The Social Amoeba Polysphondylium pallidum Loses Encystation and Sporulation, but Can Still Erect Fruiting Bodies in the Absence of Cellulose

Abstract

Amoebas and other freely moving protists differentiate into walled cysts when exposed to stress. As cysts, amoeba pathogens are resistant to biocides, preventing treatment and eradication. Lack of gene modification procedures has left the mechanisms of encystation largely unexplored. Genetically tractable Dictyostelium discoideum amoebas require cellulose synthase for formation of multicellular fructifications with cellulose-rich stalk and spore cells. Amoebas of its distant relative Polysphondylium pallidum (Ppal), can additionally encyst individually in response to stress. Ppal has two cellulose synthase genes, DcsA and DcsB, which we deleted individually and in combination. Dcsa- mutants formed fruiting bodies with normal stalks, but their spores and cyst walls lacked cellulose, which obliterated stress-resistance of spores and rendered cysts entirely non-viable. A dcsa-/dcsb- mutant made no walled spores, stalk cells or cysts, although simple fruiting structures were formed with a droplet of amoeboid cells resting on an sheathed column of decaying cells. DcsB is expressed in prestalk and stalk cells, while DcsA is additionally expressed in spores and cysts. We conclude that cellulose is essential for encystation and that cellulose synthase may be a suitable target for drugs to prevent encystation and render amoeba pathogens susceptible to conventional antibiotics.

Full text

Protist,
Vol.
165,
569–579,
September
2014
http://www.elsevier.de/protis
Published
online
date
14
July
2014
ORIGINAL
PAPER
The
Social
Amoeba
Polysphondylium
pallidum
Loses
Encystation
and
Sporulation,
but
Can
Still
Erect
Fruiting
Bodies
in
the
Absence
of
Cellulose
Qingyou
Du,
and
Pauline
Schaap1
College
of
Life
Sciences,
University
of
Dundee,
MSI/WTB/JBC
complex,
Dow
Street,
Dundee,
DD15EH,
UK
Submitted
May
20,
2014;
Accepted
July
8,
2014
Monitoring
Editor:
Michael
Melkonian
Amoebas
and
other
freely
moving
protists
differentiate
into
walled
cysts
when
exposed
to
stress.
As
cysts,
amoeba
pathogens
are
resistant
to
biocides,
preventing
treatment
and
eradication.
Lack
of
gene
modification
procedures
has
left
the
mechanisms
of
encystation
largely
unexplored.
Genetically
tractable
Dictyostelium
discoideum
amoebas
require
cellulose
synthase
for
formation
of
multicellular
fructifications
with
cellulose-rich
stalk
and
spore
cells.
Amoebas
of
its
distant
relative
Polysphondylium
pallidum
(Ppal),
can
additionally
encyst
individually
in
response
to
stress.
Ppal
has
two
cellulose
syn-
thase
genes,
DcsA
and
DcsB,
which
we
deleted
individually
and
in
combination.
Dcsa-
mutants
formed
fruiting
bodies
with
normal
stalks,
but
their
spore
and
cyst
walls
lacked
cellulose,
which
obliterated
stress-resistance
of
spores
and
rendered
cysts
entirely
non-viable.
A
dcsa-/dcsb-
mutant
made
no
walled
spores,
stalk
cells
or
cysts,
although
simple
fruiting
structures
were
formed
with
a
droplet
of
amoeboid
cells
resting
on
an
sheathed
column
of
decaying
cells.
DcsB
is
expressed
in
prestalk
and
stalk
cells,
while
DcsA
is
additionally
expressed
in
spores
and
cysts.
We
conclude
that
cellulose
is
essential
for
encystation
and
that
cellulose
synthase
may
be
a
suitable
target
for
drugs
to
prevent
encystation
and
render
amoeba
pathogens
susceptible
to
conventional
antibiotics.
©
2014
The
Authors.
Published
by
Elsevier
GmbH.
This
is
an
open
access
article
under
the
CC
BY
license
(http://creativecommons.org/licenses/by/3.0/).
Key
words:
Encystation;
Amoebozoa;
Acanthamoeba
keratitis;
cellulose
synthase;
cell
wall
biosynthesis;
Polysphondylium
pallidum.
Introduction
Amoebas
and
many
other
freely
moving
proto-
zoa
differentiate
into
immobile
dormant
cysts
when
exposed
to
nutrient
depletion
or
other
forms
of
envi-
ronmental
stress.
As
cysts,
the
organisms
can
sur-
vive
adverse
conditions
from
months
to
years,
and,
1Corresponding
author;
fax
+44
1382
345386
e-mail
p.schaap@dundee.ac.uk
(P.
Schaap).
in
the
case
of
pathogenic
protozoa,
resist
the
chal-
lenges
of
antibiotic
treatment
and
immune
clear-
ance.
This
resilience
is
due
to
the
fact
that
the
cells
are
metabolically
inactive
and
surrounded
by
an
impermeable
cell
wall.
In
fungi,
the
polysaccharide
chitin
is
the
main
structural
component
of
the
cell
wall
(Free
2013),
but
in
chromalveolate
algae
and
oomycetes,
green
algae,
and
amoebozoa,
such
as
Dictyostelium
discoideum
and
Acanthamoeba
castellani,
the
structural
component
is
cellulose
http://dx.doi.org/10.1016/j.protis.2014.07.003
1434-4610/©
2014
The
Authors.
Published
by
Elsevier
GmbH.
This
is
an
open
access
article
under
the
CC
BY
license
(http://creativecommons.org/licenses/by/3.0/).

570
Q.
Du
and
P.
Schaap
(Blanton
et
al.
2000;
Dudley
et
al.
2009;
Fugelstad
et
al.
2009;
Michel
et
al.
2010;
Roberts
et
al.
2002).
In
the
social
amoeba
Dictyostelium
discoideum
(Ddis),
a
single
cellulose
synthase
gene
is
essential
for
the
construction
of
multicellular
fruiting
bod-
ies,
by
synthesizing
a
cellulose
stalk
tube
and
the
cellulose-rich
walls
of
individual
stalk
cells
and
spores
(Blanton
et
al.
2000).
Many
Dictyostelium
species,
such
as
the
genetic
model
Polysphon-
dylium
pallidum
(Ppal),
can
alternatively
encyst
as
single
cells.
Ppal
also
constructs
architecturally
more
complex
fruiting
structures
than
D.discoideum
with
multiple
regular
whorls
of
side
branches.
For
synthesis
of
the
stalk
tube,
cellulose
microfib-
rils
are
deposited
at
the
exterior
face
of
the
plasma
membrane
of
prestalk
cells
by
single
linear
arrays
of
membrane-spanning
cellulose
synthases.
While
prestalk
cells
are
maturing
into
stalk
cells,
the
long
linear
arrays
rearrange
into
multiple
parallel
rows
for
synthesis
of
the
thicker
fibrils
of
the
stalk
cell
wall
(Grimson
et
al.
1996).
The
spore
wall
consists
of
a
cellulose
layer
sandwiched
between
two
protein-
rich
layers.
Spore
coat
proteins
are
presynthesized
in
Golgi-derived
vesicles,
which
synchronously
fuse
with
the
plasma
membrane
at
the
onset
of
spore
maturation.
Cellulose
deposition
occurs
somewhat
later,
starting
at
one
pole
of
the
spore
and
travel-
ling
towards
the
other
pole.
The
spore
wall
cellulose
is
essential
for
proper
deposition
of
the
two
pro-
teinaceous
layers
of
the
spore
coat
(Zhang
et
al.
2001).
Cellulose
also
makes
up
28%
of
the
Ppal
cyst
wall
(Toama
and
Raper
1967),
but
cellulose
synthases
do
not
appear
to
form
linear
arrays
in
the
plasmamembrane
of
encysting
cells
(Erdos
and
Hohl
1980).
Acanthamoeba
castellani
is
an
opportunistic
pathogen
that
causes
vision-destroying
kerati-
tis
and
lethal
encephalitis,
with
cysts
preventing
effective
treatment
(Siddiqui
et
al.
2013).
Cell
wall
biosynthesis
is
a
major
target
for
bacte-
rial
and
fungal
antibiotics
and
herbicides
(Bush
2012;
McCormack
and
Perry
2005;
Wakabayashi
and
Böger
2002).
Acanthamoeba
encystation
was
shown
to
be
reduced
by
85%
by
0.48
mM
of
the
herbicide
2,6-dichlorobenzonitrile,
which
inhibits
plant
cellulose
synthesis
(Dudley
et
al.
2007),
and
to
50%
by
incubation
with
small
interfer-
ing
RNAs
against
the
Acanthamoeba
cellulose
synthase
(Aqeel
et
al.
2013).
Although
not
fully
penetrant,
these
treatments
show
the
potential
importance
of
cellulose
synthase
for
amoebozoan
encystation.
No
gene
knock-out
strategies
are
as
yet
available
for
Amoebozoa
outside
Dictyostelia.
The
encysting
dictyostelid
Ppal
therefore
offers
unique
opportunities
to
identify
and
assess
crucial
roles
of
cellulose
synthase
genes
in
encystation.
The
differentiation
of
spores,
stalk
cells
and
cysts
in
Dictyostelia
as
well
as
encystation
in
Acan-
thamoeba
all
require
cyclic
AMP
acting
on
PKA
(Du
et
al.
2014;
Kawabe
et
al.
2009;
Reymond
et
al.
1995;
Ritchie
et
al.
2008),
which
led
to
the
work-
ing
hypothesis
that
walled
spore
and
stalk
cells
are
evolutionary
derived
from
cysts.
Ppal
can
differen-
tiate
into
all
three
cell
types,
allowing
us
to
retrace
how
complexity
in
cell
wall
biosynthesis
emerged.
A
pilot
study
revealed
the
presence
of
two
cel-
lulose
synthase
genes
in
Ppal.
In
this
work,
we
studied
the
expression
patterns
of
both
genes
and
abrogated
the
genes
individually
and
together.
Inspection
of
the
null
mutant
phenotypes
show
both
unique
and
overlapping
roles
for
the
cellulose
syn-
thases
and
an
absolute
requirement
of
cellulose
synthesis
for
encystation
and
sporulation.
Results
Conservation
of
Cellulose
Synthase
Genes
in
Dictyostelia
The
D.
discoideum
(Ddis)
genome
contains
a
sin-
gle
cellulose
synthase
gene,
DcsA,
and
we
first
investigated
whether
DcsA
is
conserved
through-
out
the
dictyostelid
phylogeny.
The
genomes
of
species
representing
the
four
major
groups
of
Dictyostelia
and
the
solitary
amoebozoan
Acan-
thamoeba
castellani
(Acas)
(Clarke
et
al.
2013;
Eichinger
et
al.
2005;
Heidel
et
al.
2011;
Sucgang
et
al.
2011)
as
well
as
all
non-redundant
sequences
in
Genbank
were
queried
with
the
Ddis
DcsA
pro-
tein
sequence,
yielding
single
orthologues
of
DcsA
in
groups
1,
3
and
4
of
Dictyostelia
and
an
addi-
tional
gene,
DcsB,
in
A.
subglobosum
(Asub)
and
Ppal,
which
represent
the
two
major
clades
of
group
2.
The
dictyostelid
cellulose
synthase
genes
were
more
similar
to
bacterial
and
oomycete
cellulose
synthases
than
to
the
Acas
cellulose
synthase.
Phenotype
of
a
Ppal
dcsa-
Mutant
The
group
2
species
Ppal
is
the
only
encysting
dictyostelid
that
is
amenable
to
gene
knockout
pro-
cedures.
To
identify
the
respective
roles
of
DcsA
and
DcsB
in
Ppal,
we
generated
null
mutants
in
either
gene
by
transformation
with
a
floxed
neomycin
cassette
(Faix
et
al.
2004;
Kawabe
et
al.
2009)
flanked
by
∼1
kb
fragments
of
the
DcsA
or
DcsB
coding
regions.
Clones
carrying
gene
knock-
out
(KOs)
and
random
integration
(RI)
events
were
identified
by
two
PCR
reactions
and
Southern
blot
analysis
(Supplementary
Material
Figs
S1
and
S2).

Dictyostelid
Encystation
Requires
Cellulose
571
Figure
1.
Phylogeny
of
dictyostelid
cellulose
syn-
thases.
A.
Dicytostelid
phylogeny.
Genome-based
phylogeny
of
group-representative
Dictyostelium
species
and
Acanthamoeba
castellani
(Acas)
(Romeralo
et
al.
2013)
with
numbers
referring
to
the
relevant
group
or
clade.
Dpur:
D.
purpureum,
Ddis:
D.
discoideum,
Dlac:
D.
lacteum,
Ppal:
Polysphondylium
pallidum,
Asub:
Acytostelium
subglobosum,
Dfas:
D.
fasciculatum.
B.
Cellulose
synthase
phylogeny.
Amoebozoan
cellulose
synthase
genes
and
their
closest
homologs
in
other
organisms
were
retrieved
by
BlastP
search
of
Genbank
and
ongoing
D.
lac-
teum
(http://sacgb.fli-leibniz.de
and
A.
subglobosum
(http://acytodb.biol.tsukuba.ac.jp)
genome
projects,
using
Ddis
DcsA
as
bait.
The
regions
containing
the
glycosyl
transferase
domain
were
aligned
using
Clustal
Omega
(Sievers
et
al.
2011)
and
subjected
to
phylogeny
reconstruction
by
Bayesian
inference
(Ronquist
and
Huelsenbeck
2003).
The
phylogenetic
tree
is
annotated
with
the
functional
domain
architec-
ture
of
the
proteins,
as
analyzed
with
SMART
(Schultz
et
al.
1998).
The
protein
identifiers
are
color-coded
according
to
species
as
in
panel
A,
with
grey
further
indicating
the
bacteria
Leptolyngbya
sp.
(EKU97898)
and
Cyanobacterium
stanieri,
and
tan
the
oomycete
Pythium
iwayamai.
Bayesian
posterior
probabilities
of
tree
nodes
are
indicated
by
colored
dots.
Similar
to
control
RI
cells,
Ppal
dcsa-
KO
cells,
formed
fruiting
bodies
with
normal
stalks
(Fig.
2A)
that
contained
cellulose
in
their
cell
walls
(Fig.
2B
e).
However,
dcsa-
spores,
while
still
somewhat
retaining
their
elliptical
shape,
contained
little
to
no
cellulose
as
evident
by
staining
with
the
bright-
ening
agent
Calcofluor
White
that
interacts
with
cellulose
(Fig.
2B
b).
Under
submerged
conditions,
Ppal
amoebas
encyst
individually
when
starved,
and
encystation
is
accelerated
by
high
osmolar-
ity.
The
Ppal
dcsa-
cells
rounded
off
and
lost
their
amoeboid
shape
when
starved
under
these
conditions,
but
unlike
RI
cells
(Fig.
2C
a),
they
did
not
produce
the
cellulose
cell
wall
(Fig.
2C
b).
To
confirm
that
these
phenotypes
were
caused
by
loss
of
DcsA,
the
neomycin
cassette
was
deleted
from
dcsa-
cells
by
transformation
with
Cre
recom-
binase
and
the
resulting
dcsa-neo-
cells
were
transformed
with
the
DcsA
coding
region
and
1.6
kb
5intergenic
sequence
(Supplementary
Material
Fig.
S1).
This
construct,
1.6p::DcsA,
restored
cellu-
lose
deposition
in
spore
walls
(Fig.
2B
c),
but
not
in
cyst
walls
(Fig.
2C
c).
We
therefore
prepared
a
sec-
ond
construct,
3.0p::DcsA,
with
3.0
kb
intergenic
sequence,
which
also
restored
cellulose
synthesis
in
cysts
(Fig.
2C
d).
These
data
show
that
DcsA
is
essential
for
cellulose
synthesis
in
spores
and
cysts
and
that
DcsA
expression
in
either
cell
type
is
regulated
by
different
promoter
regions.
Overall,
the
data
show
that
Ppal
DcsA
is
required
for
spore
and
cyst
wall
synthesis,
but
not
stalk
wall
synthe-
sis.
Phenotypes
of
dcsb-
and
dcsa-/dcsb-
Mutants
We
next
disrupted
the
DcsB
gene,
but
surpris-
ingly
the
dcsb-
cells
made
normal
cellulose-rich
spore,
stalk
and
cyst
cell
walls
(Fig.
3A).
This
suggests
that
DcsB
and
DcsA
have
overlapping
roles
in
stalk
wall
formation
and
to
test
this
hypothesis,
we
generated
a
double
dcsa-/dcsb-
mutant.
The
phenotype
of
the
dcsa-/dcsb-
mutant
was
much
more
severe
than
that
of
the
dcsa-
mutant.
The
dcsa-/dcsb-
mutant
showed
normal
aggregation
and
formation
of
the
primary
sorogen
(Fig.
3B
e,
f).
The
mutant
did
manage
to
erect
stalked
fruiting
structures
(Fig.
3B
g-i),
which
often
showed
the
pinched-off
cell
masses
(Fig.
3B
h),
that
give
rise
to
the
whorls
of
side
branches
in
wild
type
Ppal
(Fig.
3B
c,
d).
These
cell
masses
never
developed
into
side-branches
and
the
ter-
minal
fruiting
structures
usually
consisted
of
a
single
mass
of
cells
on
top
of
an
irregularly
shaped
stalk
(Fig.
3B
i).
The
cells
at
the
inte-
rior
of
the
“spore”
mass
were
amoeboid
and
did
not
stain
with
Calcofluor
White
(Fig.
3C
e).
The
dcsa-/dcsb-
“spores”
were
also
more
isodiametric
(length/diameter
ratio
1.1
±
0.1)
than
dcsa-
spores
(1.4
±
0.2)
and
wild-type
spores
(1.8
±
0.13),
sug-
gesting
that
DcsB
still
contributes
somewhat
to
spore
wall
integrity
and
shape
maintenance.
The
cells
at
the
periphery
of
the
dcsa-/dcsb-
“spore”
mass
appeared
to
be
lysed
and
showed
weak
staining
throughout,
which
is
probably
caused
by
interaction
of
Calcofluor
White
with
intracellular
polysaccharides.
The
stalk
consisted
of
a
fibrous
sheath,
that
was
initially
filled
with
cell
material
(Fig.
3C
c),
but

572
Q.
Du
and
P.
Schaap

Dictyostelid
Encystation
Requires
Cellulose
573
seemed
empty
in
more
mature
structures
(Fig.
3C
d).
There
was
none
or
very
weak
staining
with
Cal-
cofluor
White.
Since
wild-type
stalk
cells
also
die
and
leave
little
else
behind
than
their
walls,
the
dcsa-/dcsb-
stalk
cells
may
just
be
following
their
normal
death
programme.
Even
without
a
cellulose-
rich
tube
the
progression
of
stalk
formation
in
dcsa-/dcsb-
sorogens
was
similar
as
in
wild-type
sorogens,
with
newly
formed
stalk
cells
descending
from
the
tip
through
the
center
of
the
cell
mass
to
form
the
stalk
(Fig.
3C
a,
b).
Similar
to
dcsa-
cells,
the
dcsa-/dcsb-
cells
also
did
not
form
cyst
walls
(Fig.
3C
f).
The
results
indicate
that
DcsA
is
the
primary
enzyme
for
spore
and
cyst
wall
cellulose
synthesis,
and
that
DcsB
has
an
overlapping
role
with
DcsA
in
cellulose
synthesis
for
the
stalk
tube
and
the
walls
of
the
stalk
cells.
Expression
Patterns
of
DcsA
and
DcsB
We
next
investigated
whether
the
apparent
func-
tional
specialization
of
DcsA
and
DcsB
is
reflected
by
the
expression
pattern
of
their
genes.
The
1.6
and
3.0
kb
DcsA
promoter
fragments
and
2.7
kb
DcsB
promoter
fragment
(Supplementary
Material
Fig.
S2)
were
fused
to
the
LacZ
reporter
gene
in
plasmid
pDd17
gal
and
transformed
into
Ppal
wild-type
cells.
Developing
structures
were
stained
with
X-gal
to
visualize
activity
of
the
cognate
LacZ
gene
product,
␤-galactosidase.
The
DcsA
3.0
kb
promoter
activated
LacZ
expression
in
most
cells
in
aggregates
(Fig.
4A
a)
and
in
both
early
and
late
sorogens
(Fig.
4A
b,
c),
although
X-gal
staining
tended
to
be
somewhat
more
intense
at
the
utmost
tip
and
stalk.
DcsA
promoter
activity
disappeared
completely
from
mature
spores,
but
not
from
the
stalks
(Fig.
4A
d).
The
3.0
kb,
but
not
the
1.6
kb
DcsA
promoter,
was
also
active
in
encysting
cells
(Fig.
4C
a,
b).
Cells
expressing
DcsB::LacZ
first
appeared
scat-
tered
throughout
late
aggregates
(Fig.
4B
a),
but
expression
became
rapidly
restricted
to
the
emerging
tips
(Fig.
4B
b).
In
sorogens,
DcsB
promoter
activity
was
high
in
the
tip
and
stalk
and
some
scattered
cells
throughout
the
soro-
gens
(Fig.
4B
c,
d).
There
was
no
DcsB
promoter
activity
in
encysting
cells
(Fig.
4C
c).
The
low
or
lacking
expression
of
DcsB
in
prespore
and
cyst
cells,
respectively,
is
in
good
agreement
with
the
fact
that
DcsB
is
not
required
for
spore
and
cyst
differentiation.
The
absence
of
LacZ
expres-
sion
from
the
DcsA
1.6
kb
promoter
in
cysts
also
explains
why
expression
of
DcsA
from
the
1.6
kb
fragment
does
not
restore
encystation.
A
more
distal
region
contained
in
the
3.0
kb
fragment
is
likely
to
mediate
cyst-specific
expression
of
DcsA.
Viability
of
Spores
and
Cysts
in
Single
and
Double
Cellulose
Synthase
Knockouts
We
next
assessed
how
loss
of
DcsA
and/or
DcsB
affected
spore
and
cyst
viability.
Spores
were
har-
vested
from
the
sori
of
mature
fruiting
bodies,
while
cysts
were
obtained
by
incubating
cells
for
4
days
in
encystation
medium.
At
this
point,
wild-type,
dcsb-
and
DcsA
RI
cells
had
fully
encysted.
The
cells
were
counted
and
shaken
for
10
min
in
the
presence
and
absence
of
0.1%
Triton-X100
before
plating
on
Klebsiella
lawns’
and
after
three
days
the
emerging
colonies
were
counted.
About
70-80%
of
plated
wild-type,
DcsA
RI
and
dcsb-
spores
formed
colonies,
regardless
of
detergent
treatment
(Fig.
5).
The
dcsa-
and
dcsa-/dcsb-
spore
equivalents
still
formed
80
and
60%
colonies,
respectively,
in
the
absence
of
detergent
treatment,
but
none
after
detergent
treatment.
Detergent
treatment
caused
a
small
(∼10%)
decrease
in
the
number
of
colonies
formed
by
wild-type,
random
integrant
and
dcsb-
cysts
(80-90%
of
plated
cells).
However,
both
the
dcsa-
and
dcsa-/dcsb-
cyst
equivalents
formed
hardly
any
colonies
in
the
absence
of
detergent
treatment
and
none
in
its
presence.
Apparently,
the
dcsa-
and
dcsa-/dcsb-
spore
equivalents
are
viable,
but
not
detergent
resistant
in
the
absence
of
cellulose,
while
the
dcsa-
and
dcsa-/dcsb-
cyst
equivalents
are
entirely
non-viable.
➛
Figure
2.
Phenotype
of
a
dcsa-
mutant.
A.
DcsA
knockout
(KO)
and
control
random
integrant
(RI)
cells
were
plated
on
PB
agar
and
incubated
until
fruiting
bodies
had
formed.
Bar:
200
␮m.
B.
Fruiting
bodies
of
DcsA
KO6
(a,
d)
and
RI5
(b,
e)
cells,
and
of
dcsa-neo-
cells,
transformed
with
the
1.6p::DcsA
expression
cassette
(c)
were
transferred
to
0.001%
Calcofluor
White
on
a
slide
glass.
Spores
and
stalks
were
photographed
under
phase
contrast
(left
panels),
and
under
UV,
combined
with
faint
phase
contrast
illumination.
Bar:
10
␮m.
C.
DcsA
KO6
(a)
and
RI5
cells
(b)
and
dcsa-
cells
transformed
with
the
1.6::DcsA
(c)
or
3.0p::DcsA
(d)
cassettes
were
incubated
in
encystation
medium.
Calcofluor
White
was
added
to
0.001%
after
4
days
and
cells
were
photographed.
Bar:
10
␮m.

574
Q.
Du
and
P.
Schaap

Dictyostelid
Encystation
Requires
Cellulose
575
Discussion
Gene
Duplication
Followed
by
Functional
Specialization
of
Group
2
Cellulose
Synthases
Cellulose
is
a
component
of
several
structural
fea-
tures
of
D.
discoideum,
such
as
the
slime
sheath
that
surrounds
the
migrating
slug,
the
walls
of
spore,
stalk
and
basal
disc
cells
and
the
support-
ive
tube
that
surrounds
the
stalk
cells.
A
single
enzyme,
DcsA,
produces
cellulose
for
all
these
fea-
tures
and
its
deletion
prevents
the
formation
of
viable
spores
and
of
a
stalk
to
lift
the
sorus
from
the
substratum
(Blanton
et
al.
2000;
Zhang
et
al.
2001).
Among
the
four
dictyostelid
taxon
groups,
the
group
2
species
Ppal
and
Asub
have
a
sec-
ond
cellulose
synthase
gene,
DscB.
This
gene
most
likely
emerged
by
duplication
of
DcsA,
since
it
is
more
similar
to
DcsA
than
to
any
gene
outside
Dic-
tyostelia
(Fig.
1).
Our
data
indicate
that
in
group
2
the
two
genes
have
started
to
acquire
specialized
functions.
DcsA
null
mutants
show
severe
defects
in
spore
and
cyst
wall
formation,
but
the
stalk
cell
wall
and
stalk
tube
are
still
normally
formed.
While
the
walled
cell
types
and
multicellular
structures
of
dcsb-
mutants
are
not
markedly
different
from
those
of
wild-type
Ppal,
stalk
formation
becomes
severely
defective
in
a
dcsa-/dcsb-
mutant,
indicat-
ing
that
DcsA
and
DcsB
have
an
overlapping
role
in
stalk
formation.
The
expression
patterns
of
the
two
genes
reflect
this
partial
specialization.
DcsB
is
only
expressed
in
prestalk
and
stalk
cells,
while
DcsA
is
additionally
expressed
in
prespore
cells
and
from
a
separate
distal
promoter
element
in
the
cysts.
The
group
2
cellulose
synthases
seem
to
be
on
an
evo-
lutionary
trajectory
to
perform
specialized
roles
in
cell
wall
synthesis.
The
P.
pallidum
Stalk
is
Rigid
Without
Cellulose
Unlike
Ddis
dcsa-
sorogens,
which
entire
fail
to
form
a
stalk
(Blanton
et
al.
2000),
the
Ppal
dcsa-/dcsb-
sorogens
still
form
a
stalk
tube-like
structure
with
sufficient
rigidity
to
keep
an
apical
cell
mass
airborne
(Fig.
3B
h,
i).
While
dictyostelid
genomes
do
not
contain
chitin
synthases
(personal
BLAST
search),
D.
discoideum
has
two
conserved
extracellular
matrix
proteins,
EcmA
and
EcmB,
which
consist
of
over
20
copies
of
a
24-amino-acid
long
repeat
with
5
cysteine
residues
each.
By
forming
extensive
disulfide
bridges
these
proteins
contribute
to
the
rigidity
of
the
matrix,
and
EcmA
was
shown
to
enhance
the
tensile
strength
of
the
slime
sheath
(Morrison
et
al.,
1994).
At
least
three
homologs
of
EcmA
and
EcmB
are
present
in
the
P.
pallidum
genome
(Genbank
IDs:
EFA80374,
EFA79535
and
EFA82732).
It
is
plausible
that
the
group
2
Polysphondylids
with
their
habitually
long
thin
stalks
(Romeralo
et
al.
2013)
have
a
larger
abundance
of
these
matrix
proteins
than
D.discoideum
with
its
shorter
thicker
stalks,
and
that
this
abundance
allows
the
dcsa-/dcsb-
mutant
to
form
a
cellulose-free
stalk.
Similar
to
Ddis
dcsa-
prestalk
cells
(Blanton
et
al.
2000),
the
Ppal
dcsa-/dcsb-
prestalk
cells
still
descend
into
the
center
of
the
cell
mass
attempting
to
form
the
stalk
(Fig.
3C
b).
However,
they
never
form
a
cell
wall,
and
unlike
Ddis
dcsa-
stalk
cells,
never
vacuolate
properly.
Cellulose
Synthesis
is
Essential
for
the
Differentiation
of
Viable
Cysts
The
loss
of
dcsa-
alone
from
Ppal
is
sufficient
to
pre-
vent
any
viable
cysts
from
being
formed,
highlight-
ing
an
absolutely
essential
role
for
cellulose
in
cyst
differentiation.
While
Ppal
and
most
dictyostelids
are
harmless
soil
inhabitants,
this
is
not
the
case
for
other
Amoebozoa
such
as
Acanthamoeba
and
Bal-
amuthia
sp.
which
can
cause
blinding
keratitis
and
lethal
amoebic
encephalitis
(Trabelsi
et
al.
2012;
Visvesvara
2010).
Even
the
encysting
dictyostelid
D.
polycephalum
was
shown
to
be
responsible
for
a
case
of
keratitis
(Reddy
et
al.
2010).
These
infections
resist
antibiotic
treatment,
because
the
amoeba
encyst
in
response
to
the
perceived
stress
response.
Eradication
of
the
cysts
requires
months
of
painful
treatment
with
a
cocktail
of
antiseptics
and
antibiotics.
The
use
of
cellulose
synthase
as
a
target
for
weed
killers
(Wakabayashi
and
Böger
➛
Figure
3.
Phenotypes
of
dcsb-
and
dcsa-/dcsb-
mutants.
A.
Dcsb-
cells
were
developed
to
fruiting
bodies
on
PB
agar
and
to
cysts
in
400
mM
sorbitol.
Fruiting
bodies
were
photographed
in
situ
(bar:
200
␮m),
stalk
cells,
spores
and
cysts
were
stained
with
Calcofluor
White
and
photographed
under
UV
illumination.
Bar:
10
␮m.
B.
Wild-type
P.
pallidum
and
the
dcsa-/dcsb-
mutant
were
incubated
on
PB
agar
and
photographed
at
the
indicated
time
points.
Bar:
200
␮m.
C.
dcsa-/dcsb-
and
wild
type
sorogens
and
fruiting
bodies
were
submerged
in
situ
in
0.001%
Calcofluor
White,
placed
under
a
coverslip
and
photographed
under
phase
contrast
and
UV
illumination.
Ca,b
Bar:
100
␮m;
Cc,d,e,f
Bar:
10
␮m.

576
Q.
Du
and
P.
Schaap
Figure
4.
Expression
patterns
of
DcsA
and
DcsB.
A/B.
Ppal
wild-type
cells
transformed
with
the
DcsA3.0::LacZ
(A)
and
DcsB::LacZ
(B)
constructs
were
plated
on
nitrocellulose
filters
supported
by
PB
agar.
Emerging
aggregates
and
early
and
late
sorogens
were
fixed
and
stained
with
X-
gal
to
visualize
␤-galactosidase
activity.
Bar:
50
␮m.
C.
Cells
transformed
with
DcsA1.6::LacZ,
DcsA3.0::LacZ
and
DcsB::LacZ
were
incubated
for
two
days
in
encystation
medium.
Cells
were
then
fixed
and
stained
with
X-gal,
counterstained
with
Calcofluor
White
to
identify
cysts,
and
photographed
under
UV
and
brightfield
illumination.
Bar:
10
␮m.

Dictyostelid
Encystation
Requires
Cellulose
577
Figure
5.
Spore
and
cyst
viability
of
cellulose
syn-
thase
null
mutants.
Wild-type,
DcsA
RI,
dcsa-,
dcsb-
and
dcsa-/dcsb-
cells
were
harvested
from
mature
spore
heads
or
from
400
mM
sorbitol
after
4
days
of
incubation.
Cells
were
counted
and
shaken
for
10
min
with
and
without
0.1%
Triton-X100
before
being
plated
at
500
cells/plate
on
Klebsiella
lawns.
After
3
days
the
emerging
colonies
were
counted.
The
number
of
colonies
as
percentage
of
plated
cells
are
shown,
and
the
data
are
present
means
and
SD
of
two
experi-
ments
with
duplicate
plates
for
each
variable.
2002),
shows
that
these
enzymes
can
be
effec-
tively
inhibited.
However,
existing
plant
cellulose
synthase
inhibitors
do
not
always
inhibit
Amoebo-
zoan
cellulose
synthesis
(Kiedaisch
et
al.
2003)
and
may
have
unwanted
side
effects.
By
a
developing
an
effective,
non-toxic
inhibitor
for
the
amoebozoan
cellulose
synthase,
and
combining
this
compound
with
standard
antibiotics,
the
treatment
of
amoeba
keratitis
could
be
fast,
effective
and
painless.
Conclusions
The
encysting
dictyostelid
P.
pallidum
has
two
cellu-
lose
synthase
genes.
DcsB
is
expressed
in
prestalk
and
stalk
cells
and
synthesizes
stalk
wall
cellulose,
together
with
DcsA.
DcsA
is
additionally
expressed
in
prespore
cells
and,
from
a
more
distal
promoter
element,
in
cysts.
DcsA
is
required
for
production
of
spore
and
cyst
wall
cellulose
and
is
essential
for
spore
and
cyst
viability.
P.
pallidum
is
the
first
genetically
tractable
model
organism
for
systematic
analysis
of
amoebozoan
encystation,
a
process
that
renders
amoebozoan
pathogens
impervious
to
immune
attack
and
antibi-
otics.
The
essential
role
for
cellulose
synthase
in
cyst
formation
shown
here,
identifies
this
enzyme
as
a
potential
target
for
therapeutics
to
prevent
encystation.
Methods
Cell
culture:
Ppal
strain
PN500
was
grown
in
association
with
Klebsiella
aerogenes
at
22 ◦C
on
LP
or
1/5th SM
agar.
For
mul-
ticellular
development,
cells
were
harvested
from
growth
plates
in
10
mM
Na/K-phosphate,
pH
6.5
(PB)
and
incubated
at
106
cells/cm2on
PB
agar
(1.5%
agar
in
PB).
DcsA
and
DcsB
single
and
double
knock-out
mutants:
To
obtain
a
DcsA
knock-out
plasmid,
KO
fragments
DcsA
I
and
II
(Supplementary
figure
S1)
were
amplified
from
Ppal
PN500
genomic
DNA
using
primer
pairs
DcsAI5/DcsAI3and
DcsAII5/DcsAII3(Supplementary
Material
Ta b l e
S1),
respec-
tively,
introducing
XbaI/BglII
sites
on
fragment
I
and
HindIII/XhoI
sites
on
fragment
II.
The
fragments
were
sequentially
inserted
into
the
XbaI/BamHI
and
HindIII/XhoI
digested
plasmid
pLox-
NeoIII
(Kawabe
et
al.
2012)
yielding
plasmid
pDcsA_KO.
Correct
insertion
was
validated
by
DNA
sequencing.
For
a
DcsB
knock-out
plasmid,
DcsB
KO
fragments
I
and
II
(Supplementary
Material
Fig.
S2)
were
similarly
ampli-
fied
with
primer
pairs
DcsBI5/DcsBI3and
DcsBII5/DcsBII3
and
inserted
in
pLox-NeoIII,
yielding
plasmid
pDcsB_KO.
The
XbaI/XhoI
inserts
from
the
pDcsA_KO
and
pDcsB_KO
plasmids
were
excised
and
5
␮g
of
either
insert
was
transformed
into
2.5
x
106Ppal
cells
together
with
2
nanomoles
of
its
flanking
primers
(Kuwayama
et
al.
2008).
For
transformation,
Ppal
cells
were
harvested
from
growth
plates,
incubated
for
5
hours
in
HL5
at
2.5
x
106cells/ml
and
electroporated
in
ice-cold
H-50
buffer
with
two
pulses
at
a
5
s
interval
of
0.65
kV/25
␮Fd
from
a
GenPulser2
(BioRad),
followed
by
selection
of
transformants
on
autoclaved
K.aerogenes
at
300
␮g/ml
G418
(Kawabe
et
al.
1999).
Knock-
out
clones
were
diagnosed
by
two
PCR
reactions
and
Southern
blot
analysis
as
illustrated
in
Supplementary
Material
Figures
S1
and
S2.
To
generate
a
dcsa-/dcsb-
double
knock-out
mutant,
the
floxed
A6neo
cassette
was
first
removed
from
dcsa-
KO6
by
transformation
with
vector
pA15NLS.Cre
for
transient
expres-
sion
of
Cre
recombinase
(Faix
et
al.
2004).
Transformed
clones
were
replica-plated
onto
autoclaved
K.
aerogenes
on
LP
agar
plates
with
and
without
300
␮g/ml
G418
for
negative
selection.
The
dcsa-neo-
cells
were
subsequently
transformed
with
the
XbaI/XhoI
insert
from
pDcsB_KO
and
screened
for
knock-out
of
DcsB
as
described
above.
DcsA
expression
constructs:
To
express
DcsA
from
its
own
promoter,
a
4.59
kb
genomic
fragment
including
the
DcsA
coding
region
and
1.59
kb
5to
the
startcodon
(Supplemen-
tary
Material
Fig.
S1)
was
amplified
by
PCR
using
primer
DcsAPro1_5and
DcsA3,
which
include
XbaI
and
HindIII
restriction
sites,
respectively
(Supplementary
Material
Ta b l e
S1).
After
XbaI/HindIII
digestion,
the
fragment
was
ligated
into
similarly
digested
plasmid
pExp5
(Meima
et
al.
2007),
yielding
plasmid
1.6p::DcsA,
and
validated
by
DNA
sequencing.
The
plasmid
was
transformed
into
dcsa-neo-
cells,
but
only
par-
tially
restored
the
dcsa-
phenotype.
Therefore,
a
longer
6.19
kb
fragment
including
2.99
kb
5to
the
start
AT G
was
amplified,
using
DcsAPro2_5(Supplementary
Material
Ta b l e
S1)
as
the
5primer,
and
inserted
in
pExp5,
yielding
3.0p::DcsA.

578
Q.
Du
and
P.
Schaap
DcsA
and
DcsB
promoter-LacZ
constructs:
The
1.6
and
3
kb
DcsA
promoter
fragments
and
a
2.7
kb
DcsB
promoter
fragment
(Supplementary
Material
Fig.
S2)
were
amplified
from
Ppal
genomic
DNA
using
primer
pairs
DcsaPro1_5’/DcsApro3,
DcsaPro2_5/DcsApro3and
DcsB
Pro5/DcsBpro3(Supple-
mentary
Material
Ta b l e
S1),
respectively.
The
5and
3
primers
contain
XbaI
and
BamHI
restriction
sites,
respectively,
which
were
used
to
insert
the
constructs
into
the
BglII/XbaI
digested
vector
pDdGal17
(Harwood
and
Drury
1990).
This
generated
plasmids
pDcsA_1.6::LacZ,
pDcsA_3.0::LacZ
and
pDcsB::LacZ
with
the
LacZ
coding
sequence
fused
at
its
5end
to
either
of
the
three
promoters.
The
plasmids
were
transformed
into
Ppal
wild-type
cells
and
␤-galactosidase
activity
was
visu-
alized
with
X-gal
as
described
previously
(Kawabe
et
al.
2009).
All
plasmids
and
knock-out
mutants
that
were
generated
in
this
study
have
been
deposited
in
the
Dictyostelium
Stock
Centre
(http://dictybase.org/StockCenter/)
or
are
available
on
request.
Cyst
and
spore
germination
assay:
To
obtain
spores,
Ppal
wild-type
cells
and
mutants
were
harvested
from
growth
plates
and
incubated
at
22 ◦C
on
PB
agar
for
4
days
until
mature
fruiting
bodies
had
fully
formed.
For
cysts,
cells
were
resuspended
in
encystation
medium
(PB
with
400
mM
sorbitol)
and
incubated
for
4
days
in
the
dark
until
wild-type
cells
had
formed
mature
cysts.
Spores
and
cysts,
harvested
from
fruiting
bodies
and
encystation
medium,
respectively,
were
resuspended
in
80
mM
sucrose
in
PB
(Zhang
et
al.
2001)
and
counted.
Triton-X100
(or
an
equivalent
volume
of
water)
was
added
to
a
concentration
of
0.1%,
cells
were
shaken
for
10
min.
and
then
diluted
at
least
100x
in
80
mM
sucrose
for
plating
with
K.aerogenes
on
1/5th
SM
agar
plates
at
500
cells
per
15
cm
plate.
Colony
numbers
were
counted
after
3
days
of
culture
at
22 ◦C.
Acknowledgements
We
acknowledge
Dr.
Christina
Schilde
(Dundee)
for
detecting
DcsB
in
the
Ppal
genome.
This
research
was
funded
by
Wellcome
Trust
grants
090276
and
100293
and
by
BBSRC
grant
BB/K000799.
Appendix
A.
Supplementary
data
Supplementary
data
associated
with
this
arti-
cle
can
be
found,
in
the
online
version,
at
http://dx.doi.org/10.1016/j.protis.2014.07.003.
References
Aqeel
Y,
Siddiqui
R,
Khan
NA
(2013)
Silencing
of
xylose
isom-
erase
and
cellulose
synthase
by
siRNA
inhibits
encystation
in
Acanthamoeba
castellanii.
Parasitol
Res
112:1221–1227
Blanton
RL,
Fuller
D,
Iranfar
N,
Grimson
MJ,
Loomis
WF
(2000)
The
cellulose
synthase
gene
of
Dictyostelium.
Proc
Natl
Acad
Sci
USA
97:2391–2396
Bush
K
(2012)
Antimicrobial
agents
targeting
bacterial
cell
walls
and
cell
membranes.
Rev
Sci
Tech
31:43–56
Clarke
M,
Lohan
AJ,
Liu
B,
Lagkouvardos
I,
Roy
S,
Zafar
N,
Bertelli
C,
Schilde
C,
Kianianmomeni
A,
Burglin
TR,
Frech
C,
Turcotte
B,
Kopec
KO,
Synnott
JM,
Choo
C,
Paponov
I,
Finkler
A,
Heng
Tan
CS,
Hutchins
AP,
Weinmeier
T,
Rat-
tei
T,
Chu
JS,
Gimenez
G,
Irimia
M,
Rigden
DJ,
Fitzpatrick
DA,
Lorenzo-Morales
J,
Bateman
A,
Chiu
CH,
Tang
P,
Hege-
mann
P,
Fromm
H,
Raoult
D,
Greub
G,
Miranda-Saavedra
D,
Chen
N,
Nash
P,
Ginger
ML,
Horn
M,
Schaap
P,
Caler
L,
Lof-
tus
BJ
(2013)
Genome
of
Acanthamoeba
castellanii
highlights
extensive
lateral
gene
transfer
and
early
evolution
of
tyrosine
kinase
signaling.
Genome
Biol
14:R11
Du
Q,
Schilde
C,
Birgersson
E,
Chen
ZH,
McElroy
S,
Schaap
P
(2014)
The
cyclic
AMP
phosphodiesterase
RegA
critically
regulates
encystation
in
social
and
pathogenic
amoebas.
Cel-
lular
Signalling
26:453–459
Dudley
R,
Alsam
S,
Khan
NA
(2007)
Cellulose
biosynthe-
sis
pathway
is
a
potential
target
in
the
improved
treatment
of
Acanthamoeba
keratitis.
Appl
Microbiol
Biotechnol
75:
133–140
Dudley
R,
Jarroll
EL,
Khan
NA
(2009)
Carbohydrate
analysis
of
Acanthamoeba
castellanii.
Exp
Parasitol
122:338–343
Eichinger
L,
Pachebat
JA,
Glöckner
G,
Rajandream
MA,
Sucgang
R,
Berriman
M,
Song
J,
Olsen
R,
Szafranski
K,
Xu
Q,
Tunggal
B,
Kummerfeld
S,
Madera
M,
Konfortov
BA,
Rivero
F,
Bankier
AT,
Lehmann
R,
Hamlin
N,
Davies
R,
Gaudet
P,
Fey
P,
Pilcher
K,
Chen
G,
Saunders
D,
Soder-
gren
E,
Davis
P,
Kerhornou
A,
Nie
X,
Hall
N,
Anjard
C,
Hemphill
L,
Bason
N,
Farbrother
P,
Desany
B,
Just
E,
Morio
T,
Rost
R,
Churcher
C,
Cooper
J,
Haydock
S,
van
Driess-
che
N,
Cronin
A,
Goodhead
I,
Muzny
D,
Mourier
T,
Pain
A,
Lu
M,
Harper
D,
Lindsay
R,
Hauser
H,
James
K,
Quiles
M,
Madan
Babu
M,
Saito
T,
Buchrieser
C,
Wardroper
A,
Felder
M,
Thangavelu
M,
Johnson
D,
Knights
A,
Loulseged
H,
Mungall
K,
Oliver
K,
Price
C,
Quail
MA,
Urushihara
H,
Her-
nandez
J,
Rabbinowitsch
E,
Steffen
D,
Sanders
M,
Ma
J,
Kohara
Y,
Sharp
S,
Simmonds
M,
Spiegler
S,
Tivey
A,
Sug-
ano
S,
White
B,
Walker
D,
Woodward
J,
Winckler
T,
Tanaka
Y,
Shaulsky
G,
Schleicher
M,
Weinstock
G,
Rosenthal
A,
Cox
EC,
Chisholm
RL,
Gibbs
R,
Loomis
WF,
Platzer
M,
Kay
RR,
Williams
J,
Dear
PH,
Noegel
AA,
Barrell
B,
Kuspa
A
(2005)
The
genome
of
the
social
amoeba
Dictyostelium
dis-
coideum.
Nature
435:43–57
Erdos
EW,
Hohl
HR
(1980)
Freeze-fracture
examination
of
the
plasma
membrane
of
the
cellular
slime
mould
Polysphondylium
pallidum
during
microcyst
formation
and
germination.
Cytobios
29:7–16
Faix
J,
Kreppel
L,
Shaulsky
G,
Schleicher
M,
Kimmel
AR
(2004)
A
rapid
and
efficient
method
to
generate
multiple
gene
disruptions
in
Dictyostelium
discoideum
using
a
single
selectable
marker
and
the
Cre-loxP
system.
Nucleic
Acids
Res
32:e143
Free
SJ
(2013)
Fungal
cell
wall
organization
and
biosynthesis.
Adv
Genet
81:33–82
Fugelstad
J,
Bouzenzana
J,
Djerbi
S,
Guerriero
G,
Ezcurra
I,
Teeri
TT,
Arvestad
L,
Bulone
V
(2009)
Identification
of
the
cellulose
synthase
genes
from
the
oomycete
Saproleg-
nia
monoica
and
effect
of
cellulose
synthesis
inhibitors
on
gene
expression
and
enzyme
activity.
Fungal
Genet
Biol
46:
759–767
Grimson
MJ,
Haigler
CH,
Blanton
RL
(1996)
Cellulose
microfibrils,
cell
motility,
and
plasma
membrane
protein
organization
change
in
parallel
during
culmination
in
Dic-
tyostelium
discoideum.
J
Cell
Sci
109:3079–3087

Dictyostelid
Encystation
Requires
Cellulose
579
Harwood
AJ,
Drury
L
(1990)
New
vectors
for
expression
of
the
E.
coli
lacZ
gene
in
Dictyostelium.
Nucleic
Acids
Res
18:4292
Heidel
A,
Lawal
H,
Felder
M,
Schilde
C,
Helps
N,
Tung-
gal
B,
Rivero
F,
John
U,
Schleicher
M,
Eichinger
L,
Platzer
M,
Noegel
A,
Schaap
P,
Glöckner
G
(2011)
Phylogeny-wide
analysis
of
social
amoeba
genomes
highlights
ancient
ori-
gins
for
complex
intercellular
communication.
Genome
Res
:1882–1891
Kawabe
Y,
Enomoto
T,
Morio
T,
Urushihara
H,
Tanaka
Y
(1999)
LbrA,
a
protein
predicted
to
have
a
role
in
vesicle
trafficking,
is
necessary
for
normal
morphogenesis
in
Polyspho-
ndylium
pallidum.
Gene
239:75–79
Kawabe
Y,
Morio
T,
James
JL,
Prescott
AR,
Tanaka
Y,
Schaap
P
(2009)
Activated
cAMP
receptors
switch
encystation
into
sporulation.
Proc
Natl
Acad
Sci
USA
106:7089–7094
Kawabe
Y,
Weening
KE,
Marquay-Markiewicz
J,
Schaap
P
(2012)
Evolution
of
self-organisation
in
Dictyostelia
by
adap-
tation
of
a
non-selective
phosphodiesterase
and
a
matrix
component
for
regulated
cAMP
degradation.
Development
139:1336–1345
Kiedaisch
BM,
Blanton
RL,
Haigler
CH
(2003)
Characteriza-
tion
of
a
novel
cellulose
synthesis
inhibitor.
Planta
217:922–930
Kuwayama
H,
Yanagida
T,
Ueda
M
(2008)
DNA
oligonucleotide-assisted
genetic
manipulation
increases
transformation
and
homologous
recombination
efficiencies:
evidence
from
gene
targeting
of
Dictyostelium
discoideum.
J
Biotechnol
133:418–423
McCormack
PL,
Perry
CM
(2005)
Caspofungin:
a
review
of
its
use
in
the
treatment
of
fungal
infections.
Drugs
65:2049–2068
Meima
ME,
Weening
KE,
Schaap
P
(2007)
Vectors
for
expres-
sion
of
proteins
with
single
or
combinatorial
fluorescent
protein
and
tandem
affinity
purification
tags
in
Dictyostelium.
Protein
Expr
Purif
53:283–288
Michel
G,
Tonon
T,
Scornet
D,
Cock
JM,
Kloareg
B
(2010)
The
cell
wall
polysaccharide
metabolism
of
the
brown
alga
Ecto-
carpus
siliculosus.
Insights
into
the
evolution
of
extracellular
matrix
polysaccharides
in
Eukaryotes.
New
Phytol
188:82–97
Morrison
A,
Blanton
RL,
Grimson
M,
Fuchs
M,
Williams
K,
Williams
J
(1994)
Disruption
of
the
gene
encoding
the
EcmA,
extracellular
matrix
protein
of
Dictyostelium
alters
slug
morphol-
ogy.
Dev
Biol
163:457–466
Reddy
AK,
Balne
PK,
Garg
P,
Sangwan
VS,
Das
M,
Krishna
PV,
Bagga
B,
Vemuganti
GK
(2010)
Dictyostelium
polycephalum
infection
of
human
cornea.
Emerg
Infect
Dis
16:1644–1645
Reymond
CD,
Schaap
P,
Vero n
M,
Williams
JG
(1995)
Dual
role
of
cAMP
during
Dictyostelium
development.
Experientia
51:1166–1174
Ritchie
AV,
van
Es
S,
Fouquet
C,
Schaap
P
(2008)
From
drought
sensing
to
developmental
control:
evolution
of
cyclic
AMP
signaling
in
social
amoebas.
Mol
Biol
Evol
25:
2109–2118
Roberts
AW,
Roberts
EM,
Delmer
DP
(2002)
Cellulose
synthase
(CesA)
genes
in
the
green
alga
Mesotaenium
cal-
dariorum.
Eukaryot
Cell
1:847–855
Romeralo
M,
Skiba
A,
Gonzalez-Voyer
A,
Schilde
C,
Lawal
H,
Kedziora
S,
Cavender
JC,
Glöckner
G,
Urushi-
hara
H,
Schaap
P
(2013)
Analysis
of
phenotypic
evolution
in
Dictyostelia
highlights
developmental
plasticity
as
a
likely
consequence
of
colonial
multicellularity.
Proc
Biol
Sci
280:20130976
Ronquist
F,
Huelsenbeck
JP
(2003)
MrBayes
3:
Bayesian
phylogenetic
inference
under
mixed
models.
Bioinformatics
19:1572–1574
Schultz
J,
Milpetz
F,
Bork
P,
Ponting
CP
(1998)
SMART,
a
simple
modular
architecture
research
tool:
identifica-
tion
of
signaling
domains.
Proc
Natl
Acad
Sci
USA
95:
5857–5864
Siddiqui
R,
Aqeel
Y,
Khan
NA
(2013)
Killing
the
dead:
chemotherapeutic
strategies
against
free-living
cyst-forming
protists
(Acanthamoeba
sp.
and
Balamuthia
mandrillaris).
J
Eukaryot
Microbiol
60:291–297
Sievers
F,
Wilm
A,
Dineen
D,
Gibson
TJ,
Karplus
K,
Li
W,
Lopez
R,
McWilliam
H,
Remmert
M,
Soding
J,
Thompson
JD,
Higgins
DG
(2011)
Fast,
scalable
generation
of
high-quality
protein
multiple
sequence
alignments
using
Clustal
Omega.
Mol
Syst
Biol
7:539
Sucgang
R,
Kuo
A,
Tian
X,
Salerno
W,
Parikh
A,
Feasley
CL,
Dalin
E,
Tu
H,
Huang
E,
Barry
K,
Lindquist
E,
Shapiro
H,
Bruce
D,
Schmutz
J,
Salamov
A,
Fey
P,
Gaudet
P,
Anjard
C,
Babu
MM,
Basu
S,
Bushmanova
Y,
van
der
Wel
H,
Katoh-Kurasawa
M,
Dinh
C,
Coutinho
PM,
Saito
T,
Elias
M,
Schaap
P,
Kay
RR,
Henrissat
B,
Eichinger
L,
Rivero
F,
Putnam
NH,
West
CM,
Loomis
WF,
Chisholm
RL,
Shaulsky
G,
Strassmann
JE,
Queller
DC,
Kuspa
A,
Grigoriev
IV
(2011)
Comparative
genomics
of
the
social
amoebae
Dic-
tyostelium
discoideum
and
Dictyostelium
purpureum.
Genome
Biol
12:R20
Toama
MA,
Raper
KB
(1967)
Microcysts
of
the
cellular
slime
mold
Polysphondylium
pallidum.
II.
Chemistry
of
the
microcyst
walls.
J
Bacteriol
94:1150–1153
Trabelsi
H,
Dendana
F,
Sellami
A,
Sellami
H,
Cheikhrouhou
F,
Neji
S,
Makni
F,
Ayadi
A
(2012)
Pathogenic
free-living
amoebae:
epidemiology
and
clinical
review.
Pathol
Biol
(Paris)
60:399–405
Visvesvara
GS
(2010)
Amebic
meningoencephalitides
and
keratitis:
challenges
in
diagnosis
and
treatment.
Curr
Opin
Infect
Dis
23:590–594
Wakabayashi
K,
Böger
P
(2002)
Target
sites
for
herbi-
cides:
entering
the
21st
century.
Pest
Manag
Sci
58:
1149–1154
Zhang
P,
McGlynn
AC,
Loomis
WF,
Blanton
RL,
West
CM
(2001)
Spore
coat
formation
and
timely
sporulation
depend
on
cellulose
in
Dictyostelium.
Differentiation
67:72–79
Available
online
at
www.sciencedirect.com
ScienceDirect