Eddie Dominguez
Micro 790
March 5 2015
Today’s adventure entails…
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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
Wikipedia
Ingestion of cysts by the patient
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
Wikipedia
Ingestion of cysts by the patient
Colon Tissue
iupui.edu
UNSW Embryology
Glycoforum. 1999
Extracellular Matrix
Frantz et al. J Cell Sci 2010
Matrix Metalloproteinase (MMP)
Tan and Liu. American Journal of Physiology – Renal Physiology 2012
MMPs
IL-1β
Wikipedia
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 αvβ3 integrin on
colonocytes (colon epithelial cells) and
triggers a NFκB-mediated proinflammatory response (TNF-α and IL1β)
Thibeaux et al. Cellular Microbiology 2012
Thibeaux et al. Cellular Microbiology 2012
Techniques
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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
Model for E. histolytica tissue invasion
MMPs
MMP-3
MMP-1
Thibeaux et al. Cellular Microbiology 2012
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