Microbiology (2005), 151, 81–89
DOI 10.1099/mic.0.27333-0
Generation and functional in vivo characterization
of a lipid kinase defective phosphatidylinositol
3-kinase Vps34p of Candida albicans
Juliane Günther,1 Monika Nguyen,1 Albert Härtl,1 Waldemar Künkel,2
Peter F. Zipfel1 and Raimund Eck1
1
Hans-Knöll-Institute for Natural Products Research, Department of Infection Biology,
Beutenbergstrasse 11, D-07745 Jena, Germany
Correspondence
Raimund Eck
2
[email protected]
University of Applied Sciences, Tatzendpromenade 1b, D-07745 Jena, Germany
Received 14 May 2004
Revised 30 July 2004
Accepted 14 September 2004
The phosphatidylinositol (PI) 3-kinase Vps34p of Candida albicans has lipid kinase and
autophosphorylation activity and is involved in virulence and vesicular protein transport. In order
to characterize the roles of lipid kinase activity, a chimeric Vps34 protein was created which
lacks lipid kinase but retains autophosphorylation activity. To this end, six amino acids within
the putative lipid-binding site of Vps34p were replaced by the homologous region of the PI
3-kinase-like C. albicans Tor protein. The resulting chimeric Vps34T protein was recombinantly
expressed in Escherichia coli and shown to lack lipid kinase activity. The corresponding chimeric
VPS34TOR gene was inserted into the genome of C. albicans, and this lipid-kinase-defective
strain had a distinctive phenotype compared to those of the wild-type strain SC5314 and the
vps34 null mutant. The lipid-kinase-defective strain was non-virulent, and showed altered hyphal
growth, reduced adherence, as well as defective vacuole morphology and endosomal vesicle
transport. These results demonstrate an important role for the lipid kinase activity of Vps34p in
virulence and vesicular protein transport. On the other hand, the lipid-kinase-defective strain and
the vps34 null mutant differ in their temperature- and osmotic-stress response. This indicates a
possible role for activities different from the lipid kinase function of Vps34p.
INTRODUCTION
Candida albicans is the major systemic fungal pathogen in
humans (Odds, 1994). This polymorphic yeast is capable of
causing life-threatening infections in immunocompromised
patients as well as a variety of mucosal infections in healthy
individuals. Recent experimental data suggest that the
virulence of C. albicans is dependent on several properties,
including the ability of the yeast to switch between different
morphogenetic forms, host epithelial and endothelial cell
recognition and adhesion, as well as the ability to secrete
proteinases and phospholipases (Cutler, 1991; Köhler &
Fink, 1996). A number of virulence factors of C. albicans
have been characterized; however, the mechanisms that
enable this opportunistic fungus to become pathogenic have
not yet been unraveled.
temperature and hyperosmotic stress and exhibits reduced
adherence to human cells. In addition, the vps34 null mutant
displays enlarged and electron-transparent vacuoles and is
avirulent in a mouse model of systemic candidiasis (Eck
et al., 2000; Bruckmann et al., 2000; 2001).
In C. albicans, the phosphatidylinositol (PI) 3-kinase
Vps34p is involved in virulence. The C. albicans vps34
null mutant is unable to form hyphae on various solid
media, shows a significantly delayed yeast to hyphae
transition in liquid media, is hypersensitive to high
PI 3-kinases control a wide variety of cellular processes in
eukaryotic cells, including mitogenesis, protection from
apoptosis, growth factor receptor down-regulation, stimulation of glucose uptake, endocytosis, actin cytoskeleton
rearrangement and intracellular protein/membrane trafficking (DeCamilli et al., 1996; Toker & Cantley, 1997). PI
3-kinases are subdivided into three classes, based on
sequence similarities, substrate specificity and regulatory
properties (Vanhaesebroeck et al., 1997). Class I PI 3-kinases
represent dual-specificity enzymes, and display both lipid
kinase and protein kinase activity. The generation of a lipidkinase-defective human PI 3-kinase c with a defective
putative PI-binding domain was used to show the transphosphorylation of the PI 3-kinase adapter protein p101
and the MAP protein kinase MEK-1 in vitro (Stoyanov et al.,
1995; Bondeva et al., 1998; Bondev et al., 1999).
Abbreviations: FCS, fetal calf serum; HBEC, human buccal epithelial
cells; PI, phosphatidylinositol.
The class III PI 3-kinase Vps34p (Vps: vacuolar protein
sorting) represents the only PI 3-kinase activity in the yeast
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J. Günther and others
Saccharomyces cerevisiae (Schu et al., 1993; Vanhaesebroeck
et al., 1997). ScVps34p regulates intracellular protein
trafficking to the vacuole, retrograde endosome-to-Golgi
transport, autophagocytosis and vacuole acidification
(Herman & Emr, 1990; Zhou et al., 1995; Burda et al.,
2002; Kihara et al., 2001; Munn & Riezman, 1994). Two
distinct Vps34p-containing protein complexes were identified in S. cerevisiae. (Kihara et al., 2001). ScVps34p shows
lipid kinase and autophosphorylation activity, and the
protein is predominantly phosphorylated on serine residues
(Stack & Emr, 1994). ScVps34 proteins containing mutated
residues in the putative lipid kinase domain (amino acid
positions 731, 735, 736 and 749) are defective in both
autophosphorylation and lipid kinase function (Stack &
Emr, 1994; Schu et al., 1993).
In this study, we generated a Vps34T mutant protein of C.
albicans with specifically inactivated lipid kinase activity. A
Candida mutant strain containing the lipid-kinase-defective
gene was generated. The phenotype of this strain indicated
that the lipid kinase activity is essential for the virulence,
dimorphism, adhesion, vesicular protein transport and
vacuole morphology of C. albicans. Additional activities of
Vps34p, which are mainly involved in the stress response,
were shown in vivo.
METHODS
Strains and growth conditions. The C. albicans strains and plasmids used are listed in Table 1. Strains were grown in YPD medium
(2 % glucose, 2 % peptone, 1 % yeast extract), Sabouraud medium
(2 % glucose, 1 % peptone from casein) and SD medium [0?7 %
yeast nitrogen base without amino acids (Difco), 2 % glucose] at
30 uC. SD medium was supplemented with 20 mg uridine ml21 for
ura2 strains. Cell numbers were determined using a haemocytometer. Hyphal growth was induced in liquid culture by diluting lateexponential-phase cultures grown at 30 uC tenfold in fresh YPD
medium supplemented with 15 % fetal calf serum (FCS) at 37 uC.
On solid medium, hyphal growth was induced by nutrient
limitation. Cells were grown overnight in YPD at 30 uC, washed,
diluted and spread on Spider plates. Between 20 and 100 cells per
plate were incubated at 37 uC for 14 days. Sensitivities of the mutant
strains to KCl and NaCl were assayed on YPD plates. Escherichia coli
XL-1 Blue, supE44 hsdR17 recA1 endA1 gyrA46 thi relA1 lac
F9[proAB+ lacIq M15 Tn10(Tetr)] (Stratagene), was used for cloning,
whereas E. coli M15 (Qiagen) was used for protein expression.
Construction of expression plasmid pVTPEX and integration
plasmid pKEUT. To insert a KpnI restriction site in the lipid-
binding domain of VPS34, a 380 bp C-terminal fragment of VPS34
was amplified using the following primers (coding sequences are
underlined): 59-CGGGCGGCCGTGACCCTAAACCTTTGGTACCATTA-39 (KpnI site indicated in bold type; upstream) and 59GCCTGCAGTTAAGCTCTCCAATACTGTGCTAA-39 (downstream).
The XmaIII and PstI restriction sites generated by the primers were
used to clone the fragment into the Vps34p–6His-tag expression
plasmid pVPEX1 (Eck et al., 2000), yielding pVPEX2. Then, the
VPS34 coding sequence between the XmaIII and KpnI restriction
sites was replaced by synthetic double-stranded DNA consisting of
the oligonucleotides 59-GGCCGTGCAATATTACGTGAGAAGTATCCAGAGAGGGTAC-39 and 59-CCTCTCT GGATACTTCTCACGTAATATTGCAC-39 (CaTOR coding sequences – underlined – are
found in Stanford’s Candida albicans sequencing project Assembly
19, ORF19?1903 on Contig135; URL: http://sequence-www.stanford.
edu/group/candida/index.html) to generate the chimeric VPS34T
gene containing the TOR PI head-group interaction site (Fig. 1). For
the integration of the chimeric VPS34T gene in the C. albicans
genome, an integration vector was constructed. To this end, the Cterminal sequence of the VPS34T gene of pVTPEX was amplified
using the following primers (pVTPEX sequences are underlined):
59-CGGGCGGCCGTGCAATATTAC-39 (upstream) and 59-CGGGCACGTGACATATATTTATTTTTGCTCTGTTCATTTTAAGCTCTCCAATACTGTGCTAAAC-39 (downstream). The 450 bp fragment was
cloned into VPS34 reintegration plasmid pKEU1 (Bruckmann et al.,
2000) using XmaIII and PmaCI restriction sites generated by the
primers, yielding the VPS34TOR gene.
Expression
of recombinant
chimeric
CaVps34T
protein.
Induction of high-level protein expression, purification of the 6Histagged protein and Western blots with His-tag antibodies were carried out according to the standard protocols of the manufacturer
(Qiagen).
Table 1. Strains and plasmids used in this study
Strain or plasmid
C. albicans strain
SC5314
CAV3
CAV4
CAV5
CAV9
Plasmid
pKE2
pKEU1
pKEU1T
pVPEX1
pVPEX2
pVTEX
82
Genotype or description
Reference or source
Wild-type
Dvps34 : : hisG/Dvps34 : : hisG-URA3-hisG, Dura3 : : imm434/Dura3 : : imm434
Dvps34 : : hisG/Dvps34 : : hisG, Dura3 : : imm434/Dura3 : : imm434
Dvps34 : : hisG/VPS34 : : URA3, Dura3 : : imm434/Dura3 : : imm434
Dvps34 : : hisG/VPS34TOR : : URA3, Dura3 : : imm434/Dura3 : : imm434
Fonzi & Irwin (1993)
Bruckmann et al. (2000)
Bruckmann et al. (2000)
Bruckmann et al. (2000)
This work
pUC18 containing a 4?9 kb C. albicans genomic fragment bearing VPS34
As pKE2, but containing URA3
As pKEU1, but containing chimeric VPS34TOR
pQE9 containing 3?1 kb fragment with complete VPS34
As pVPEX1, but with KpnI restriction site
As pVPEX2, but with chimera VPS34T
Bruckmann et al. (2000)
Bruckmann et al. (2000)
This work
Eck et al. (2000)
This work
This work
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Microbiology 151
In vivo functions of CaVps34p
for 30 min by adding 17 ml 46 sample buffer according to Laemmli
(1970). The proteins were separated on SDS-polyacrylamide gels, dried
and autoradiographic signals were analysed using a phosphoimager.
Integration of the chimeric VPS34TOR gene in C. albicans.
The chimeric VPS34TOR gene was introduced into the vps34 null
mutant strain CAV4 (ura2) using the 6?0 kb BbvI/HindIII insert of
pKEUT (Fig. 2). This insert contains the chimeric VPS34TOR gene,
and includes upstream and downstream sequences to allow homologous recombination, as well as the URA3 gene as a selectable
marker. Transformations were performed by electroporation, as
described by DeBacker et al. (1999). Chromosomal DNA isolated
from selected clones was digested with HindIII/KpnI and analysed
by Southern hybridization with an [a-32P]dCTP-labelled EcoRI/
HindIII 4?9 kb insert of plasmid pKE2 (Bruckmann et al., 2000).
Fluorescent labelling and microscopy. Labelling with FM4-64
Fig. 1. Generation of a lipid-kinase-defective Vps34 protein.
(a) Vps34p of C. albicans contains three homology regions
(HR) and a linker region (L). The positions are indicated. The
chimeric Vps34T protein was generated by replacing the putative PI head-group interaction site of Vps34p (grey box) with
the corresponding domain of the Tor protein of C. albicans
without a lipid kinase activity, using XmaIII and KpnI restriction
sites. The changed sequence is marked black in the Vps34Tp
sequence. Identical amino acids in the three sequences are
boxed. (b) His-tagged Vps34p and Vps34Tp were expressed,
purified, identified by Western blot (W) and incubated with
[c-32P]ATP to show the autophosphorylation of both Vps34p
and Vps34Tp. (c) Lipid kinase activity of recombinant Vps34p
and chimeric Vps34T proteins. Recombinant proteins were
expressed as His-tag fusion proteins in E. coli, purified by
Ni-chelation and incubated with [c-32P]ATP and phosphatidylinositol. Phosphatidylinositol 3-phosphate (PI 3-P) separated by
TLC is indicated.
Enzyme assays. PI 3-kinase assays were carried out essentially as
described by Schu et al. (1993). Approximately 0?15 mg of recombinant protein was assayed in 50 ml reactions containing 20 mM
HEPES, pH 7?5, 10 mM MgCl2, 0?2 mg sonicated phosphatidylinositol ml21, 60 mM ATP and 0?1 mCi [c-32P]ATP ml21. The mixtures
were incubated at 30 uC for 15 min, reactions were terminated by
the addition of 80 ml 1 M HCl and lipids were extracted with 160 ml
chloroform/methanol (1 : 1). The organic phase was dried, samples
were resuspended in chloroform and spotted onto Silica gel 60 TLC
plates (Merck), and the plates were developed in a borate buffer
system (Walsh et al., 1991). Labelled phosphoinositides were analysed
using a phosphoimager.
Autophosphorylation assays were carried out as described by
Czupalla et al. (2003). Recombinant protein (200 ng) was assayed in
50 ml reactions containing 0?1 % BSA, 1 mM EGTA, 0?2 mM EDTA,
7 mM MgCl2, 10 mM MnCl2, 100 mM NaCl, 40 mM HEPES, pH 7?4,
1 mM DTT, 1 mM b-glycerophosphate, 25 mM ATP and 0?1 mCi
[c-32P]ATP ml21. The reaction was stopped after incubation at 30 uC
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[N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl)
pyridinium dibromide] (Molecular Probes) was performed as described by Vida & Emr (1995). Candida cells were grown at 30 uC to
an OD600 of 0?8 to 1?6. Cells were harvested and resuspended at
OD600 20–40 in YPD medium. The dye FM4-64 (16 nM) was added,
and the cells were incubated for 15 min at 30 uC, followed by incubation in YPD without dye at OD600 10–20 for 1 h. For microscopic
analysis, cells were placed on slides covered with a thin 1 % agarose
film. Samples were observed for fluorescence using a filter of
546 nm excitation and 575–640 nm emission (Zeiss).
Adherence assay. The ability of C. albicans strains SC5314, CAV3
and CAV9 to adhere to human buccal epithelial cells (HBEC)
in vitro were examined by a visual assay (Bailey et al., 1995). The
Candida strains were grown, washed and counted according to the
method of Bailey et al. (1995). HBEC from a male volunteer were
washed with 16 PBS (Invitrogen) and counted. A total of 107
Candida cells were incubated with 105 HBEC in 16 PBS at 37 uC
for 2 h. The cells were then transferred to microscope slides.
Adherence was expressed as the percentage of HBEC with adhering
Candida cells.
studies. Male outbred NMRI mice (HarlanWinkelmann), 6 weeks old, were housed five per cage and checked
daily. C. albicans cells were grown in Sabouraud dextrose broth at
28 uC until late-exponential phase. Cells were washed three times
and resuspended in 0?9 % NaCl. Portions (200 ml) of suspensions
containing 56106, 56105 and 56104 cells were used to infect
immunocompetent mice by intravenous injection into the lateral tail
vein. Survival was monitored for 21 days. To quantify kidney colonization by C. albicans, mice were sacrificed 3 days and 21 days after
injection, and kidneys were homogenized in 3 ml physiological NaCl
buffer. Serially diluted suspensions were then plated on YPD agar.
After 3 days growth at 28 uC, numbers of Candida colonies were
counted. Homogenized kidney material was also fixed with 10 %
formaldehyde and stained with 25 mg Calcofluor White ml21 to
detect C. albicans cells.
Virulence
RESULTS
Chimeric Vps34T protein lacks lipid kinase
activity but retains autophosphorylation activity
In order to distinguish effects regulated by lipid kinase
from additional activities, such as autophosphorylation, of
C. albicans Vps34p, a chimeric Vps34T protein containing
a mutated phosphatidylinositol (PI) binding domain was
generated. To this end, six amino acids of the putative PIbinding site of Vps34p were replaced by the homologous
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83
J. Günther and others
Fig. 2.
Integration
of
the
chimeric
VPS34TOR gene into vps34 null mutant
strain CAV4 (ura”) generated C. albicans
Vps34p lipid-kinase-defective strain CAV9.
(a) Restriction map of plasmid pKEU1T and
chromosomal locus of the disrupted VPS34
allele in the ura” vps34 null mutant strain
CAV4, illustrating the strategy for integration.
In the integration plasmid pKEU1T containing the VPS34 gene (thick solid arrow,
coding region; open boxes, non-coding
regions) and the URA3 gene, the putative PI
head-group interaction site between the
XmaIII and KpnI site was exchanged with
the corresponding region of C. albicans
TOR in accordance with the engineering of
the chimeric recombinant protein (Fig. 1a).
(b) Southern blot analysis of HindIII- and
KpnI-digested chromosomal DNA showed
proper integration of the VPS34TOR gene
into the chromosomal DNA of strain CAV4.
Lane 1, C. albicans wild-type strain SC5314;
lane 2, vps34 null mutant strain CAV4;
lane 3, VPS34 heterozygous revertant strain
CAV5; lane 4, CAV9 containing the lipidkinase-defective VPS34TOR gene. The blot
was hybridized with an [a-32P]dCTP-labelled
EcoRI/HindIII 4?9 kb fragment of plasmid
pKE2.
region of Torp of C. albicans, a related protein without lipid
kinase activity (Fig. 1a). The recombinant chimeric Vps34T
protein was expressed in E. coli, isolated by affinity
chromatography and detected by Western blot (Fig. 1b).
It was necessary to determine the lipid kinase activity of
chimeric Vps34Tp, and therefore the formation of PI 3phosphate (PI 3-P) by Vps34Tp was tested. Phosphorylated
lipids were separated by TLC and monitored using a
phosphoimager. The chimeric Vps34T protein did not form
PI 3-P in vitro, in contrast to wild-type Vps34p (Fig. 1c).
The autophosphorylation activity of chimeric Vps34Tp was
tested by assaying the incorporation of radioactive phosphate. Both Vps34Tp and the wild-type Vps34 protein
incorporate 32P, and thus both proteins are able to
autophosphorylate in vitro (Fig. 1b). These results prove
that the exchange of six amino acids within the putative PIbinding domain of Vps34p specifically inactivates the lipid
kinase, but preserves the autophosphorylation activity.
containing the chimeric VPS34TOR gene lacking a functional lipid kinase domain was generated. To this end, a
6?0 kb BbvI/HindIII fragment of pKEU1T containing the
chimeric VPS34TOR gene and the marker gene URA3 was
transformed to C. albicans vps34 ura2 null mutant strain
CAV4 (Fig. 2a). Southern analysis of HindIII/KpnI-digested
chromosomal DNA proved the proper integration of the
genes. The 7?0 kb fragment in lane 1 represents the VPS34
gene of C. albicans wild-type strain SC5314 (Fig. 2b). The
loss of this 7?0 kb fragment and the appearance of 2?5 and
2?7 kb fragments (Fig. 2b, lane 2) is consistent with the
replacement of both VPS34 alleles by hisG in strain CAV4.
Reintegration of VPS34 and URA3 into strain CAV4 is
demonstrated by the occurrence of an additional 8?4 kb
fragment in the heterozygous VPS34 revertant strain CAV5
(lane 3). In the Vps34p lipid-kinase-defective mutant strain
CAV9, this fragment is restricted by an additional KpnI
restriction site in the integrated chimeric VPS34TOR gene to
form 3?1 and 5?3 kb fragments (lane 4). These results prove
the correct integration of VPS34TOR.
Generating a lipid–kinase-defective Vps34pcontaining C. albicans mutant strain
The lipid kinase activity of Vps34p plays a role
in the virulence of C. albicans
In order to investigate the role of the lipid kinase activity of
Vps34p and that of the other activities, such as autophosphorylation, separately in vivo, a C. albicans mutant strain
The growth of the lipid-kinase-defective mutant strain
CAV9 was examined, as the growth rate is important for
the virulence of C. albicans. Both the CAV9 and CAV3
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Microbiology 151
In vivo functions of CaVps34p
mutant strains showed identical delayed growth. However,
the growth rates of the mutants were similar to the wildtype strain SC5314. The delayed growth of the mutants may
partly contribute to their avirulence. However, reduced
growth is not always connected to avirulence (Augsten et al.,
2002).
In order to identify whether the lipid kinase activity of
Vps34p is involved in virulence or not, we tested the lipidkinase-defective strain CAV9 in a mouse model for systemic
candidiasis. We found that mutant strain CAV9 was nonvirulent in mice. All mice infected with the mutant strain
survived for three weeks: even animals infected with a high
number of Candida cells (56106). An identical survival rate
has been shown for the vps34 null mutant strain CAV3
(Bruckmann et al., 2000). In contrast, all mice infected with
the same cell number of the wild-type strain SC5314 died
after 2 days, and 20 % of the mice survived for three weeks
after infection with 56104 cells. Nearly the same mortality
rate has been observed for mice infected with the VPS34
heterozygous revertant strain CAV5 (Bruckmann et al.,
2000). The avirulence of both the lipid-kinase-defective
Vps34p-containing mutant strain CAV9 and the vps34 null
mutant strain CAV3 shows the central role of the lipid
kinase in virulence (Fig. 3).
Systemic candidiasis is often associated with colonization
of internal organs, such as the kidneys, lung and liver. It is
known that in animal models of disseminated candidiasis
C. albicans exhibits a high predilection for the kidneys,
which leads to late fatalities in the course of the infection
(Odds, 1994). We have examined kidney colonization of
mice infected with 56105 cells of strains SC5314, CAV3
and CAV9 three days post infection (n=3). Kidneys of
mice bearing strain SC5314 exhibited a fungal burden of
1?78–3?236104 c.f.u. per gram kidney tissue, whereas
kidneys of mice infected with CAV3 or CAV9 showed
7?16101–3?16103 c.f.u. and 36101–1?96102 c.f.u., respectively. After 21 days, survivors of the virulence test were
checked for kidney colonization. We examined kidney
colonization of mice infected with 56104 cells of strain
SC5314 (n=2) and 56105 of strains CAV3 and CAV9
(n=3). Kidneys of mice bearing strain SC5314 exhibited
a fungal burden of 1?0–1?36106 c.f.u. per gram kidney
tissue, whereas kidneys of mice infected with CAV3 and
CAV9 showed 1?16103—1?56104 c.f.u. and 8?16102–
2?26103 c.f.u., respectively. In the kidneys of mice infected
with the mutant strains, only pseudohyphae were detected.
These results clearly showed reduced c.f.u. in the kidneys
of mice bearing the mutant strains CAV9 and CAV3, which
is in agreement with the non-virulence of these strains in
the virulence test.
The low c.f.u. of kidneys infected with the vps34 null mutant
and the Vps34p lipid-kinase-defective mutant may indicate
a lower ability of the mutants to adhere to endothelial cells
in vivo, resulting in rapid clearance from the blood. Thus,
the abilities of SC5314, CAV3 and CAV9 to adhere to HBEC
in vitro were determined in a visual assay (Bailey et al., 1995).
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Fig. 3. Pathogenicity of C. albicans Vps34p lipid-kinase-defective
strain CAV9. C. albicans Vps34p lipid-kinase-defective strain
CAV9 and wild-type strain SC5314 were tested in a mouse
model of systemic candidiasis. Survival of mice infected with
104 (&), 105 (.) and 106 ($) cells was monitored for
21 days (n=10, except 56105, where n=15).
The proportion of HBEC with adhering wild-type Candida
cells was 85 % (n=3). The proportion of HBEC with CAV3
or CAV9 cells on the surface was lower, at 45 and 50 %,
respectively.
Lipid kinase activity of CaVps34p is required for
hyphal growth
The switch between yeast and hyphae is considered
important to the virulence of C. albicans. The Vps34p
lipid-kinase-defective strain CAV9 was examined in liquid
medium and on solid medium under different hyphaeinducing conditions in order to test the influence of Vps34p
lipid kinase activity on hyphal growth.
First, hyphal growth was induced in liquid YPD medium
supplemented with serum. Under these conditions, the
mutant strain CAV9 showed delayed hyphal formation.
Moreover, approximately 80 % of the hyphae were
pseudohyphae. The delayed hyphal formation was more
pronounced in the vps34 null mutant strain CAV3 (Fig. 4a).
Identical results were obtained in liquid Spider medium
(data not shown).
Second, hyphal formation was examined on solid Spider
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medium containing mannitol as carbon source. The
lipid-kinase-defective strain CAV9 did not show hyphal
development. The wild-type strain SC5314 formed normal
mycelial colonies, which are indicative of filamentous
growth. Nearly the same phenotype was observed for
colonies of the VPS34 revertant strain CAV5 (Bruckmann
et al., 2000). In general, the mutant strain CAV3 also showed
a defect in hyphal growth. However, the mutant strains
showed differences in colony morphology. The mutant
strain CAV9 formed smooth colonies and CAV3 mutant
colonies exhibited a rough surface (Fig. 4b). The same
differences were observed on solid YPD plates supplemented
with serum (data not shown). Microscopic examination
showed an identical yeast form morphology of CAV9 and
CAV3 cells (data not shown).
The differences in hyphal growth observed between the
lipid-kinase-defective strain CAV9 and the wild-type strain
indicate that the lipid kinase activity of Vps34p regulates the
hyphal formation of C. albicans.
Lipid kinase activity of Vps34p is involved in
transport of prevacuolar endocytic
compartments to the vacuole and vacuole
morphology
In the yeasts S. cerevisiae and C. albicans, Vps34p is involved
in the transport of prevacuolar vesicles to the vacuole during
the overlapping late steps of endocytosis and the CPY
(carboxypeptidase Y) protein transport pathway from the
late Golgi to the vacuole (Wurmser & Emr, 1998;
Bruckmann et al., 2001). This function may be analysed
by following the distribution of the fluorescent lipophilic
dye FM4-64. In yeast, this dye allows endocytotic uptake and
vesicle-mediated transport to the vacuole to be monitored
(Vida & Emr, 1995). In order to investigate the influence of
the lipid kinase activity of Vps34p in this process, the
endocytic transport of the dye was analysed in the mutant
strain CAV9, containing the lipid-kinase-defective Vps34T
protein, and was compared to the distribution in the C.
albicans wild-type strain and the vps34 null mutant strain
CAV3 by fluorescence microscopy (Fig. 5). The mutant
strain CAV9 showed weak fluorescent staining of the
vacuole membrane, but prevacuolar endocytic compartments in the cytoplasm were stained. In the wild-type strain
SC5314, the dye had reached the vacuole, and a typical ring
staining pattern was observed. This difference indicates that
the lack of lipid kinase activity in the mutant strain CAV9
results in defects in the transport/fusion of prevacuolar
endocytic vesicles to the vacuole. Thus, the lipid kinase
activity of Vps34p seems necessary for a late step in
endocytic protein transport to the vacuole.
In addition, enlarged vacuoles were observed in the Vps34p
lipid-kinase-defective strain CAV9. In contrast, the wildtype strain showed normal vacuoles. This difference
indicates that the lipid kinase activity of Vps34p is involved
in maintaining normal vacuole morphology. This was also
shown by the identical enlarged vacuoles in both mutant
strains (Fig. 5).
Activities of Vps34p different from the lipid
kinase function are involved in hightemperature and osmotic stress response
Fig. 4. Hyphal growth of C. albicans Vps34p lipid-kinasedefective strain CAV9, wild-type strain SC5314 and vps34 null
mutant strain CAV3. (a) Hyphal growth of C. albicans strains in
liquid medium supplemented with 15 % FCS at 37 6C. A value
of 100 % hyphae means that all yeast cells have either formed
true hyphae or pseudohyphae. (b) Phenotypes of colonies of
the Vps34p lipid-kinase-defective strain CAV9, the wild-type
strain SC5314 and the vps34 null mutant strain CAV3 using
solid Spider medium containing mannitol as carbon source.
86
The C. albicans vps34 null mutant strain CAV3 is more
sensitive to high temperature and hyperosmotic stress than
the wild-type strain SC5314 (Bruckmann et al., 2000).
Therefore, the contribution of the lipid kinase activity of
Vps34p to high-temperature and osmotic stress responses
was determined. The growth of the C. albicans Vps34p lipidkinase-defective strain CAV9, the wild-type strain SC5314,
the VPS34 revertant strain CAV5 and the vps34 null mutant
strain CAV3 was assayed on YPD plates at 30, 38 and 40 uC.
The osmotic sensitivity was examined on solid YPD medium
containing NaCl or KCl at 1?2 M or 1?5 M. The mutant
strain CAV9 showed a reduced stress resistance compared
to the wild-type strain and the revertant strain CAV5.
However, the vps34 null mutant strain CAV3 was clearly
more sensitive to high-temperature and osmotic stress than
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Microbiology 151
In vivo functions of CaVps34p
little is known about the specific role of each of the two
enzymic functions in vivo (Bondeva et al., 1998).
Here, we describe the exchange of six amino acids from the
putative PI-interacting domain of Vps34p with the homologous region of the PI 3-kinase-like Tor protein, resulting
in the chimeric Vps34T protein. Probably, the exchange of
the interaction domain blocks binding of the substrate PI to
Vps34Tp, while autophosphorylation is not influenced. This
is surprising, because the change of asparagine, localized
eight amino acids upstream from the first amino acid of the
exchanged region, causes the loss of autophosphorylation
activity in Vps34p of S. cerevisiae (Stack & Emr, 1994).
Fig. 5. Characterization of the endocytic protein transport pathway by following the distribution of the lipophilic dye FM4-64
in C. albicans Vps34p lipid-kinase-defective strain CAV9,
vps34 null mutant strain CAV3 and wild-type strain SC5314.
Cells were pulsed for 30 min with the fluorescent endocytic
dye FM4-64, and then incubated for 60 min without dye.
Differential interference contrast (DIC) and fluorescence microscopy (FM4-64) showed that the lipophilic dye FM4-64 accumulated in the mutant strains CAV9 and CAV3 in prevacuolar
compartments (arrows) in the cytoplasm. The wild-type strain
SC5314 showed a typical ring staining pattern of the vacuole
membrane.
the CAV9 strain (Fig. 6). These findings suggest that an
activity distinct from the lipid kinase activity of Vps34p
plays a role in the stress response of C. albicans.
DISCUSSION
C. albicans is one of the major human pathogenic fungi.
The phosphatidylinositol (PI) 3-kinase Vps34p was characterized as important for virulence in a mouse model for
systemic candidiasis. Therefore, we were interested to assay
which activity of Vps34p is necessary for virulence.
In general, manipulations of the lipid-binding domain of
the PI 3-kinase Vps34 interfere with both lipid kinase and
protein kinase activity (Wymann et al., 1996; Stack & Emr,
1994). In human cells, a lipid-kinase-defective PI 3-kinase c
with remaining protein kinase function was constructed, but
http://mic.sgmjournals.org
The chimeric Vps34T protein lacking lipid kinase activity
but retaining autophosphorylation activity allows the
separate study of the role of the lipid kinase activity of
the PI 3-kinase Vps34p. A C. albicans lipid-kinase-defective
strain, CAV9, was generated, which has the chimeric
Vps34TOR gene integrated. This gene encodes a protein
which specifically lacks lipid kinase activity. Otherwise, it is
identical to the wild-type Vps34p. Thus, the Vps34p lipidkinase-defective strain CAV9 and the VPS34 revertant strain
CAV5, which shows a phenotype nearly identical to that
of the wild-type strain SC5314, differ in only six amino acids
in the VPS34 gene; this causes the lack of Vps34Tp lipid
kinase activity in the mutant strain CAV9, although the
autophosphorylation activity is maintained. The differences in the phenotypes observed here between the mutant
strain CAV9 and the CAV5 or wild-type strain indicate
an important role for the lipid kinase activity of Vps34p,
as most likely this activity was specifically eliminated.
Therefore, the phenotypic differences suggest that lipid
kinase activity plays a role in virulence, hyphal growth and
adhesion, as well as in vacuole morphology and vesicular
protein transport to the vacuoles. This conclusion is
confirmed by several defective features of the vps34 null
mutant strain CAV3 which are identical to the phenotypes
observed for the mutant strain CAV9. These phenotypes
are likely connected to the lipid kinase activity of Vps34p,
because both strains lack this activity.
However, minor differences exist between the Vps34p lipidkinase-defective strain CAV9 and the vps34 null mutant
strain CAV3 regarding hyphal formation. The delay of
hyphal development of the mutant strains CAV9 and CAV3
may be connected to delayed growth. However, the delay
of hyphal growth is lower for CAV9 than for CAV3. In
addition, although both mutant strains do not form hyphae
on hyphae-inducing solid agar, their colony morphology
differs clearly. As both CAV9 and CAV3 are avirulent in a
mouse model of systemic candidiasis, the differences in
hyphae growth and colony morphology do not seem to be
important for virulence.
The different phenotypes observed between the Vps34p
lipid-kinase-defective strain CAV9 and the vps34 null
mutant strain CAV3 are indicative of additional activities
for the Vps34 protein in vivo because the lipid kinase activity
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87
J. Günther and others
30 ºC
1.2 M KCl
38 ºC
40 ºC
1.5 M KCl
SC
CAV3
5314
1.2 M NaCl
1.5 M NaCl
CAV9 CAV5
Fig. 6. Stress sensitivity of C. albicans
Vps34p lipid-kinase-defective strain CAV9.
The growth of lipid-kinase-defective strain
CAV9 was compared with that of the wildtype strain SC5314, the VPS34 heterozygous revertant strain CAV5, as well as
vps34 null mutant strain CAV3, on YPD
plates at 30, 38 and 40 6C and on plates
incubated at 30 6C containing NaCl or KCl
(1?2 M or 1?5 M).
was absent in both CAV9 and CAV3. These phenotypes are
connected to stress response.
Bailey, A., Wadsworth, E. & Calderone, R. (1995). Adherence of
The generation of Vps34Tp, which lacks lipid kinase but
retains autophosphorylation activity, allowed the assay of
the role of lipid kinase activity in vivo. Moreover, the
functions of lipid-kinase-independent activities in the stress
response were shown. These results indicate that the lipid
kinase activity of Vps34p plays a role in virulence, and will
help to identify the proper signalling pathways required for
virulence.
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Candida albicans to human buccal epithelial cells: host-induced
protein synthesis and signaling events. Infect Immun 63, 569–572.
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ACKNOWLEDGEMENTS
The authors thank B. Frais, U. Stöckel and B. Weber for technical
assistance in the virulence test. This work was supported by the
Deutsche Forschungsgemeinschaft.
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