the plasma membrane Infected macrophages killed by influx of Infected macrophages killed by influx of
gentamicingentamicin
(Portnoy et al., 1994)
WT LM LM w/PFO LLO-LM
Escape LLO
What role does LLO play in cell to What role does LLO play in cell to cell spread?cell spread?
LLO-negative bacteria incubated with LLO-negative bacteria incubated with nickel ionsnickel ions
Incubated LLO-negative bacteria with six-Incubated LLO-negative bacteria with six-His-tagged listeriolysin O (HisLLO).His-tagged listeriolysin O (HisLLO).
LLO-negative LM+ +
++++
+ +
++++
Escape LLO
WT and LLO-negative LM spread to secondary cells after 5.5 hours
WT and LLO-negative LM after 8.5 hours
(Gedde et al., 2000)
Escape LLO
LLO-negative LM LLO-negative LM found in double found in double membrane vacuolesmembrane vacuoles
LLO-,PlcA-,PlcB- LLO-,PlcA-,PlcB- LM found in multiple LM found in multiple membrane vacuolesmembrane vacuoles
LLO is necessary for LLO is necessary for intracellular life intracellular life cycle of LMcycle of LM
(Gedde et al., 2000) Size bar = 0.5μm
Escape LLO
(Portnoy et al., 2002)
LLO
PFO
SLO
HISLLO
PKC
Vb β-strand
NF-κB
Escape LLO Escape PLC
LM SoldiersLM Soldiers
LM
PI-PLC
PC-PLC
LLO
Escape PLC
PLC’s!PLC’s!After LLO breaks down the front lineAfter LLO breaks down the front line
PI-PLC PC-PLC
LLO
Escape PLC
Google Images
What does this second What does this second wave attack do?wave attack do?
Help LM escape from the vacuoleHelp LM escape from the vacuoleUses the Pores formed by LLO to enhance Uses the Pores formed by LLO to enhance
this escapethis escapeCleave the membrane lipid PI (PI-PLC)Cleave the membrane lipid PI (PI-PLC)Decrease the affinity of LM for GPI-Decrease the affinity of LM for GPI-
anchored proteinsanchored proteinsActivate NF-Activate NF-κκB geneB gene
Escape PLC
Are PLCs doing the dirty work, or Are PLCs doing the dirty work, or making their troops?making their troops?
Escape PLC
Google Images
PI-PLC and PKC in LM escapePI-PLC and PKC in LM escape
LM
PI-PLC
Cleave PI
Inositol phosphate
Diacylglycerol (DAG)
Protein kinase C (PKC)
?????
Escape PLC
How is PKC related to PLC’s and How is PKC related to PLC’s and escape?escape?
DAG is a product in the cleavage of DAG is a product in the cleavage of membrane lipid PImembrane lipid PI
DAG can activate PKC’sDAG can activate PKC’s
The PKC The PKC ββ was shown to increase the was shown to increase the percent escape LMpercent escape LM
Escape PLC
Without Without PKC PKC ββ there is there is limited escapelimited escape
Escape PLC
PI-PLC helps the LM escape PI-PLC helps the LM escape By using PKCBy using PKC
LM
PI-PLC
Cleave PI
Inositol phosphate
Diacylglycerol (DAG)
Protein kinase C (PKC)
LM escape
Escape PLC
Is less more?Is less more? LM PI-PLC’s lack Vb LM PI-PLC’s lack Vb ββ-strand-strand
The Vb The Vb ββ-strand gives a contact for the -strand gives a contact for the glycan linker of GPI-anchored proteinsglycan linker of GPI-anchored proteins
PI-PLC’s ≠ PI-PLC’s ≠ Vb Vb ββ-strand -strand
Escape PLC
Why this missing LINKer?Why this missing LINKer?
The LM lacking the Vb β-strand showed a significant increase in the percentage of LM escape
Escape PLC
PI-PLC – Vb PI-PLC – Vb ββ-strand = ESCAPE!-strand = ESCAPE!
LM evolved this absence or loss to enhance LM evolved this absence or loss to enhance the growth inside the host cellthe growth inside the host cell
LM
Vb β-Strand
Escape PLC
Are PLCs doing the dirty work, or Are PLCs doing the dirty work, or making their troops?making their troops?
Escape PLC
They are delegating the work!!!Google Images
What else do PLC’s do What else do PLC’s do after escape?after escape?
LM after escape
Now what?
Escape PLC
PLC’s have been seen to regulate PLC’s have been seen to regulate NF- NF- κκB activationB activation
• NF-κB is a transcription factor which is regulated by IκBβ
•When IκBβ is degraded, NF-κB becomes active
•Degradation levels of IκBβ are seen in correlation with expression of PLC’s in cells
exposed to LM infection
NF-κB
Iκ-Bβ
PLC’s secreted by LM
degradedactivation
Escape PLC
(Portnoy et al., 2002)
PKC
Vb β-strand
NF-κB
ActA+Arp 2/3 complex
(PtdIns(4,5)P2)
Escape PLC Actin Based Motility
The Act A Cast
dhttp://olenka.med.virginia.edu/mcsg/images/structures/1ZPVx500r.jpg
-Made of 610 amino acids containing:
• A Charged N-terminal end.
• Proline rich repeats
• A C-terminal end anchoring the protein to the Listeria
Act A protein runs the show.Act A protein runs the show.Actin Based Motility
Act A Arp 2/3 complex
Actin-based motility
Why is Act A important?Why is Act A important?
http://medicine.ucsf.edu/labs/brown/jun05/marinum_actin.jpg
Actin Based Motility
Act A directly interacts with Arp2/3 Act A directly interacts with Arp2/3 complex.complex.
http://biophysics.jhu.edu/classes/250.106-300-306/2005/documents/EXAMPLE7.PDF
Act A – Arp 2/3 complex interaction
“Normal” model of Arp 2/3 complex in a cell
Actin Based Motility
Deceptive Role of Act ADeceptive Role of Act A
-The active WASP family proteins bind to the Arp 2/3 complex.
-Act A protein of Listeria has similar regions with the same abilities (CA and T regions).
http://www.blackwell-synergy.com/doi/abs/10.1046/j.1462-5822.2000.00053.xgoogleimages
Actin Based Motility
Actin-based motilityActin-based motility
Actin molecules are added to the free barbed ends of filaments.
Capping proteins “funnel or channel” the filament formation.
Actin Based Motility
Made By Josh Haas
Some support for the theory.Some support for the theory.
A control group compared with Knock outs and reimplementation of Arp2/3.
http://mcb.berkeley.edu/labs/welch/Yarar%20et%20al%201999.pdf
Arp2/3 complex is key for actin-based motility!!!!!!!
Actin Based Motility
http://mcb.berkeley.edu/labs/welch/Yarar%20et%20al%201999.pdf
Act A
Arp2/3 complex
Actin polymerization
Actin-based Motility
Tie it togetherTie it togetherActin Based Motility
Movement between cellsMovement between cells
Filopods are the way to go.
http://www.umanitoba.ca/faculties/medicine/medical_microbiology/Courses/MicroPath97.705/DOC/Listeria%20summary.doc
Actin Based Motility
Filopod FormationFilopod Formation
-Listeria is propelled into the membrane
-The forces pushes on the cell membrane.
-Filopods form using tightly packed/ un-branched bundles of actin at the leading end of the bacteria.
http://www.jbc.org/cgi/reprint/280/12/11379
Actin Based Motility
Key components.Key components.
Phosphatidylinositol (4,5)-bisphosphate
Phosphatidylinositol (3,4,5)-trisphosphate
googleimages
googleimages
-Both (PtdIns(4,5)P2) and (PtdIns(3,4,5)P3) are found in areas of newly polymerized actin.
Other important moleculesOther important molecules
googleimages
Actin Based Motility
-AKT-PH-GFP inhibits (PtdIns(4,5)P2)
-PLCα-PHGFP inhibits (PtdIns(3,4,5)P3)?
What supports this theory?What supports this theory?
Actin Based Motility
-When the PI-3Kinase was removed full recovery was observed.
-Used LY294002
Recovery of filopod formationRecovery of filopod formation
Actin Based Motility
(Portnoy et al., 2002)
ActA+Arp 2/3 complex
(PtdIns(4,5)P2)
Actin Based Motility
(Portnoy et al., 2002)
How to Treat your How to Treat your ListeriaListeria
AmpicillinAmpicillinGentamicinGentamicinChloramphenicol Chloramphenicol Pluchea quitocPluchea quitoc
Treatments
http://tfp.killbots.com/scans/078_prof-stethoscope.gif
Ampicillin Ampicillin
Preferred agent Preferred agent
Used since the 1960’s Used since the 1960’s
Treatments
How it works… Inhibits the third and final stage of bacterial cell wall
synthesisMade by Solmaz
Wikipedia.com
GentamicinGentamicin
Antibiotic that can treat many different Antibiotic that can treat many different types of bacterial infectionstypes of bacterial infections
It prevents or reduces irritation and It prevents or reduces irritation and swelling. swelling.
How it works…
It inhibits metabolic It inhibits metabolic activities in the activities in the bacteria. It binds to a bacteria. It binds to a site on the bacterial site on the bacterial ribosome, causing the ribosome, causing the genetic code to be genetic code to be misread. misread.
Treatments
http://images.google.com/imgres?imgurl=http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/icu8/images/antibiotics/gentamicin.gif&imgrefurl=http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/icu8/antibiotics/protein.html&h=250&w=260&sz=3&tbnid=pMkb1fZOCbZD5M:&tbnh=102&tbnw=107&hl=en&start=2&prev=/images%3Fq%3DGentamicin%26svnum%3D10%26hl%3Den%26lr%3D%26safe%3Doff%26sa%3DG
Ampicillin + Gentamicin Ampicillin + Gentamicin
Often ampicillin is combined with Often ampicillin is combined with gentamicin for synergy gentamicin for synergy
Interaction of two or more agents or forces Interaction of two or more agents or forces so that their combined effect is greater so that their combined effect is greater than the sum of their individual effects. than the sum of their individual effects.
Treatments
ChloramphenicolChloramphenicol
Treatments
NOT preferred treatmentNOT preferred treatment Serious side effects in humansSerious side effects in humans The WHO uses this treatment in many The WHO uses this treatment in many
third world countriesthird world countries
How it works…Stops bacterial growth by
inhibiting the enzyme “peptide transferase,”.
Wikipedia.com
Experimental Treatments via Experimental Treatments via PlantsPlants
South American traditional medicine South American traditional medicine Used for the treatment of digestive diseasesUsed for the treatment of digestive diseases Research has demonstrated strong anti-Research has demonstrated strong anti-
inflammatory and antioxidant activitiesinflammatory and antioxidant activities
Pluchea quitoc
http://images.google.com/imgres?imgurl=http://www.herbotecnia.com.ar/images/pluchea002.jpg&imgrefurl=http://www.herbotecnia.com.ar/aut-lucera.html&h=178&w=177&sz=13&tbnid=CAR3_qOKF1fIJM:&tbnh=95&tbnw=94&hl=en&start=1&prev=/images%3Fq%3DPluchea%2Bquitoc%2B%26svnum%3D10%26hl%3Den%26lr%3D%26sa%3DN
Treatments
Did Did Pluchea quitoc Help? Help? Mice Infected with Listeria
Treated with 3 different doses of Pluchea quitoc extracts
Treatments
Queiroz et al.
Did Did Pluchea quitoc Help? Help? Normal Mice
Treated with the same 3 doses of Pluchea quitoc extracts
Treatments
Queiroz et al.
Investigating the Trojan horse: Investigating the Trojan horse: Future StudiesFuture Studies
Discover the mechanism in how Pluchea quitoc operates
Pluchea quitoc as a possible remedy for humans
Treatments
Another Trojan horse story: NEW Another Trojan horse story: NEW MECHANISM FOR ENTRY OF LMMECHANISM FOR ENTRY OF LM
IGFIIR is a novel IGFIIR is a novel receptor for LM entryreceptor for LM entry
Works independently or Works independently or in combination with in combination with known mechanismsknown mechanisms
New research avenue!New research avenue!
Entry
Protect YourselfProtect Yourself
Government organizationsGovernment organizations
Practice good hygiene Practice good hygiene
L. monocytogenes L. monocytogenes can proliferate at can proliferate at refrigerator temperatures (Re-heat)refrigerator temperatures (Re-heat)
Prevention
clipart
Goggle Images
Protection in progress Protection in progress
Irradiation has been Irradiation has been approved as a method approved as a method of reducing the risk of of reducing the risk of LMLM
Not favored by the Not favored by the public. public.
Alteration of Alteration of appearance and taste appearance and taste
Prevention
(Portnoy et al., 2002)
InlA
E-cadherin
α & β catenin
Actin
PKC
Vb β-strand
NF-κB
LLO
PFO
SLO
HISLLO
ActA+Arp 2/3 complex
(PtdIns(4,5)P2)
Putting it all together!!!
We are grateful to:We are grateful to:Shubhik K. DebBurman for his support Shubhik K. DebBurman for his support
and guidance throughout this endeavorand guidance throughout this endeavorKatrina Brandis and Crystal Lester for Katrina Brandis and Crystal Lester for
helping us improve our presentationhelping us improve our presentationMichael Zorniak, Michael Wollar, and Michael Zorniak, Michael Wollar, and
Jenny Riddle for valuable advice Jenny Riddle for valuable advice You for bearing with us!You for bearing with us!