FEMS MicrobiologyLetters 77 (1991) 187-194 187

1991 Federation of European MicrobiologicalSocieties0378-1097/91/$03.50

ADONIS 037810979100081L
FEMSLE 04260

Effect of calcium ion uptake on Candida albicans morphology

A n n R. H o l m e s , R i c h a r d D. C a n n o n a n d M a x w e l l G. S h e p h e r d

Experimental Oral Biology Unit, School of Dentistry, Universityof Otago, Dunedin, New Zealand

Received 14 August 1990

Accepted 20 August 1990

Key words: Candida albicans; Calcium; Morphogenesis

1. S U M M A R Y 2. I N T R O D U C T I O N

Candida albicans is an opportunistic pathogen

In liquid culture using a synthetic medium,
which is polymorphic, existing as either a yeast
added magnesium but not calcium was required
form, a hyphal form, an apparently intermediate
for exponential growth of Candida albicans yeast
pseudohyphal form or as chlamydospores. Fre-
cells. However, medium without added divalent
quently it is the hyphal form that is seen in
cations supported 2 - 3 generations of yeast growth
diseased tissue and the yeast form which pre-
or germ tube induction. The addition of calcium
dominates in commensal colonisation [1]. There is
ions (1.0 mM) at any stage during the induction of
also evidence that the hyphal form is more adher-
germ tube formation caused reversion to a yeast
ent to host tissues [2], which may be a factor in the
mode of growth, in contrast to the effect of zinc
pathogenesis of candidosis.
and cobalt ions which were toxic to all growth.
The yeast and mycelial growth forms are inter-
Inhibition of germ tube formation by calcium was
convertible both in vivo and in vitro. The induc-
not observed in the presence of either magnesium
tion of the yeast to mycelial transition in vitro has
(10/~M) or manganese (100/~M). The presence of
been the subject of extensive study both as a
either of these ions caused inhibition of 45Ca up-
model of eukaryotic differentiation and because
take in yeast cultures. We conclude that unre-
the transition may have a role in pathogenesis [3].
stricted calcium uptake resulted in the specific
A number of chemical and environmental factors
inhibition of C. albicans mycelial growth, indicat-
induce the yeast to mycelial transition [3], and the
ing a critical role for calcium in the regulation of
use of gratuitous inducers [4,5] is an indication
C. albicans morphogenesis.
that signal transduction is involved. In two other
dimorphic fungi, Sporothrix schenckii and Cerato-
cystis ulmi, a calcium signal for morphogenesis has
been proposed [6,7]. The involvement of a
Correspondence to: A.R. Holmes, Experimental Oral Biology c a l c i u m / c a l m o d u l i n interaction in the yeast-
Unit, School of Dentistry, University of Otago, P.O. Box 647, mycefial transition of C. albieans has also been
Dunedin, New Zealand. suggested [8,9].
188
3. MATERIALS A N D M E T H O D S
3.1. Yeasts
The strains used in this study were as follows:
C. albicans strains ATCC 10261, N C P F 3153, A72
(serotype A) and MEN (serotype B). All strains
were propagated and maintained on yeast ex-
t r a c t / p e p t o n e / d e x t r o s e agar slopes. A simple de-
fined medium (GSB) was used for growth of C.
aIbicans in liquid culture. GSB contained the fol-
lowing (g 1-1): glucose, 10.0; (NH4)2SO4, 1.0;
KH2PO4, 2.0; MgSO 4 7H20, 0.05; CaC12 2H20,
0.05; and biotin, 0.05 mg 1-1. For examination of
the effect of divalent cations on growth and mor-
phology, a divalent cation deprived (DCD)
medium, equivalent to GSB medium without ad-
ded Mg z+ or Ca 2+, was used. The p H was ad-
justed to 6.5 with KOH. For all media, analytical
grade reagents and high grade deionised water
were used to minimise possible divalent cation
contamination. D C D medium contained < 3 # M
of Ca 2+ and Mg 2+ (combined total) as de-
termined by an EDTA titrimetric method.
3.2. Conditions for yeast and myceIial growth
Cultures grew in the yeast morphology when
shaken in GSB medium at 30C and germ tube
formation was induced in GSB at 37C and p H
6.5 after glucose deprivation, as described previ-
ously [10]. In some experiments, N-acetylglu-
cosamine (GlcNAc; Sigma) (2.5 mM) and proline
(10 mM) replaced glucose and ammonium as in-
ducers. For the determination of the effects of
divalent cations or the ionophore A23187 (Sigma)
on yeast and mycelial growth, cells were washed
twice in deionised water, and resuspended in D C D
medium with or without added divalent cations or
lonophore at the concentrations given in the text,
at the temperature of incubation for 20 rain before
addition of glucose. Yeast growth was followed by
absorbance measurement at 54Onm. Germ tube
formation was assessed by phase contrast mi-
croscopy as described previously [11].
3.3. Scanning electron microsopy
Cells were harvested by centrifugation, washed
twice in deionised water and resuspended in water
at a cell concentration of 5 106 cells m1-1. Drops
were placed on either 1 cm 2 pieces of plastic cut
from microtitre trays (Nunc) or on 13 mm nitro-
cellulose filters (0.2 /~m) and the cells allowed to
attach at 4C overnight. The attached cells were
then fixed (0.1 M sodium cacodylate buffer, p H
7.4, containing 2.5% ( v / v ) ghitaraldehyde) for 90
min at 4C, washed four times in 0.1 M sodium
cacodylate and post-fixed for 1 h at room temper-
ature in 1% osmium tetroxide. After dehydration
through a graded ethanol series (30%, 50%, 70%,
95%, 2 absolute) specimens were dried using
liquid CO 2 in a Polaron E3000 critical point dry-
ing apparatus. Specimens were sputter coated with
gold and examined in a Cambridge stereoscan 360
scanning electron microscope at an accelerating
voltage of 20 kV.
3.4. Determination of morphology index
The morphology index (MI) for cells was
calculated according to the formula MI = 2+
1.78 log10 [lsd -2] where / = l e n g t h of cell, s=
diameter at septal junction and d = maximum dia-
meter [12].
3.5. Determination of uptake of 45Cainto yeast cells
The uptake of 45Ca was assayed using a modifi-
cation of a method used with Saecharomyces
cerevisiae [13]. Mid-exponential phase yeast cells
were washed twice in deionised water and resus-
pended in 45 m M Tris/succinate buffer, p H 6.0,
at a final cell concentration of 1 108 cells m V 1.
A volume of 0.6 ml was preincubated at 30C for
20 min in the presence of 10 m M glucose, before
addition of 6 /~1 of 45Ca (Amersham, 0.22 mCi
ml -~, 1.8 mCi mg C a - l ; 7.77 MBq m1-1, 62.7
MBq mg Ca -~) final concentration 1.25 /~M. At
15-s intervals 100 /~1 samples were removed into
5.0 ml ice cold 10 m M CaC12 before collection of
ceils on Whatman G F C filters. Filters were washed
twice with distilled water and radioactivity de-
termined by scintillation counting.
4. RESULTS
4.1. Effect of added divalent cations on germ tube
formation and yeast growth
The time course and degree of germ tube for-
mation in D C D medium was the same as that in

GSB medium over a 3-h period. Table 1 shows
that in DCD medium, germ tube formation in
strains of both serotypes was abolished by the
addition of the chloride salts (1 raM) of the diva-
lent cations calcium, zinc or cobalt. Calcium
sulphate also inhibited germ tube formation.
Mycelial growth in two other strains, A72 and
NCPF 3153, was inhibited in the presence of
calcium ions and germ tube formation induced in
the presence of either GlcNAc or proline was also
calcium-sensitive.
It was observed that although germ tube forma-
tion was inhibited in the presence of calcium,
there was an increase in the A540 of these cultures,
therefore the effect of divalent cations on yeast
growth at 30C Was examined (Fig. 1). A limited
degree of growth (2-3 generations) of a washed
inoculum was observed in DCD medium. Yeast
growth in DCD medium with magnesium (1 raM)
and calcium (1 mM) was identical to that in DCD
medium with magnesium alone (1 mM). In both
cases growth was halted by glucose exhaustion, as
addition of glucose resulted in further growth,
whereas the growth observed in DCD medium
alone was not glucose limited. In contrast to the
inhibitory effect of calcium on mycelial growth,
yeast growth in DCD medium was not affected by
the addition of 1.0 mM calcium, although no
growth was observed when either zinc or cobalt (1
mM) were added to cultures in DCD medium.
We also examined the effect of the ionophore
A23187 on germ tube formation and yeast growth.
Table 1
The effect of divalent cations on germ tube formation a in
D C D medium
Cation source Strain
added (1 mM)
10261 MEN
None + +
NaC1 + +
MgC12 + +
MnC12 + +
CaC12 -- --
ZnC12 - _
CoC12 - -
a Using glucose/ammonium induction system. Results ex-
pressed as % cells with germ-tubes: + 80-100%, - < 10%.
189
6"0
iI
il
5"0 I /
3"0: :% "
z
,,
se
u~ 2 ' 0
1'0
, f , I , I , I , ~ '1I I
2 4 6 8 10 21
TIME ( h )
Fig. 1. Effect of divalent cation content of medium on yeast
growth of C. albicans, strain 10261. Glucose was added at time
zero and later to some cultures ( - - - - - ) after cessation of
growth. D C D medium was used to which divalant cations (1
raM) were added. (A) Mg 2+ and Ca2+; (zx) Mg 2+ only; ( 0 )
Ca 2+ only; (B) Zn 2+ only; (e) none. Values shown are the
mean of triplicate determinations, the S.E. did not exceed 10%.
Roy and Datta [8] reported that A23187 (4.0/~M)
inhibited germ tube formation and this inhibition
was reversed by the addition of calcium (10 raM).
We found that this concentration of A23187 in-
hibited both germ tube formation and yeast growth
in GSB medium, and the inhibition could not be
reversed by calcium ions (10 raM).
4. 2. Effect of delayed addition of calcium on germ
tube formation in the absence of magnesium
Glucose-starved yeast cultures in DCD medium
at 37C, formed germ tubes within 30 rain of the
addition of glucose as inducer, and growth con-
tinued in the mycelial morphological form for up
to 3 h (Fig. 2a). If calcium was present at induc-
tion, however, growth was in the yeast mor-
phology at a growth rate similar to that observed
at 30C. If calcium was added subsequent to the
initiation of germ tube formation, there was an
apparent reversion to a yeast mode of growth,
which took two forms; either a bud shape formed
on the end of the hypha (Fig. 2b) or the hyphal tip
swelled to a club shape (Fig. 2b,c). This effect was
190

observed when calcium was added at either 30
min, 1 h or 2 h after initiation of germ tube
formation. If incubation in the presence of calcium
was continued, separate yeast cells accumulated in
the culture. If, however, after 1 h in calcium-con-
taining DCD medium the cultures were washed,
glucose-starved and reincubated with inducer,
fresh germ tubes emerged from clubs o r mother
yeast cells. Another addition of calcium (1 mM)
again caused reversion from mycelial to yeast
growth and resulted in the appearance of some
apparent double clubs (Fig. 2d). The morphology
index (MI) [12] for a number of individual cells
was determined using measurements taken from
photoelectronmicrographs. The MI values for cells
of different morphological forms were as follows:
mother yeast cells, 1.3-1.4; hyphal cells, 4.2-4.3;
club shaped cells resulting from calcium treat-
ment, 2.7-2.8; buds emerging from hyphae after
calcium treatment, 1.7-1.8.

(a) (b)

(c) (d)
Fig. 2. Scanning electron photomicrographs of C. albicans strain 10261 showing the effect of calcium added after germ tube
formation was induced. Bars represent 5/~m. (a) Germ tube cell from 3 h culture in D C D medium. (b) Cell showing both club shaped
germ tube and a yeast cell budding from the end of a germ tube and (c) club shaped germ tube, from 3 h cultures in D C D medium to
which Ca 2+ 'was added at l h . (d)Mycelial element from culture to which Ca 2+ was added both at 1 h after initiation of germ tube
formation and after a second induction of mycelial growth. In a and d samples were fixed on nitrocellulose filters which give the
granular background on the photoelectronmicrographs.

4.3. Protective effect of magnesium and manganese
on the inhibition of mycelial growth by calcium
We have shown [10] that GSB medium can be
used for induction of mycelial growth. The con-
centration of calcium in this medium is 0.34 mM.
When germ tube formation was induced in DCD
medium to which this concentration of calcium
was added, there was a 40-60% inhibition of
germ-tube formation. Table 2 shows that the in-
hibition of germ tube formation by calcium (1.0
mM) was prevented by the addition of either
magnesium or manganese at concentrations 10-
100-fold lower than the calcium concentration.
Magnesium was protective at lower concentrations
than manganese. We determined the uptake of
calcium in experiments using 45Ca (Fig. 3). Uptake
was measured in the presence of glucose [13].
There was very little uptake in cultures on ice, the
cell associated counts under these conditions
probably represented binding to the cell surface.
Calcium uptake was inhibited by magnesium in a
concentration-dependent manner (Fig. 3). Manga-
nese at a concentration of 100/~M gave the same
degree of inhibition as 10/~M magnesium (results
not shown).
5. DISCUSSION
This paper descFibes a specific effect of calcium
ions on C. albicans morphogenesis; addition of
Table 2
Prevention of the inhibitory effect of Ca 2+ on germ tube
formation in D C D m e d i u m by Mg 2+ and M n 2 +
Cation added Concentration G e r m tube
(1 m M Ca a+ present) (raM) formation a
None m depleted, whereas in the present study mycelial
Mg 2+ 1.0 + growth was initiated within 30 min. Sabie and
0.1 + Gadd [9] reported a stimulatory effect of calcium
0.01 +/ -
0.01
M n 2+ 1.0 +
0.1 +/-
0.01
a Strain M E N using g l u c o s e / a m m o n i u m induction system.
Results expressed as % cells with germ-tubes: + 8 0 - 1 0 0 % ,
+/- 10-80%, - < 10%.
191
10
9
8
7
6
x
5
n
(3 4
3
2
1
15 30 45 60 75
TIME ( s e c )
Fig. 3. Uptake of 45Ca into yeast cultures of C. albicans strain
10261. (11) in Tris/succinate buffer only on ice; (O) in
Tris/succinate buffer only at 30C; (zx) 100/zM Mg 2+ present
at 30C; (A) 10/~M Mg 2+ present at 30C.
calcium to cultures in DCD medium caused a
switch from mycelial to yeast growth. The con-
centration of magnesium (0.20 raM) present in
GSB growth medium prevented the inhibitory ef-
fect of calcium on germ tube formation, which
explains why this observation has not been re-
ported previously. Magnesium was required for
growth, although cell associated reserves were suf-
ficient for germ tube formation or 2-3 generations
of yeast growth in a divalent cation deprived
medium (Table 1; Fig. 1). A previous study indi-
cated a requirement for magnesium ions in germ
tube formation [14]. However, with the induction
system used in that study germ tube formation
occurred only after a 2 h incubation when cell
associated magnesium reserves would probably be
on mycelial growth. However, the effect was only
observed under a specific set of conditions (tem-
perature, calcium concentration, and initial pH
and inoculum size) which in our laboratory pro-
duced only yeast growth in all strains tested.
A reversion from the mycelial to the yeast
mode of growth occurred when calcium was added
192
to 1 h germ tube cultures in D C D medium, indi-
cating that the effect of calcium was not to inhibit
growth per se but was a specific effect on growth
morphology. The switch to the yeast m o d e of
growth resulted in either a regular, spherical yeast
cell forming at the end of the hyphal element or
the formation of club shaped cells (Fig. 2b). The
formation of both types of cell is in accord with a
model of wall expansion proposed by Staebell and
Soll [15], in which cell shape depends on the ratio
of general to apical wall expansion. If the addition
of calcium changed the ratio in favour of general
wall expansion, then club-shaped cells would re-
sult if calcium was added some time after septum
formation, or yeast-shaped buds if calcium ad-
dition was synchronous with septum formation.
The MI values for yeast and mycelial cells were in
agreement with those reported by Merson-Davies
and Odds [12]. Intermediate values were obtained
for club-shaped cells. However this is due to the
transition along individual club ceils from a
mycelial to a yeast shape, which results in a mor-
phology that is distinct from pseudohyphae. Simi-
lar club-shaped "revertant phenotypes" have been
observed in mycelial cultures in which the p H of
the medium was shifted to lower values which did
not permit germ tube formation [16]. The effect of
calcium was reversible; alternate cycles of the
mycelial and yeast modes of growth could be
induced in the same fungal element by sequential
addition and removal of calcium. To date, most
studies of morphogenesis in C. albicans have ex-
amined the yeast to mycelial transition; the use of
calcium and D C D medium enables the reverse
transition to be studied in the absence of p H or
temperature changes.
In S. cerevisiae there is a common carrier for
divalent cations (although there may be other
specific carriers) [17] and the relative affinities are
magnesium > manganese > calcium. Thus if a sim-
ilar common divalent cation uptake system occurs
in C. albicans, initial calcium influx would be
greater in D C D medium than in magnesium con-
taining growth medium. This hypothesis is sup-
ported by the observation that both magnesium
and manganese inhibited uptake of 45Ca into yeast
cells (Fig. 3), a n d they abolished the inhibitory
effect of calcium o n mycelial growth, Magnesium
was more effective than manganese both for in-
hibition of calcium uptake and for the prevention
of calcium inhibition of germ tube formation.
In both yeasts and filamentous fungi there is
evidence that calcium/calmodufin interactions are
involved in site selection for wall growth and
therefore in the regulation of morphogenesis
[7,18-20]. Calcium is also important for the func-
tioning of of the yeast cytoskeleton [21] which in
turn may affect growth morphology [22]. Actin
localisation in hyphae of C. albieans differs from
that in yeast cells [23]. We propose that under
conditions in which the extracellular medium is
deprived of other divalent cations, an influx of
calcium ions disrupts the intracellular calcium bal-
ance such that general wall expansion is favoured
over apical growth. Our results indicate a role for
calcium in the control of the growth morphology
of C. albicans.
ACKNOWLEDGEMENTS
This work was supported in part by the Medi-
cal Research Council of New Zealand and by the
Wellcome Trust, London, U.K. We are indebted
to Mr K.N. Goldie for the operation of the scan-
ning electron microsope and for preparation of
samples.
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