Sabrina Jedlicka
In collaboration with:Prof. Kristen Jellison, Prof. Frank Zhang, Dr. Xia Luo,
Tooba Sarkhosh, Elissa Mayerberger, Lu Luo, and Graeme Nichols
Flagellates:• Giardia lamblia• Dientamoeba fragilis• Chilomastix mesnili• Trichomonas hominis• Enteromonas hominis• Retortamonas intestinalisAmeba:• Entamoeba histolytica• Entamoeba dispar• Entamoeba coli• Entamoeba hartmanni• Endolimax nana• Iodamoeba bütschliiApicomplexa:• Cryptosporidium hominis• Cryptosporidium parvum• Cyclospora cayetanensis• Isospora belliOther:• Blastocystis hominis• Balantidium coli
Intestinal Protozoaunicellular eukaryotic organisms
Source: MMWR – Summary of Notifiable Diseases –
U.S., 2011
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2716703/
CryptosporidiumLife Cycle • Infectious form = oocyst
• Sporozoites ‘invade’
intestinal epithelial cells• Merogony
• produce merozoites• Gametogony
• produce micro- and macrogametes
• Sporogony• produce sporozoites• completed on host cell• thin (autoinfection) or
thick walled oocysts
Image Credits: http://www.hominis.mic.vcu.edu/Cparvum_sporozoites.html; http://www.esemag.com/archive/0199/crypto.htm; Current & Blagburn, 1990
Ingested
Exits Host
OocystSporozoite Type I Meront
Merozoite
Type II Meront
MerozoitesMicrogamont
Macrogamont
Zygote
Thin-walledOocysts
Thick-walledOocysts
Auto-infection
Host Enterocytes
SexualCycle
1. Filtration
2. Elution
3. Immunomagnetic Separation (IMS)
4. Immunofluorescent Assay (IFA)
(Helmi et al. 2008; Howe et al. 2002; Rogers & Keevil 1995; Searcy et al. 2006; Wolyniak-DiCesare et al. 2012; Wolyniak et al. 2009; Wolyniak et al. 2010)
Mature biofilm at different shear
Mature biofilm by season
http://www.marvistavet.com/assets/images/single_cryptosporidium_oocyst.gif
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1) Little lehigh River - Light
Neq=217.58 oos/cm2
Kd=0.12hr-1
R2=0.80
# oo
cyst
s/un
it su
rfac
e ar
ea (
oos/
cm2 ) 2) Little lehigh River - Dark
Neq=260.04 oos/cm2
Kd=0.017hr-1
R2=0.80
Time (hr)Time (hr)
# oo
cyst
s/un
it su
rfac
e ar
ea (
oos/
cm2 )
Neq= 214.3 oos/cm2
Kd=0.087hr-1
R2=0.86
3) Monocacy Creek - Light 4) Monocacy Creek - Dark
Neq=232.9 oos/cm2
Kd=0.027hr-1
R2=0.97
Conceptual model of oocyst deposition onto biofilm due to calcium bridging with carboxylic groups.
Magnesium may form an outer sphere surface complex with oocysts and biofilm surfaces to slightly enhance oocyst deposition by charge neutralization.
Cryptosporidium Oocyst Surface Properties
Force Spectroscopy to Measure Binding Forces
Study of oocyst adhesion force to different surface models
(hydroxyl, amine and carboxyl) with atomic force microscopy
(AFM) method.
A powerful tool to measure the mechanical properties of macromolecules, and to investigate the forces and motions
associated with molecular activities at single molecule level
Schematic drawing of AFM operation
Approach
Contact
Retraction
Separation
oocyst
Surfacemodel
AFMprobe
Force curves
Detachment(rupture)force
Approach
Contact
Retraction
Separation
Three sample AFM curves showing theinteraction between oocysts andmodel surfaces
1.Nointeraction
2.Intermediateruptureforce
3.Strongestadhesion
*
0.00E+00
1.00E-09
2.00E-09
3.00E-09
4.00E-09
5.00E-09
6.00E-09
7.00E-09
week1 week2 week3 week4 week5 week6 week7 week8
Force(N)
OH
NH2
COOH
*
*
**
*
*
*
*
*
** *
**
*
#
#
#
#
#
#
#
#
#
#
#
^
^
^^
^
^^
^
0.00E+00
2.00E-10
4.00E-10
6.00E-10
8.00E-10
1.00E-09
1.20E-09
week 1 week 2 week 3 week 4 week 5 week 6 week 7 week 8
Force(N)
OH
NH2
COOH***
##
With Divalent Cations Without Divalent Cations
0.00E+00
2.00E-10
4.00E-10
6.00E-10
8.00E-10
1.00E-09
1.20E-09
week1 week2 week3 week4 week5 week6 week7 week8
Force(N)
without divalent ions
with divalent ions
0.00E+00
1.00E-09
2.00E-09
3.00E-09
4.00E-09
5.00E-09
6.00E-09
7.00E-09
8.00E-09
9.00E-09
week1 week2 week3 week4 week5 week6 week7 week8
Force(N)
without divalent ions
with divalent ions
0.00E+00
5.00E-10
1.00E-09
1.50E-09
2.00E-09
2.50E-09
3.00E-09
week1 week2 week3 week4 week5 week6 week7 week8
Force(N)
without divalent ions
with divalent ions
*
*
**
*
*
*
*
*
*
**
*
*
*
(a)
(b)
(c)
(d)
https://pubs.rsc.org/en/content/articlelanding/2015/py/c4py01415d/unauth#!divAbstract
Hyaluronic Acid, Chondroitin Sulfate, Heparin, Many others