1. A Ride with Listeria monocytogenes: The Trojan Horse
Presented By:
Josh Haas
Krista Kusinski
Shruti Pore
Solmaz Shadman
Mithaq Vahedi

2. Everything you’d want know about Listeriosis
(Portnoy et al., 2002)
Background
Entry
Escape With LLO
Escape With PLC
Actin Base Motility
Treatments
Prevention

3. History and Epidemiology
First discovered in 1924 in England by E.G.D. Murray, R.A. Webb, and M.B.R. Swann.
First reported disease in humans, in Denmark in 1929.
Outbreak of Listeriosis in California, in 1985, killed 18 people and 30 fetuses.
LM used as a model organism to study immune response to intracellular pathogens.

4. Some Facts
Listeriosis is an bacterial infection caused by a gram-positive bacterium, Listeria monocytogenes (LM)
Effects epithelial cells, hepatocytes, and neurons

5. Who Is most susceptible?
Pregnant women, and fetus
20 times more than healthy people
People with immune deficiencies
People with AIDS
300 times more at risk than healthy people
The elderly

6. Listeriosis: the way to the porcelain 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

7. The Workings of the Trojan Horse
The main route of acquisition of Listeria is through the ingestion of contaminated food products
Isolated from raw meat, dairy products, vegetables, and seafood
Soft cheeses and unpasteurized milk/dairy products should be AVOIDED
Google images

8. How the Trojan Horse got in: LM ENTRY MECHANISMS

9. (Portnoy et al., 2002)
InlA
E-cadherin
α & β catenin
Actin

10. Internalin (InlA) is involved in LM entry
Clip Art
Clip Art
Lecuit et al., 1997
Yes, LM entry depends on Internalin (InlA)

11. E-Cadherin is the receptor for InlA that is involved in entry of LM
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
Describe E-cadherin---- ecto +endoE- cadherin… is important.. Ecto-adhesion…. Endo---entry

12. α and β-catenins are important in LM uptake
Entry
α and β-catenins are important in LM uptake
Yes, E-cadherin binds β-catenin which binds α-catenin
Lecuit et al., 2000
Well established it binds.. Still…

13. Actin is important in LM uptake
Entry
Actin is important in LM uptake
Lecuit et al., 2000
Actin is recruited during LM entry

14. Myosin VIIa is recruited at the site of entry of LM
BDM----2,3-Butanedione monoxime (BDM) (Sigma, St Louis, MO) 0.5 M (Cramer and Mitchison, 1995).
Yes!

15. PUTTING IT ALL TOGETHER…
Entry
PUTTING IT ALL TOGETHER…
Reproduced with modifications from Sousa et al., 2004

16. Entry Escape LLO InlA E-cadherin α & β caterin Actin LLO PFO SLO
(Portnoy et al., 2002)
InlA
E-cadherin
α & β caterin
Actin
LLO
PFO
SLO
HISLLO

17. Listeriolysin O (LLO) Pore-forming toxin, Coded by hly gene
Escape LLO
Listeriolysin O (LLO)
Pore-forming toxin,
Coded by hly gene
Member of thiol-activated toxins
*Perfringolysin O (PFO), Streptolysin O (SLO), etc.
Works with Phospholipases to lyse vacuoles
(Vazquez et al., 2001)
LLO acting on a sheep erythrocyte (bar=100 nanometers)

18. Is LLO unique in its ability to lyse the phagolysosome?
Escape LLO
Is LLO unique in its ability to 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

19. Is LLO more active at a lower pH?
Escape LLO
Is LLO more active at a lower pH?
Purified Protein injected in cuvette containing erythrocytes
Hemolysis measured as decrease in right-angle scatter
Portnoy et al., 1992
Dashed lines represent activity at pH 5.5, while solid lines represent activity at pH 7.0

20. Can PFO confer virulence?
Escape LLO
Can PFO confer virulence?
Expressed PFO in Listeria monocytogenes
PFO mediated escape from vacuole
Disrupted the plasma membrane
Infected macrophages killed by influx of gentamicin
(Portnoy et al., 1994)
WT LM
LM w/PFO
LLO-LM

21. What role does LLO play in cell to cell spread?
Escape LLO
What role does LLO play in cell to cell spread?
LLO-negative bacteria incubated with nickel ions
Incubated LLO-negative bacteria with six-His-tagged listeriolysin O (HisLLO).
Mention that this is non covalent bonding +
LLO-negative LM

22. WT and LLO-negative LM spread to secondary cells after 5.5 hours
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)

23. LLO-negative LM found in double membrane vacuoles
Escape LLO
(Gedde et al., 2000)
Size bar = 0.5μm
LLO-negative LM found in double membrane vacuoles
LLO-,PlcA-,PlcB- LM found in multiple membrane vacuoles
LLO is necessary for intracellular life cycle of LM

24. Escape LLO Escape PLC PKC Vb β-strand NF-κB LLO PFO SLO HISLLO
(Portnoy et al., 2002)
PKC
Vb β-strand
NF-κB
LLO
PFO
SLO
HISLLO

25. Escape PLC
LM Soldiers LM
LLO
PI-PLC
PC-PLC

26. PLC’s! After LLO breaks down the front line
Escape PLC
PLC’s! After LLO breaks down the front line
Google Images
PI-PLC
PC-PLC
LLO

27. What does this second wave attack do?
Escape PLC
What does this second wave attack do?
Help LM escape from the vacuole
Uses the Pores formed by LLO to enhance this escape
Cleave the membrane lipid PI (PI-PLC)
Decrease the affinity of LM for GPI-anchored proteins
Activate NF-κB gene

28. Are PLCs doing the dirty work, or making their troops?
Escape PLC
Are PLCs doing the dirty work, or making their troops?
Google Images

29. PI-PLC and PKC in LM escape
Escape PLC
PI-PLC and PKC in LM escape LM
Inositol phosphate
PI-PLC
Cleave PI
Diacylglycerol (DAG)
Protein kinase C (PKC)
?????

30. How is PKC related to PLC’s and escape?
Escape PLC
How is PKC related to PLC’s and escape?
DAG is a product in the cleavage of membrane lipid PI
DAG can activate PKC’s
The PKC β was shown to increase the percent escape LM

31. Without PKC β there is limited escape
Escape PLC
Without PKC β there is limited escape
The RO AND go 6983 are PCK inhibitors, and the Y axis is percent escape from the phagosome

32. PI-PLC helps the LM escape By using PKC
Escape PLC
PI-PLC helps the LM escape By using PKC LM
Inositol phosphate
PI-PLC
Cleave PI
Diacylglycerol (DAG)
Protein kinase C (PKC)
LM escape

33. Is less more? LM PI-PLC’s lack Vb β-strand
Escape PLC
Is less more? LM PI-PLC’s lack Vb β-strand
The Vb β-strand gives a contact for the glycan linker of GPI-anchored proteins
PI-PLC’s ≠ Vb β-strand

34. Why this missing LINKer?
Escape PLC
Why this missing LINKer?
The LM lacking the Vb β-strand showed a significant increase in the percentage of LM escape

35. PI-PLC – Vb β-strand = ESCAPE!
Escape PLC
PI-PLC – Vb β-strand = ESCAPE!
LM evolved this absence or loss to enhance the growth inside the host cell LM
Vb β-Strand

36. Are PLCs doing the dirty work, or making their troops?
Escape PLC
Are PLCs doing the dirty work, or making their troops?
Google Images
They are delegating the work!!!

37. What else do PLC’s do after escape?
Escape PLC
What else do PLC’s do after escape?
Now what?
LM after escape

38. PLC’s have been seen to regulate NF- κB activation
Escape PLC
PLC’s have been seen to regulate NF- κB 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
PLC’s secreted by LM
Iκ-Bβ
Once NF-κB is activated LM can use this as a means to go into the nucleous of the host cell and carry out replication
degraded
activation
NF-κB

39. Escape PLC Actin Based Motility PKC Vb β-strand NF-κB
(Portnoy et al., 2002)
PKC
Vb β-strand
NF-κB
ActA+Arp 2/3 complex
(PtdIns(4,5)P2)

40. Act A protein runs the show.
Actin Based Motility
The Act A Cast
-Made of 610 amino acids containing:
A Charged N-terminal end.
Proline rich repeats
A C-terminal end anchoring the protein to the Listeria
dhttp://olenka.med.virginia.edu/mcsg/images/structures/1ZPVx500r.jpg

41. Why is Act A important? Act A Arp 2/3 complex Actin-based motility

42. Act A directly interacts with Arp2/3 complex.
Actin Based Motility
Act A directly interacts with Arp2/3 complex.
“Normal” model of Arp 2/3 complex in a cell
Act A – Arp 2/3 complex interaction

43. -The active WASP family proteins bind to the Arp 2/3 complex.
Deceptive Role of Act A
Actin Based Motility
googleimages
-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).

44. Actin-based motility
Actin Based Motility
Made By Josh Haas
Actin molecules are added to the free barbed ends of filaments.
Capping proteins “funnel or channel” the filament formation.

45. Some support for the theory.
Actin Based Motility
A control group compared with Knock outs and reimplementation of Arp2/3.
Arp2/3 complex is key for actin-based motility!!!!!!!

46. Tie it together Actin-based Motility Arp2/3 complex Act A
Actin polymerization

47. Movement between cells
Actin Based Motility
Movement between cells
Filopods are the way to go.

48. Filopod Formation -Listeria is propelled into the membrane
Actin Based Motility
Filopod 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.

49. Key components. Phosphatidylinositol (4,5)-bisphosphate
googleimages
Phosphatidylinositol (3,4,5)-trisphosphate
googleimages

50. Other important molecules
Actin Based Motility
Other important molecules
-Both (PtdIns(4,5)P2) and (PtdIns(3,4,5)P3) are found in areas of newly polymerized actin.
googleimages

51. What supports this theory?
Actin Based Motility
What supports this theory?
-AKT-PH-GFP inhibits (PtdIns(4,5)P2)
-PLCα-PHGFP inhibits (PtdIns(3,4,5)P3)?

52. Recovery of filopod formation
Actin Based Motility
Recovery of filopod formation
-When the PI-3Kinase was removed full recovery was observed.
-Used LY294002

53. Actin Based Motility ActA+Arp 2/3 complex (PtdIns(4,5)P2)
(Portnoy et al., 2002)
ActA+Arp 2/3 complex
(PtdIns(4,5)P2)

54. (Portnoy et al., 2002)

55. How to Treat your Listeria
Treatments
How to Treat your Listeria
Ampicillin
Gentamicin
Chloramphenicol
Pluchea quitoc

56. Inhibits the third and final stage of bacterial cell wall synthesis
Treatments
Ampicillin
Preferred agent
Used since the 1960’s
Wikipedia.com
which ultimately makes the cell wall lysis. Make the cell wall of a bacterium leaky and that eventually kills the bacteria
How it works…
Inhibits the third and final stage of bacterial cell wall synthesis
Made by Solmaz

57. Treatments
Gentamicin
Antibiotic that can treat many different types of bacterial infections
It prevents or reduces irritation and swelling.
It inhibits metabolic activities in the bacteria. It binds to a site on the bacterial ribosome, causing the genetic code to be misread.
How it works…

58. Ampicillin + Gentamicin
Treatments
Ampicillin + Gentamicin
Often ampicillin is combined with gentamicin for synergy
Interaction of two or more agents or forces so that their combined effect is greater than the sum of their individual effects.

59. Chloramphenicol NOT preferred treatment Serious side effects in humans
The WHO uses this treatment in many third world countries
Wikipedia.com
which is and enzyme found in ribosomes that helps form peptide links between adjacent amino acids during translation process of protein biosynthesis
Stops bacterial growth by inhibiting the enzyme “peptide transferase,”.
How it works…

60. Experimental Treatments via Plants
Pluchea quitoc
South American traditional medicine
Used for the treatment of digestive diseases
Research has demonstrated strong anti-inflammatory and antioxidant activities
such as damage to bone marrow , reserved as a last resort for life-threatening infections, due to its availability and it is a cheaper alternative.

61. Did Pluchea quitoc Help?
Treatments
Did Pluchea quitoc Help?
 Mice Infected with Listeria
 Treated with 3 different doses of Pluchea quitoc extracts
(with infected mice) The number of CFU-GM (granulocyte-macrophage progenitor cells) in bone marrow of LM was reduced in the initial stage, most of the reduction occurred in the first 48hrs. Then in the 72h recovery was seen, but the number was lower than that of the control mice.
P. quitoc extract has a significant stimulatory effect on the production of leukocytes in the blood of infected mice as well as normal mice. This treatment with this extract may be helpful in host defense against intracellular bacterial infection.
Queiroz et al.

62. Did Pluchea quitoc Help?
Treatments
Did Pluchea quitoc Help?
 Normal Mice
 Treated with the same 3 doses of Pluchea quitoc extracts
same results were seen, and increase in the colonies with all three doses.
Queiroz et al.

63. Investigating the Trojan horse: Future Studies
Treatments
Investigating the Trojan horse: Future Studies
Discover the mechanism in how Pluchea quitoc operates
Pluchea quitoc as a possible remedy for humans
Mechanism for how this extract works is unknown

64. Another Trojan horse story: NEW MECHANISM FOR ENTRY OF LM
IGFIIR is a novel receptor for LM entry
Works independently or in combination with known mechanisms
New research avenue!

65. Protect Yourself Government organizations Practice good hygiene
Prevention
Protect Yourself
Government organizations
Practice good hygiene
L. monocytogenes can proliferate at refrigerator temperatures (Re-heat)
Goggle Images
Much extreme percussion has been taken to reduce the risk a listeria outbreak. Government organizations have created programs to enhance and ensure the safety of food products.
clipart

66. Protection in progress
Prevention
Protection in progress
Irradiation has been approved as a method of reducing the risk of LM
Not favored by the public.
Alteration of appearance and taste
If these side effects of irradiation can be overcome then it could ensure the safety of food products to consumers.

67. Putting it all together!!!
InlA
E-cadherin
α & β catenin
Actin
(Portnoy et al., 2002)
PKC
Vb β-strand
NF-κB
LLO
PFO
SLO
HISLLO
ActA+Arp 2/3 complex
(PtdIns(4,5)P2)

68. We are grateful to:
Shubhik K. DebBurman for his support and guidance throughout this endeavor
Katrina Brandis and Crystal Lester for helping us improve our presentation
Michael Zorniak, Michael Wollar, and Jenny Riddle for valuable advice
You for bearing with us!