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A Ride with Listeria monocytogenes : The Trojan Horse

A Ride with Listeria monocytogenes : The Trojan Horse. Presented By: Josh Haas Krista Kusinski Shruti Pore Solmaz Shadman Mithaq Vahedi. (Portnoy et al., 2002). Everything you’d want know about Listeriosis. Background Entry Escape With LLO Escape With PLC Actin Base Motility

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A Ride with Listeria monocytogenes : The Trojan Horse

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

  2. (Portnoy et al., 2002) Everything you’d want know about Listeriosis 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) • Effectsepithelial cells, hepatocytes, and neurons http://encyclopedia.laborlawtalk.com/wiki/images/thumb/6/66/250px-Listeria.jpg

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

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

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

  11. Entry Lecuit et al., 2000 The E-cadherin ectodomain is important in cell adhesion, and cytoplasmic E-cadherin is required for LM entry E-Cadherin is the receptor for InlA that is involved in entry of LM Clip Art Clip Art

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

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

  14. Entry Myosin VIIa is recruited at the site of entry of LM Yes!

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

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

  17. Escape LLO LLO acting on a sheep erythrocyte (bar=100 nanometers) (Vazquez et al., 2001) 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

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

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

  20. Escape LLO LM w/PFO WT LM LLO-LM (Portnoy et al., 1994) 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

  21. 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). LLO-negative LM

  22. 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. Escape LLO Size bar = 0.5μm (Gedde et al., 2000) • 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 (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. Escape PLC PLC’s!After LLO breaks down the front line Google Images PI-PLC PC-PLC LLO

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

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

  30. 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. Escape PLC Without PKC β there is limited escape

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

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

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

  38. 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 degraded activation NF-κB

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

  40. Actin Based Motility Act A protein runs the show. 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. Actin Based Motility Why is Act A important? Act A Arp 2/3 complex Actin-based motility http://medicine.ucsf.edu/labs/brown/jun05/marinum_actin.jpg

  42. “Normal” model of Arp 2/3 complex in a cell Act A – Arp 2/3 complex interaction Actin Based Motility Act A directly interacts with Arp2/3 complex. http://biophysics.jhu.edu/classes/250.106-300-306/2005/documents/EXAMPLE7.PDF

  43. Actin Based Motility Deceptive Role of Act A http://www.blackwell-synergy.com/doi/abs/10.1046/j.1462-5822.2000.00053.x 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. http://mcb.berkeley.edu/labs/welch/Yarar%20et%20al%201999.pdfhttp://mcb.berkeley.edu/labs/welch/Yarar%20et%20al%201999.pdf Actin Based Motility Some support for the theory. http://mcb.berkeley.edu/labs/welch/Yarar%20et%20al%201999.pdf A control group compared with Knock outs and reimplementation of Arp2/3. Arp2/3 complex is key for actin-based motility!!!!!!!

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

  47. Actin Based Motility Filopods are the way to go. Movement between cells http://www.umanitoba.ca/faculties/medicine/medical_microbiology/Courses/MicroPath97.705/DOC/Listeria%20summary.doc

  48. 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. http://www.jbc.org/cgi/reprint/280/12/11379

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

  50. 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

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