Presentation on theme: "A Ride with Listeria monocytogenes: The Trojan Horse"— Presentation transcript:
1A Ride with Listeria monocytogenes: The Trojan Horse Presented By:Josh HaasKrista KusinskiShruti PoreSolmaz ShadmanMithaq Vahedi
2Everything you’d want know about Listeriosis (Portnoy et al., 2002)BackgroundEntryEscape With LLOEscape With PLCActin Base MotilityTreatmentsPrevention
3History 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.
4Some FactsListeriosis is an bacterial infection caused by a gram-positive bacterium, Listeria monocytogenes (LM)Effects epithelial cells, hepatocytes, and neurons
5Who Is most susceptible? Pregnant women, and fetus20 times more than healthy peoplePeople with immune deficienciesPeople with AIDS300 times more at risk than healthy peopleThe elderly
6Listeriosis: the way to the porcelain throne Flu-like symptoms : fever, muscle aches, nausea, and diarrheaPregnant women treatment can prevent a spontaneous abortionfever and chills (stillbirth)Stiff neck, loss of balance, and seizures can occur if the infection spreads to the CNSMeningitisClip Art
7The Workings of the Trojan Horse The main route of acquisition of Listeria is through the ingestion of contaminated food productsIsolated from raw meat, dairy products, vegetables, and seafoodSoft cheeses and unpasteurized milk/dairy products should be AVOIDEDGoogle images
9(Portnoy et al., 2002)InlAE-cadherinα & β cateninActin
10Internalin (InlA) is involved in LM entry Clip ArtClip ArtLecuit et al., 1997Yes, LM entry depends on Internalin (InlA)
11E-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 entryLecuit et al., 2000Clip ArtClip ArtDescribe E-cadherin---- ecto +endoE- cadherin… is important.. Ecto-adhesion…. Endo---entry
12α and β-catenins are important in LM uptake Entryα and β-catenins are important in LM uptakeYes, E-cadherin binds β-catenin which binds α-cateninLecuit et al., 2000Well established it binds.. Still…
13Actin is important in LM uptake EntryActin is important in LM uptakeLecuit et al., 2000Actin is recruited during LM entry
14Myosin 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!
15PUTTING IT ALL TOGETHER… EntryPUTTING IT ALL TOGETHER…Reproduced with modifications from Sousa et al., 2004
17Listeriolysin O (LLO) Pore-forming toxin, Coded by hly gene Escape LLOListeriolysin O (LLO)Pore-forming toxin,Coded by hly geneMember 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)
18Is LLO unique in its ability to lyse the phagolysosome? Escape LLOIs LLO unique in its ability to lyse the phagolysosome?Control bacteria after 3 and 5 hoursB. Subtilis expressing LLO after 3 and 5 hoursB. Subtilis expressing PFO after 3 and 5 hoursPortnoy et al., 1992
19Is LLO more active at a lower pH? Escape LLOIs LLO more active at a lower pH?Purified Protein injected in cuvette containing erythrocytesHemolysis measured as decrease in right-angle scatterPortnoy et al., 1992Dashed lines represent activity at pH 5.5, while solid lines represent activity at pH 7.0
20Can PFO confer virulence? Escape LLOCan PFO confer virulence?Expressed PFO in Listeria monocytogenesPFO mediated escape from vacuoleDisrupted the plasma membraneInfected macrophages killed by influx of gentamicin(Portnoy et al., 1994)WT LMLM w/PFOLLO-LM
21What role does LLO play in cell to cell spread? Escape LLOWhat role does LLO play in cell to cell spread?LLO-negative bacteria incubated with nickel ionsIncubated LLO-negative bacteria with six-His-tagged listeriolysin O (HisLLO).Mention that this is non covalent bonding+LLO-negative LM
22WT and LLO-negative LM spread to secondary cells after 5.5 hours Escape LLOWT and LLO-negative LM spread to secondary cells after 5.5 hoursWT and LLO-negative LM after 8.5 hours(Gedde et al., 2000)
23LLO-negative LM found in double membrane vacuoles Escape LLO(Gedde et al., 2000)Size bar = 0.5μmLLO-negative LM found in double membrane vacuolesLLO-,PlcA-,PlcB- LM found in multiple membrane vacuolesLLO is necessary for intracellular life cycle of LM
26PLC’s! After LLO breaks down the front line Escape PLCPLC’s! After LLO breaks down the front lineGoogle ImagesPI-PLCPC-PLCLLO
27What does this second wave attack do? Escape PLCWhat does this second wave attack do?Help LM escape from the vacuoleUses the Pores formed by LLO to enhance this escapeCleave the membrane lipid PI (PI-PLC)Decrease the affinity of LM for GPI-anchored proteinsActivate NF-κB gene
28Are PLCs doing the dirty work, or making their troops? Escape PLCAre PLCs doing the dirty work, or making their troops?Google Images
29PI-PLC and PKC in LM escape Escape PLCPI-PLC and PKC in LM escapeLMInositol phosphatePI-PLCCleave PIDiacylglycerol (DAG)Protein kinase C (PKC)?????
30How is PKC related to PLC’s and escape? Escape PLCHow is PKC related to PLC’s and escape?DAG is a product in the cleavage of membrane lipid PIDAG can activate PKC’sThe PKC β was shown to increase the percent escape LM
31Without PKC β there is limited escape Escape PLCWithout PKC β there is limited escapeThe RO AND go 6983 are PCK inhibitors, and the Y axis is percent escape from the phagosome
32PI-PLC helps the LM escape By using PKC Escape PLCPI-PLC helps the LM escape By using PKCLMInositol phosphatePI-PLCCleave PIDiacylglycerol (DAG)Protein kinase C (PKC)LM escape
33Is less more? LM PI-PLC’s lack Vb β-strand Escape PLCIs less more? LM PI-PLC’s lack Vb β-strandThe Vb β-strand gives a contact for the glycan linker of GPI-anchored proteinsPI-PLC’s ≠ Vb β-strand
34Why this missing LINKer? Escape PLCWhy this missing LINKer?The LM lacking the Vb β-strand showed a significant increase in the percentage of LM escape
35PI-PLC – Vb β-strand = ESCAPE! Escape PLCPI-PLC – Vb β-strand = ESCAPE!LM evolved this absence or loss to enhance the growth inside the host cellLMVb β-Strand
36Are PLCs doing the dirty work, or making their troops? Escape PLCAre PLCs doing the dirty work, or making their troops?Google ImagesThey are delegating the work!!!
37What else do PLC’s do after escape? Escape PLCWhat else do PLC’s do after escape?Now what?LM after escape
38PLC’s have been seen to regulate NF- κB activation Escape PLCPLC’s have been seen to regulate NF- κB activationNF-κB is a transcription factor which is regulated by IκBβWhen IκBβ is degraded, NF-κB becomes activeDegradation levels of IκBβ are seen in correlation with expression of PLC’s in cells exposed to LM infectionPLC’s secreted by LMIκ-BβOnce NF-κB is activated LM can use this as a means to go into the nucleous of the host cell and carry out replicationdegradedactivationNF-κB
39Escape PLC Actin Based Motility PKC Vb β-strand NF-κB (Portnoy et al., 2002)PKCVb β-strandNF-κBActA+Arp 2/3 complex(PtdIns(4,5)P2)
40Act A protein runs the show. Actin Based MotilityThe Act A Cast-Made of 610 amino acids containing:A Charged N-terminal end.Proline rich repeatsA C-terminal end anchoring the protein to the Listeriadhttp://olenka.med.virginia.edu/mcsg/images/structures/1ZPVx500r.jpg
41Why is Act A important? Act A Arp 2/3 complex Actin-based motility
42Act A directly interacts with Arp2/3 complex. Actin Based MotilityAct A directly interacts with Arp2/3 complex.“Normal” model of Arp 2/3 complex in a cellAct A – Arp 2/3 complex interaction
43-The active WASP family proteins bind to the Arp 2/3 complex. Deceptive Role of Act AActin Based Motilitygoogleimages-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).
44Actin-based motilityActin Based MotilityMade By Josh HaasActin molecules are added to the free barbed ends of filaments.Capping proteins “funnel or channel” the filament formation.
45Some support for the theory. Actin Based MotilityA control group compared with Knock outs and reimplementation of Arp2/3.Arp2/3 complex is key for actin-based motility!!!!!!!
46Tie it together Actin-based Motility Arp2/3 complex Act A Actin polymerization
47Movement between cells Actin Based MotilityMovement between cellsFilopods are the way to go.
48Filopod Formation -Listeria is propelled into the membrane Actin Based MotilityFilopod 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.
55How to Treat your Listeria TreatmentsHow to Treat your ListeriaAmpicillinGentamicinChloramphenicolPluchea quitoc
56Inhibits the third and final stage of bacterial cell wall synthesis TreatmentsAmpicillinPreferred agentUsed since the 1960’sWikipedia.comwhich ultimately makes the cell wall lysis. Make the cell wall of a bacterium leaky and that eventually kills the bacteriaHow it works…Inhibits the third and final stage of bacterial cell wall synthesisMade by Solmaz
57TreatmentsGentamicinAntibiotic that can treat many different types of bacterial infectionsIt 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…
58Ampicillin + Gentamicin TreatmentsAmpicillin + GentamicinOften ampicillin is combined with gentamicin for synergyInteraction of two or more agents or forces so that their combined effect is greater than the sum of their individual effects.
59Chloramphenicol NOT preferred treatment Serious side effects in humans The WHO uses this treatment in many third world countriesWikipedia.comwhich is and enzyme found in ribosomes that helps form peptide links between adjacent amino acids during translation process of protein biosynthesisStops bacterial growth by inhibiting the enzyme “peptide transferase,”.How it works…
60Experimental Treatments via Plants Pluchea quitocSouth American traditional medicineUsed for the treatment of digestive diseasesResearch has demonstrated strong anti-inflammatory and antioxidant activitiessuch as damage to bone marrow , reserved as a last resort for life-threatening infections, due to its availability and it is a cheaper alternative.
61Did Pluchea quitoc Help? TreatmentsDid 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.
62Did Pluchea quitoc Help? TreatmentsDid Pluchea quitoc Help? Normal Mice Treated with the same 3 doses of Pluchea quitoc extractssame results were seen, and increase in the colonies with all three doses.Queiroz et al.
63Investigating the Trojan horse: Future Studies TreatmentsInvestigating the Trojan horse: Future StudiesDiscover the mechanism in how Pluchea quitoc operatesPluchea quitoc as a possible remedy for humansMechanism for how this extract works is unknown
64Another Trojan horse story: NEW MECHANISM FOR ENTRY OF LM IGFIIR is a novel receptor for LM entryWorks independently or in combination with known mechanismsNew research avenue!
65Protect Yourself Government organizations Practice good hygiene PreventionProtect YourselfGovernment organizationsPractice good hygieneL. monocytogenes can proliferate at refrigerator temperatures (Re-heat)Goggle ImagesMuch 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
66Protection in progress PreventionProtection in progressIrradiation has been approved as a method of reducing the risk of LMNot favored by the public.Alteration of appearance and tasteIf these side effects of irradiation can be overcome then it could ensure the safety of food products to consumers.
67Putting it all together!!! InlAE-cadherinα & β cateninActin(Portnoy et al., 2002)PKCVb β-strandNF-κBLLOPFOSLOHISLLOActA+Arp 2/3 complex(PtdIns(4,5)P2)
68We are grateful to:Shubhik K. DebBurman for his support and guidance throughout this endeavorKatrina Brandis and Crystal Lester for helping us improve our presentationMichael Zorniak, Michael Wollar, and Jenny Riddle for valuable adviceYou for bearing with us!