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

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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

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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!