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Eddie Dominguez Micro 790 March 5 2015

Eddie Dominguez Micro 790 March 5 2015. Today’s adventure entails…. Overview of Entamoeba histolytica Colon epithelial tissue Extracellular Matrix Matrix Metalloproteinase Paper Previous work Techniques. Entamoeba histolytica. Parasite Disease: Amebiasis

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Eddie Dominguez Micro 790 March 5 2015

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  1. Eddie Dominguez Micro 790 March 5 2015

  2. Today’s adventure entails… • Overview of Entamoebahistolytica • Colon epithelial tissue • Extracellular Matrix • Matrix Metalloproteinase • Paper • Previous work • Techniques

  3. Entamoebahistolytica • Parasite • Disease:Amebiasis • Key Source: contaminated food/drinking water • Deaths/year: 70,001 worldwide • Incdence in U.S.: 1,800/year, mainly travelers/immigrants • Symptoms: diarrhea, dysentery, amebic colitis, liver abscesses

  4. Atlas of Human Infectious Diseases

  5. E. histolyticaLife Cycle Ingestion of cysts Excystation in small intestine Division of quadrinucleate cyst into 4 and then 8 trophozoites Trophozoites move to colonize the colon Excystation Excretion of cysts Wikipedia Ingestion of cysts by the patient

  6. E. histolyticaLife Cycle Ingestion of cysts Excystation in small intestine Division of quadrinucleate cyst into 4 and then 8 trophozoites Trophozoites move to colonize the colon Excystation Excretion of cysts Wikipedia Ingestion of cysts by the patient

  7. Colon Tissue iupui.edu UNSW Embryology Glycoforum. 1999

  8. Extracellular Matrix Frantz et al. J Cell Sci2010

  9. Matrix Metalloproteinase (MMP) Tan and Liu. American Journal of Physiology – Renal Physiology 2012

  10. MMPs IL-1β Wikipedia

  11. Previous findings include… • Well documented that cysteine proteases are a requirement in intestinal barrier disruption (Que and Reed, 2000; Moncadaet al. 2006) • 86 genes encoding putative proteases are present in the E. histolyticagenome (Tillacket al., 2007) • Trophozoites silenced for CP-A5 failed to trigger a host inflammatory response or invade the colonic lamina propria (Banselet al., 2009) • Pro-mature CP-A5 binds colonocyte and triggers cytokine secretion (Houet al., 2010) • MMPs-1 and -3 were overexpressed in patients with intestinal amoebiasis (Peterson et al., 2011)

  12. Cysteine protease A5 (CP-A5) • Secreted protease by the trophozoite • Cleaves the major mucus component mucin 2 • Contains a Arg-Gly-Asp (RGD) motif that can bind directly to αvβ3integrin on colonocytes (colon epithelial cells) and triggers a NFκB-mediated pro-inflammatory response (TNF-α and IL-1β) Thibeaux et al. Cellular Microbiology 2012

  13. Thibeaux et al. Cellular Microbiology 2012

  14. Techniques • Western Blot • qRT-PCR • Immunofluorescence and confocal microscopy • Multiphoton (MPM) and second-harmonic generation imaging microscopy (SHIM) • Collagen degradation assays

  15. Second-Harmonic Generation Imaging Microscopy Scientific Volume Imaging

  16. Big Question Are human MMPs involved in the destruction of the ECM of the lamina propria during intestinal invasion by E. histolytica?

  17. Does Human MMP play a role in E. histolyticainvasion?

  18. Human MMP enzymatic activity plays a role in E. histolytica invasion • Trophozoites pretreated with marimastat results in tissue invasion • Tissue pretreated with marimastat results in no tissue invasion

  19. Is Human MMP activity responsible for collagen remodeling? Fibrillar collagen: Green Endogenous cells: Red WT trophozoites: Purple

  20. Human MMP activity responsible for collagen remodeling Fibrillar collagen: Green Endogenous cells: Red WT trophozoites: Purple • Trophozoites pretreated with marimastat results in collagen remodelling • Tissue pretreated with marimastat results in no collagen remodelling

  21. Is CP-A5 involved in MMP expression?

  22. CP-A5 is involved in MMP expression

  23. In what tissue is MMP-3 and -1 localized upon amoebic invasion?

  24. MMP-3 and -1 are localized at the epithelial cell layer and lamina propria

  25. Active MMP-3 and -1 is present during colonic invasion

  26. Active MMP-1 is present during colonic invasion

  27. C119 is important for catalytic activity in CP-A5

  28. Who activates MMP-1?

  29. CP-A5 does not activate MMP-1

  30. Who activates MMP-1 and MMP-3?

  31. CP-A5 activates Pro-MMP-3 which in turn activates Pro-MMP-1

  32. WT-CP-A5 restores invasion

  33. Role of CP-A5

  34. Model for E. histolyticatissue invasion MMPs MMP-3 MMP-1 Thibeaux et al. Cellular Microbiology 2012

  35. Summary • E. histolytica utilizes the hosts immune system during intestinal invasion • CP-A5 instigates this process by eliciting the -secretion of pro-inflamatory cytokines (THF-α and IL-1β), which then triggers the synthesis of MMPs • CP-A5 then activates MMP-3 who in turn activates MMP-1 which then degrades the ECM. • Upon degradation of the ECM, trophozoites are able to invade into the lamina propria

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