Transcript Culturing A. castellanii - College of Science

Acanthamoeba castellanii life habits and mechanisms of self preservation
Deborah Moore, Wendy Trzyna, David Neff, Michael Norton, Hongwei D. Yu, and John Barry
Departments of Biochemistry, Chemistry, and Biology at Marshall University and MU School of Medicine (JCESOM)
Huntington, WV
Spring 2016
Introduction: Ongoing work in the Trzyna lab at Marshall U. is focused on
characterizing phenotypic properties and stress response mechanisms of the
protozoa Acanthamoeba castellanii. These eukaryotes of clade Amoebozoa are
a species of gymnamoeba (naked amoeba) that fills niches in diverse aquatic and
soil environments. These organisms are also opportunistic pathogens of humans
and are associated with certain pathogenic and non-pathogenic bacterial species
such as Pseudomonas and E. coli. Of particular interest here are strains of
Pseudomonas aeuroginosa that have been selectively cultured in the Yu lab to be
more or less pathogenic and to express green fluorescent protein (GFP) to allow
fluorescence imaging of the bacteria as they interact with amoebae.
Specifically, we studied the feeding behaviours of A. castellanii, their relationship
with bacteria both as food source and endosymbiont, and the process of
encystment which protects the organism from death during times of food
shortage or environmental stress. These cysts are essentially encapsulated in a
double walled capsule containing cellulose.
During the spring semester of 2016, we used scanning electron microscopy (SEM)
and confocal laser scanning microscopy (CSLM) to study both the free living
metabolically active trophozoite form and dormant cyst form. We imaged fixed
and dehydrated trophozoites and cysts with SEM. Trophozoites were imaged with
CSLM in transmitted light and using fluorescent emissions from GFP. Cysts were
imaged with CSLM after being stained with cellulose specific fluorescent dye
calcofluor white.
e- source
(–) relative to anode
Culturing A. castellanii: To maintain axenic (i.e. without bacteria)
cultures, Acanthamoeba are kept in logarithmic growth to a mid-log
phase of ~2x10^6 cells/mL in PGY (Proteose Glucose Yeast) liquid media
in 125mL Erlenmeyer flasks shaking at 200rpm and set to 30⁰C. These
cultures were sub-cultured every 3 days to maintain healthy logarithmic
cultures. Green fluorescent protein expressing bacteria were also fed to
amoeba in liquid culture to observe feeding behavior and the
morphological details of the amoeba/bacteria symbiotic relationship.
Alternatively, motile ameobae in trophic form were cultured by feeding
them bacteria immobilized on a nutrient-poor agar gel plate (as seen in
figure 4 at right).
To induce encystment, logarithmic cultures of amoebae were washed and
transferred in NEM (Neff Encystment Media) media, which is a special
media used for the sole purpose of inducing encystment. Once set in the
media, cultures were placed in a shaking incubator at 200rpm and which
was set at 30⁰C.
Figure 2. SEM image (right) of a
mature fixed and dehydrated cyst
(glutaraldehyde followed by
ethanol dehydration series).
Image of the cell was captured
using the SEM at 20kV and 5000x.
Scale bar = 5 micrometers.
Figure 4. Migration assay on agar plate with
bacterial lawn. Amoebae migrate up bacterial end of
migration assay
smear (marked with black vertical lines) on
plate seen at right with advancing front of
proliferation seen as a dense band of
amoebae feeing while moving upwards with
less dense populations left behind where food
start
sources are depleted. This front of
proliferation can be seen at higher
magnification in the 3 images directly below.
This assay is being used to determine
virulence of bacterial
species of
interest based
on relative
migration rate
of amoebae.
The particular
strains studied
Arrow represents
24 hours of migration,
here are
~0.8cm
important in the
pathology of
cystic fibrosis.
4.5cm
direction of movement
Figure 5. SEM image (left) of a fixed and
dehydrated trophozoite (glutaraldehyde
followed by ethanol series). Imaged with SEM
at 20kV and 5000x magnification. Panels
below A-C show the interaction of
Acanthamoeba castellanii trophozoites with
Pseudomonas aeruginosa strain PAO1V Δaroa,
while bottom panels D-F show interaction with
a different less virulent strain PAO1V.
Micrographs were taken approx. 45 minutes
after first contact of bacterial cells with
amoeba. Imaging
was done with a
confocal scanning
laser microscope
with blue Argon
laser excitation and
green narrow band
emissions collected.
Scale bar =10um for
all images. Greyscale
transmitted light
images shown for
context. Contractile
vacuole is outlined in
dashed red lines,
and cell nucleus in
white.
24h
anode
relatively (+)
here 20,000V
48h
Figure 1. Top photos show two of the scanning microscopes housed at
Marshall University, left is the Leica SP5 CSLM and right is JEOL5310-LV SEM.
Bottom row shows diagrams of each indicating that the CSLM scans a
focused laser beam to elicit fluorescence which can be selected for imaging
based on wavelength while the SEM scans an electron beam across the
sample to generate electron signal.
MU College of Science
http://www.marshall.edu/cos/
MU Molecular and Biological
Imaging Center
http://www.marshall.edu/mbic/
Joan C. Edwards Scool of Medicine-MU
https://jcesom.marshall.edu/
Figure 3. Top pictures show a maturing cyst in brightfield (left) and
brightfield with calcofluor white fluorescent emission overlay (right)
after 24 hours post NEM. Bottom pictures show a near mature cyst after
48 hours NEM. Long UV (405nm) laser excitation and blue narrow band
emissions collected with Leica SP5 CSLM. Scale bar = 5 micrometers.
At left is a time lapse image
series of a single A.
castellanii trophozoite
feeding on GFP expressing
Peudomonas bacteria in
liquid media over the
course of about 1 hour.
The frame was shifted 2-3
times during this hour to
keep the moving amoeba in
field.
References: Cordingley, J. S., & Trzyna, W. C. (2008). Multiple factors
affecting growth and encystment of Acanthamoeba castellanii in axenic
culture. Acta Protozoologica, 47(4), 307-316.
Cometa, I., Schatz, S., Trzyna, W., Rogerson, A. (2011) Tolerance of naked
amoebae to low oxygen levels with an emphasis on the genus
Acanthamoeba. Acta Protozoologica, 50: 33-40.