Eddie Dominguez Micro 790 March

Today’s adventure entails… Overview of Entamoeba histolytica Colon epithelial tissue Extracellular Matrix Matrix Metalloproteinase Paper – Previous work – Techniques

Entamoeba histolytica 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

Atlas of Human Infectious Diseases

E. histolytica Life 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 Ingestion of cysts by the patient Wikipedia

E. histolytica Life 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 Ingestion of cysts by the patient Wikipedia

Colon Tissue Glycoforum. 1999UNSW Embryology iupui.edu

Extracellular Matrix Frantz et al. J Cell Sci 2010

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

MMPs Wikipedia IL-1β

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

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 α v β 3 integrin on colonocytes (colon epithelial cells) and triggers a NFκB-mediated pro- inflammatory response (TNF-α and IL- 1β) Thibeaux et al. Cellular Microbiology 2012

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

Second-Harmonic Generation Imaging Microscopy Scientific Volume Imaging

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

Does Human MMP play a role in E. histolytica invasion?

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

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

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

Is CP-A5 involved in MMP expression?

CP-A5 is involved in MMP expression

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

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

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

Active MMP-1 is present during colonic invasion

C119 is important for catalytic activity in CP-A5

Who activates MMP-1?

CP-A5 does not activate MMP-1

Who activates MMP-1 and MMP-3?

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

WT-CP-A5 restores invasion

Role of CP-A5

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

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