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

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Presentation on theme: "A Ride with Listeria monocytogenes: The Trojan Horse Presented By: Josh Haas Krista Kusinski Shruti Pore Solmaz Shadman Mithaq Vahedi."— Presentation transcript:

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 Background Background Entry Entry Escape With LLO Escape With LLO Escape With PLC Escape With PLC Actin Base Motility Actin Base Motility Treatments Treatments Prevention Prevention (Portnoy et al., 2002)

3 History and Epidemiology First discovered in 1924 in England by E.G.D. Murray, R.A. Webb, and M.B.R. Swann. 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 First reported disease in humans, in Denmark in Outbreak of Listeriosis in California, in 1985, killed 18 people and 30 fetuses. 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. 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) Listeriosis is an bacterial infection caused by a gram-positive bacterium, Listeria monocytogenes (LM) Effects epithelial cells, hepatocytes, and neurons Effects epithelial cells, hepatocytes, and neurons 0px-Listeria.jpg

5 Who Is most susceptible? Pregnant women, and fetus Pregnant women, and fetus 20 times more than healthy people 20 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 people 300 times more at risk than healthy people The elderly 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 The main route of acquisition of Listeria is through the ingestion of contaminated food products Isolated from raw meat, dairy products, vegetables, and seafood Isolated from raw meat, dairy products, vegetables, and seafood Soft cheeses and unpasteurized milk/dairy products should be AVOIDED Soft cheeses and unpasteurized milk/dairy products should be AVOIDED Google images

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

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

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

11 E-Cadherin is the receptor for InlA that 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

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

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

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

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

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

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

18 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 Escape LLO

19 Is LLO more active at a lower pH? Purified Protein injected in cuvette containing erythrocytes Purified Protein injected in cuvette containing erythrocytes Hemolysis measured as decrease in right- angle scatter 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 Escape LLO

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

21 What role does LLO play in cell to cell spread? LLO-negative bacteria incubated with nickel ions LLO-negative bacteria incubated with nickel ions Incubated LLO-negative bacteria with six- His-tagged listeriolysin O (HisLLO). Incubated LLO-negative bacteria with six- His-tagged listeriolysin O (HisLLO). LLO-negative LM Escape LLO

22 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

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

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

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

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

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

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

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

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

31 Without PKC β there is limited escape Escape PLC

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

33 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 The Vb β-strand gives a contact for the glycan linker of GPI-anchored proteins PI-PLC’s ≠ Vb β-strand PI-PLC’s ≠ Vb β-strand Escape PLC

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

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

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

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

38 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 NF- κB I κ-Bβ PLC’s secreted by LM degraded activation Escape PLC

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

40 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. Actin Based Motility

41 Act AArp 2/3 complex Actin-based motility Why is Act A important? Actin Based Motility

42 Act A directly interacts with Arp2/3 complex. 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

43 Deceptive 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). googleimages Actin Based Motility

44 Actin-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

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

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

47 Movement between cells Filopods are the way to go. microbiology/Courses/MicroPath97.705/DOC/Listeria %20summary.doc Actin Based Motility

48 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. Actin Based Motility

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

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

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

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

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

54 (Portnoy et al., 2002)

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

56 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 synthesis Made by Solmaz Wikipedia.com

57 Gentamicin Antibiotic that can treat many different types of bacterial infections Antibiotic that can treat many different types of bacterial infections It prevents or reduces irritation and swelling. It prevents or reduces irritation and swelling. How it works… It inhibits metabolic activities in the bacteria. It binds to a site on the bacterial ribosome, causing the genetic code to be misread. It inhibits metabolic activities in the bacteria. It binds to a site on the bacterial ribosome, causing the genetic code to be misread. Treatments icu8/images/antibiotics/gentamicin.gif&imgrefurl=http://www.bmb.leeds.ac.uk/mbiology/ug/u gteach/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

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

59 Chloramphenicol NOT preferred treatment NOT preferred treatment Serious side effects in humans Serious side effects in humans The WHO uses this treatment in many third world countries The WHO uses this treatment in many third world countries How it works… Stops bacterial growth by inhibiting the enzyme “peptide transferase,”. Wikipedia.com

60 Experimental Treatments via Plants South American traditional medicine South American traditional medicine Used for the treatment of digestive diseases Used for the treatment of digestive diseases Research has demonstrated strong anti- inflammatory and antioxidant activities Research has demonstrated strong anti- inflammatory and antioxidant activities Pluchea quitoc mgrefurl=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%26 sa%3DN Treatments

61 Did Help? Did Pluchea quitoc Help?  Mice Infected with Listeria  Treated with 3 different doses of Pluchea quitoc extracts Treatments Queiroz et al.

62 Did Help? Did Pluchea quitoc Help?  Normal Mice  Treated with the same 3 doses of Pluchea quitoc extracts Treatments Queiroz et al.

63 Investigating the Trojan horse: Future Studies Discover the mechanism in how Pluchea quitoc operates Pluchea quitoc as a possible remedy for humans Treatments

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

65 Protect Yourself Government organizations Government organizations Practice good hygiene Practice good hygiene L. monocytogenes can proliferate at refrigerator temperatures (Re-heat) L. monocytogenes can proliferate at refrigerator temperatures (Re-heat) Prevention clipart Goggle Images

66 Protection in progress Irradiation has been approved as a method of reducing the risk of LM Irradiation has been approved as a method of reducing the risk of LM Not favored by the public. Not favored by the public. Alteration of appearance and taste Alteration of appearance and taste Prevention

67 (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)P 2) Putting it all together!!!

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


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