From the Institut für Mikrobiologie und
Biologisch-Medizinisches Forschungszentrum,
Heinrich-Heine-Universität, D-40225 Düsseldorf,
Germany, § Lehrstuhl für Zellbiologie und
Pflanzenphysiologie, Universität Regensburg, 93040 Regensburg,
Germany, and the ¶ Institute for Antiinfectiva Research, Bayer
AG, 42117 Wuppertal, Germany
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ABSTRACT |
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Protein mannosylation by Pmt proteins initiates O-glycosylation in fungi. We have identified the PMT1 gene and analyzed the function of Pmt1p in the fungal human pathogen Candida albicans. Mutants defective in PMT1 alleles lacked Pmt in vitro enzymatic activity, showed reduced growth rates, and tended to form cellular aggregates. In addition, multiple specific deficiencies not known in Saccharomyces cerevisiae (including defective hyphal morphogenesis; supersensitivity to the antifungal agents hygromycin B, G418, clotrimazole, and calcofluor white; and reduced adherence to Caco-2 epithelial cells) were observed in pmt1 mutants. PMT1 deficiency also led to faster electrophoretic mobility of the Als1p cell wall protein and to elevated extracellular activities of chitinase. Homozygous pmt1 mutants were avirulent in a mouse model of systemic infection, while heterozygous PMT1/pmt1 strains showed reduced virulence. The results indicate that protein O-mannosylation by Pmt proteins occurs in different fungal species, where PMT1 deficiency can lead to defects in multiple cellular functions.
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INTRODUCTION |
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Candida albicans is the leading cause of superficial and systemic fungal infections of humans. Virulence of C. albicans is a composite of several characteristics, including adaptation to body temperature, adhesion, and penetration of epithelial cells as well as its ability to undergo morphogenetic changes, especially between a yeast and a filamentous growth form (1, 2). Conceivably, defects in protein glycosylation may have profound effects on the physical stability and function of the fungal cellular surface and may also affect the secretion efficiency and the activity of secreted soluble proteins.
Present evidence suggests that O-glycosylation proceeds
differently in fungal and in higher eukaryotic cells. The first
O-glycosylation step in the yeast Saccharomyces
cerevisiae, which is catalyzed by protein mannosyltransferases
(Pmt)1 (3), occurs in the
endoplasmic reticulum and consists of the cotranslational transfer of
mannose from Dol-P-Man to serine or threonine residues; further
mannosylation extension reactions occur in the Golgi (reviewed in Refs.
4 and 5). In C. albicans, the majority of
O-glycosyl chains consist of two or three mannoses, but they
can extend up to about seven mannoses in -glycosidic linkages (6).
Dol-P-Man-dependent O-glycosylation of secreted proteins has been observed in other yeast species and filamentous fungi
(7-9). In contrast to fungi, most O-glycosylation reactions in higher eukaryotic cells including human cells proceed in the Golgi
and commence with the attachment of N-acetylgalactosamine to
proteins (10). However, some mammalian proteins also appear to be
O-mannosylated (11, 12).
A total of seven homologous PMT genes have been identified in the genome of S. cerevisiae (13-17). The PMT1 and PMT2 gene products appear to form a heterodimer that is necessary for enzymatic activity in vitro and in vivo (18). Deletion of PMT1 does not affect viability but leads to cells that tend to aggregate, especially in a pmt2 genetic background (16, 17). Inactivation of both PMT1 and PMT2 causes defects in growth and resistance to killer toxin K1 (16), while some combinations of PMT triple mutants are inviable (19). Recently, a genomic sequence of Schizosaccharomyces pombe has been reported, which encodes a Pmt homologue (GenBankTM accession number Z99126). In Drosophila melanogaster, a gene encoding a protein with high homology to the yeast Pmt1 proteins has been described (20). Furthermore, several "expressed sequence tags" deposited at GenBankTM are derived from genes encoding putative Pmt proteins of human, mouse, rat, Caenorhabditis elegans, and rice cells.
In this report, we describe the C. albicans homologue of the S. cerevisiae PMT1 gene and characterize its function. PMT1 deletion in C. albicans leads to a lack of in vitro Pmt activity and affects two secreted proteins, Als1p and chitinase. In addition, more drastic in vivo phenotypes compared with S. cerevisiae are observed, including a complete block of morphogenesis in some inducing conditions, an increased sensitivity to some antifungal compounds, and a decreased adherence to host cells. Furthermore, PMT1 function is shown to be essential for the virulence of C. albicans in a mouse model of infection.
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MATERIALS AND METHODS |
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Strains and Growth Conditions--
PMT alleles of
C. albicans and S. cerevisiae are designated
CaPMT and ScPMT respectively, whenever necessary
for their distinction. Strains and plasmids are listed in Table
I. C. albicans strain CAI4
(21) was used for transformations and gene disruptions. Strains were
grown in YPD or SD medium (22), which for Ura strains was
supplemented with 20 µg/ml uridine. YPGal medium is identical to YPD
except that it contains 2% galactose and 0.2% glucose as carbon
sources. Growth was monitored by measuring the optical density at 600 nm of cultures (A600) using a Novaspec II
photometer (Amersham Pharmacia Biotech); cells were dispersed by
sonification (1 min) in a bath sonifier (Bandelin Sonorex TK52) before
measurements. Transformations were performed using the spheroplast or
lithium acetate methods (22). Hyphae were induced by diluting washed
cells growing exponentially at 30 °C into 5% serum at 37 °C;
alternatively, washed cells were first starved in salt base (SB)
(0.45% NaCl; 0.335% yeast nitrogen base without amino acids (Difco));
10 mM proline at pH 5.5 for 3 h, followed by dilution
(A600 = 0.5) into SB (pH 6.5) containing 2.5 mM GlcNAc at 37 °C.
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Cloning and Sequencing of PMT1--
A partial genomic DNA
library of C. albicans CAF3-1 (21) was constructed by
digesting DNA with HindIII and insertion of 5-6-kb
fragments into YEplac195 (23). E. coli DH5 transformants carrying the genomic library were analyzed by colony hybridization using a 2.5-kb EcoRV-XhoI fragment containing
the S. cerevisiae PMT1 gene (17) as a probe. Plasmid pCT1
was found to strongly hybridize to ScPMT1 and to contain a
5.5-kb C. albicans insert. Subfragments of the pCT1 insert
were ligated into pUC19 and sequenced from both ends using forward and
reverse primers or by using insert-specific oligonucleotides. The
5.5-kb HindIII CaPMT1 fragment was inserted into
the HindIII site of pRC18 (24) to generate replicating C. albicans vectors (resulting in plasmids pCT29 and pCT30
with inverse insert orientation); alternatively, the CaPMT1
fragment was inserted into the HindIII site of YEplac112 or
YCplac22 (23) to construct a high copy (pCT31) or a low copy vector
(pCT32) for transformation of S. cerevisiae. To place
CaPMT1 under transcriptional control of a defined S. cerevisiae promoter, the CaPMT1 coding region was
amplified by PCR, using the primers pcP1-9Bam
(5'-TTAGGATCCCATTCAATATGGCAA-3') and
pcP1-10Hind (5'-TTAAAGCTTAAGTTTTCATCTACAAC-3'), by which a
BamHI site (italic type) is placed 9 bp upstream of the ATG
start codon (underlined), and a HindIII site (italic type) is placed 17 bp downstream of the stop codon. The
BamHI-HindIII CaPMT1 fragment was
placed downstream of the GAL1 promoter (the EcoRI-BamHI fragment of pBM150 (25)), and the
promoter fusion was inserted, as an
EcoRI-HindIII fragment, between the
EcoRI and HindIII sites of YCplac22 or YEplac112
(23) to generate plasmids pCH1 or pCH2. pCT28 contains the 7.1-kb
BamHI-XhoI genomic ScPMT1 fragment
(17) as an SacI-SphI insert in YCplac22.
Gene Disruptions--
In preparation for disruption of
PMT1, a pUC19 derivative (pCT5) containing 2 kb of the
PMT1 promoter region and 2 kb of the coding region on a
HindIII-HincII fragment was used as template for
"divergent" PCR using the oligonucleotides A
(5'-TTAGTCGACATTGAATGGGAAACT) and B (5'-TTAAGATCTTTAGAGCCGGATTGC).
Oligonucleotide A corresponds to nucleotides 1-15 relative to the
ATG in the PMT1 sequence and also adds a SalI
site to the PCR product, while oligonucleotide B corresponds to
nucleotides 975-990 and adds a BglII site. The 6.7-kb PCR
fragment was cut with SalI and BglII and ligated
to the 4-kb SalI-BglII "URA blaster"
fragment of plasmid p5921 (26). From the resulting plasmid, pCT23, a
6.7-kb Asp718-SpeI was isolated that was used to
transform strain CAI4 to prototrophy. Correct insertion of the URA
blaster into one of the two PMT1 alleles was verified by
Southern blots on DNA of transformants, which was cut with
HindIII and probed with a 0.7-kb XbaI fragment
derived from the PMT1 promoter region or a 1.3-kb
NheI-BamHI fragment from p5921 carrying hisG.
A pmt1
::hisG-URA3-hisG/PMT1 strain was plated
out on media containing 0.02% 5-fluoroorotic acid. Spontaneous
5-fluoroorotic acid-resistant strains were analyzed for loss of the
URA3 sequence by Southern blotting. One of several identified strains, CAP1-31, of the genotype
pmt1
::hisG/PMT1 was used for a second round of
gene disruption using the original URA blaster fragment. Several
transformants had the genotype
pmt1
::hisG/pmt1
::hisG-URA3-hisG, and strain CAP1-312 was chosen to identify strains by 5-fluoroorotic acid resistance (21). Strain CAP1-3121 is a representative of mutant
strains of the genotype
pmt1
::hisG/pmt1
::hisG. The
CaPMT1 gene was reintroduced in CAP1-3121 by transformation
with plasmid pCT29 or pCT30.
Assay of Pmt1p Enzyme Activity--
Cells were grown in 50 ml of
YPD or SD to an A578 of 0.5-1.0. 40 OD units of
cells were harvested and washed once with 10 ml of ice-cold TM buffer
(50 mM Tris-HCl, pH 7.5, 0.3 mM
MgCl2). The cells were resuspended in 100 µl of TM buffer
containing protease inhibitors (1 mM phenylmethylsulfonyl
fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 50 µg/ml
N-tosyl-L-phenylalanine chloromethyl ketone,
0.25 mM
N-p-tosyl-L-lysine
chloromethyl ketone, 20 µg/ml antipain, 1 mM benzamidine)
and transferred to a 1.5-ml microcentrifuge tube. An equal volume of
glass beads was added, and the cells were lysed by vortexing eight
times in intervals of 1 min with a 1-min incubation on ice. The lysate
was collected into a fresh microcentrifuge tube. Cell debris was
pelleted by centrifugation (5 s/4 °C), and the supernatant was
transferred to a new tube. Membranes were collected by centrifugation
at 48,000 × g for 30 min at 4 °C and were then
resuspended in 100 µl of 50 mM bicine, pH 7.7, 1 mM EDTA, 33% glycerol, and protease inhibitors. Microsomal
membranes were stored in liquid nitrogen.
Adherence to Epithelial Cells-- Human colon carcinoma cells (Caco-2) (30, 31) were grown to confluency in Dulbecco's modified Eagle's medium containing 20% fetal calf serum, 0.45% glucose, 292 µg/ml glutamine, 1 mM sodium pyruvate, 1% nonessential amino acids, 50 µg/ml streptomycin, and 50 µg/ml penicillin G (Life Technologies, Inc.) at 37 °C (5% CO2). Monolayers established in culture dishes (well diameter of 60 mm) were used 2-4 days after confluency for adhesion studies. Adhesion was determined according to Rotrosen et al. (32) and Fratti et al. (33). Briefly, monolayers were washed twice with 2 ml of phosphate-buffered saline, followed by the addition of approximately 200 C. albicans cells in 1 ml of phosphate-buffered saline. Cells were incubated at 37 °C for 45 min in an atmosphere of air containing 5% CO2. Following the incubation, monolayers were washed once with 5 ml of phosphate-buffered saline to remove nonadhering cells; the monolayer in each well was then covered by 2 ml of YPD agar (1% agar). Yeast colonies appearing after 48 h of growth at 30 °C were counted. As a control, fungal cells were plated directly on YPD.
Animal Experiments-- Virulence studies were performed as described, using 8-week-old, male CFW-1 mice (Halan-Winkelmann, Paderborn, Germany) (34). Because of the aggregation of Capmt1 mutants, all strains were sonicated for 5-10 min in a bath sonifier (Bandelin Sonorex TK52) before injection; as determined by plating efficiency before and after sonication, viability was not affected by this treatment. Survival curves were calculated according to the Kaplan-Meier method using the Prism (TM) program (GraphPad Software Inc., San Diego) and compared using the log-rank test. A p value <0.05 was considered significant. To quantify colony-forming C. albicans units in kidneys, mice were sacrificed 48 h after injection, and kidneys were homogenized in 5 ml of phosphate-buffered saline, serially diluted, and plated.
Other Procedures-- S. cerevisiae PMT genes were used as heterologous probes in Southern blots with C. albicans DNA; the 2.5-kb XhoI-EcoRV fragment of pDM3 carrying PMT1 (17), the 2.2-kb BglII-XhoI fragment of Yep352-PMT2 carrying PMT2 (16), the 1.7-kb SacI-BamHI fragment of pJK10 carrying PMT3 (15), the 0.9-kb SacI-BamHI fragment of pJK9 carrying PMT4 (15), and the 1.2-kb BamHI-EcoRI fragment of pUC18/PMT5 carrying PMT5 (GenBankTM accession number X92759). The coding regions of PMT6 and PMT7 were used as probes. The known sequences of PMT6 (GenBankTM accession number Z49133) and PMT7 (GenBankTM accession number U28374) were used to amplify their coding regions by PCR and insert them into pUC18 (resulting plasmids pUC18-PMT6 and pUC18-PMT7, respectively). Filters were hybridized in 5× SSC at 50 °C and washed twice in 1× SSC at 50 °C.
RNA blots were performed as described using the 2.2-kb XbaI fragment of pCT5 carrying PMT1 or the 1.5-kb SalI-ClaI ACT1 fragment (24) as probes. For immunodetection of Als1p, cells were grown in SD medium to an A600 = 3-5 at 30 °C. Cells were harvested by centrifugation, resuspended in 10% trichloroacetic acid, 10 mM Tris-HCl (pH 8.0), 25 mM ammonium acetate, 1 mM EDTA and disrupted using glass beads (35). After centrifugation at 13,000 rpm for 30 min (4 °C), the supernatant was discarded, and the pellet was solubilized in a small volume of 8 M urea, 100 mM sodium phosphate, 10 mM Tris-HCl (pH 7.0); an equal volume of SDS-PAGE sample buffer (2×) was added, and samples were heated for 5 min at 65 °C Proteins in the solubilzed extracts were separated by SDS-PAGE (5% acrylamide) and transferred to Immobilon P (Millipore Corp.) filters. Als1p was visualized by standard procedures after reaction with anti-Als1 antiserum (1:10,000), followed by anti-rabbit IgG coupled to horseradish peroxidase and staining by the SuperSignal ULTRA substrate (Pierce). Monoclonal antibody 1B12 was kindly supplied by R. Sentandreu (36); polyclonal antibodies against the gp115 protein, the Pma1 protein, the Cdr1 protein and the Int1 protein were gifts by L. Popolo, A. Goffeau, D. Sanglard, and M. Hostetter, respectively (37-40). Chitinase was assayed essentially as described by McCreath et al. (41) using 4-methylumbelliferyl- ![]() |
RESULTS |
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Sequence of CaPMT1-- Each of the seven S. cerevisiae PMT genes was used to detect homologous genes in C. albicans genomic DNA by low stringency Southern hybridization. With genomic DNA cut by different restriction enzymes, only two patterns of hybridization were obtained. PMT1 and PMT5 probes yielded one pattern (Fig. 1A), while use of the PMT2, PMT3, and PMT6 genes resulted in an alternative pattern (Fig. 1B), and PMT4 and PMT7 did not show any hybridization. Since only a single hybridizing band was obtained in each case, these results suggested that only two PMT genes, designated PMT1 and PMT2, exist in C. albicans, although the possibility of the presence of less homologous genes could not be excluded. Previously, we had pointed out the sequence-relatedness of the S. cerevisiae PMT1 and PMT5 genes as well as the PMT2, PMT3, and PMT6 genes (13).
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CaPMT1 Complements the S. cerevisiae pmt1 Mutation--
To test if
CaPmt1p functions in S. cerevisiae, we constructed a low
copy centromeric and a high copy S. cerevisiae vector containing CaPMT1 (plasmids pCT32 and pCT31, respectively).
In addition, the CaPMT1 coding region was placed under
transcriptional control of the GAL1 promoter in low and high
copy expression vectors (pCH1 and pCH2, respectively). All plasmids
were transformed into the S. cerevisiae pmt1 mutant
B76pmt1, and transformants were grown in YPGal (to induce the
GAL1 promoter). Chitinase in the culture medium was adsorbed
to chitin, separated by SDS-PAGE, and silver-stained (42). As expected,
chitinase of the B76
pmt1 strain showed a faster electrophoretic
mobility compared with the parental wild-type strain B76 (Fig.
3), which is due to a partial defect in
O-glycosylation (17, 42). In transformants carrying either
the S. cerevisiae or the C. albicans PMT1 genes on plasmids, the wild-type migration of chitinase was restored. A
complementation of the pmt1 defect was obtained by
CaPMT1 present on low or high copy vectors and either
containing the authentic promoter or the strong S. cerevisiae
GAL1 promoter.
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Disruption of CaPMT1 Alleles--
To assess the function of
CaPMT1, we constructed strains lacking one or both
PMT1 alleles in C. albicans strain CAI4.
According to an established protocol (21), one allele in strain CAI4
was disrupted by a hisG-URA3-hisG cassette, generating
strain CAP1-3 (Fig. 4, top).
A Ura derivative, strain CAP1-31, with the
genotype PMT1/pmt1
::hisG was identified, and its remaining wild-type allele was again deleted to
construct strain CAP1-312 lacking CaPMT1 wild-type alleles (pmt1
::hisG-URA3-hisG/pmt1
::hisG).
Finally, a Ura
derivative of this strain, CAP-3121,
was isolated
(pmt1
::hisG/pmt1
::hisG). For each strain, the expected genetic configuration at the
PMT1 locus was verified by Southern blotting (Fig. 4,
bottom). In genomic DNA cut with HindIII and
EcoRV (Fig. 4A, bottom) the wild-type allele yielded a 5.5-kb band (lane 1), and the
disrupted allele resulted in a 3.4-kb band (lanes
2-5). To distinguish pmt1 alleles containing the
full-length disruption cassette from alleles only containing
hisG genomic DNA of strains was cut with HindIII
and probed with hisG (Fig. 4B,
bottom). The wild-type strain did not show a signal
(lane 6), as expected, but the allele containing the full-length disruption cassette was detectable at 8.5 kb
(lane 7), while the hisG-disrupted
allele had a fragment length of 5.5 kb (lane
8).
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pmt1 Strains Are Deficient in in Vitro Pmt Activity-- The complementation of the S. cerevisiae pmt1 defect by CaPMT1 already had suggested that CaPMT1 encodes a functional Pmt protein. To provide further evidence for this assumption, we tested membrane proteins of the parental and disruptant strains for their ability to O-mannosylate a peptide substrate using Dol-P-Man as the activated sugar (29, 43).
Membrane proteins of the wild-type strain SC5314 showed high levels of mannosylation activity, while the activity in the homozygous pmt1 mutant (CAP1-312) was low in comparison, amounting to 1-4% (Table II). Reintroduction of CaPMT1 into the pmt1 background restored in vitro O-mannosylation activity. The pmt1/PMT1 heterozygous strain showed an activity intermediate between the activities in the pmt1 mutant and wild-type cells, indicating an effect of gene dosage on enzymatic activity. Thus, the results of these in vitro enzymatic tests and the in vivo complementation (see above) strongly suggest that CaPMT1 encodes a functional Pmt protein.
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CaPmt1p Affects Growth and Secreted Proteins--
Strains carrying
deleted PMT1 alleles were viable and did not show gross
cytological defects. However, in contrast to the wild-type strain
SC5314, the heterozygous strain CAP1-3, and the retransformed mutant
CAP1-3121(pCT30), cells of the homozygous mutant CAP1-312, lacking
both functional PMT1 alleles, formed aggregates (Fig.
5), which at culture densities
A600 < 1 could be separated into single cells
by mild ultrasonic treatment using a bath sonifier. Once disaggregated,
however, pmt1 cells remained separate and did not
reaggregate, suggesting that aggregates of pmt1 strains
arise because of a defect in cell separation after cell division rather
than by autoaggregation of individual cells. Growth of the homozygous
disruptant strain CAP1-312 was reduced compared with the other
strains. For strains SC5314, CAP1-3, and CAP1-312, doubling times in
SD or YPD medium were 100 or 70 min, 100 or 70 min, and 150 or 90 min,
respectively. This phenotype differs from pmt1 mutants of
S. cerevisiae, which are not compromised for growth and do
not form aggregates in several genetic backgrounds (e.g.
B76pmt1). Simultaneous deletion of both PMT1 and
PMT2 are required to generate a comparable aggregation
phenotype in S. cerevisiae (16, 19) as in the C. albicans pmt1 strains.
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Both CaPMT1 Alleles Are Required for Hyphal
Morphogenesis--
Hyphal development in C. albicans is
induced on certain solid media after several days of growth (46) or
within minutes by positive stimuli such as serum or GlcNAc (2). As
expected, lateral hyphae began to emerge from colonies of the wild-type SC5314 and the retransformed mutant CAP1-3121(pCT30) after prolonged growth on "spider" plates (Fig. 7,
A and D). In contrast, mutant CAP1-312 was
unable to form hyphae (Fig. 7B), and the heterozygous strain
CAP1-3 showed an intermediate phenotype (Fig. 7C). Only Ura+ strains were compared in this experiment, since
Ura strains are known not to undergo morphogenesis in the
absence of uridine (47).
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Pmt1 Mutants Are Supersensitive to Some Antifungals-- Multiple pmt mutations are known to cause increased antifungal sensitivities in S. cerevisiae (19). Therefore, we tested susceptibility to several antifungal compounds of the C. albicans wild-type and the constructed heterozygous and homozygous pmt1 mutant strains (Fig. 8).
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CaPmt1p Is Required for Adherence to Epithelial Cells-- Conceivably, membrane and cell wall proteins that are O-glycosylated by CaPmt1p could be essential for C. albicans virulence, by mediating adherence to and migration across epithelial cell layers. To test this possibility, we assayed adherence of C. albicans strains to monolayers of Caco-2 epithelial cells (30, 31) (Table IV). In this assay, fungal cells, which had been briefly dispersed by a mild ultrasonification, were placed on the epithelial monolayer for 45 min, after which nonadhering cells were removed by washing; numbers of tightly adhering cells were determined by growth in a YPD agar overlay.
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Both CaPMT1 Alleles Are Required for Virulence of C. albicans-- To determine directly, if CaPMT1 is involved in pathogenicity of C. albicans, we infected mice (n = 15) intravenously with wild-type and mutant strains, and the survival of animals was tested. Because of the aggregation behavior of C. albicans pmt1 mutants, strains were briefly sonicated prior to injection to ensure that animals were infected with equal cell numbers. Determination of plating efficiencies before and after sonification indicated that this treatment did not affect cellular viability.
Infection with the wild-type strain SC5314 resulted in rapid death (mean survival of 4 days), while deletion of both PMT1 copies (strain CAP1-312) led to complete loss of virulence (Fig. 9). These characteristics correlated with kidney colonization by C. albicans strains, which amounted to 1.5 × 106 and 0 colony-forming units/kidney in the wild type and pmt1 mutant, respectively. Interestingly, although the heterozygous CAP1-3 strain almost showed wild-type kidney colonization (9.5 × 106 colony-forming units), its survival was increased significantly (mean survival of 7 days; p < 0.0001). Thus, deletion of PMT1 alleles, in a dosage-dependent manner, decreases the virulence of C. albicans. The lack of kidney colonization suggests that strains lacking both PMT1 alleles are defective in systemic spread or that they are quickly eliminated by defense mechanisms, e.g. by phagocytosis.
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DISCUSSION |
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The PMT1 gene of C. albicans is the first described PMT gene in a species other than S. cerevisiae whose function has been characterized. Other ScPMT homologues of unknown function are known in fungi. Recently, a genomic sequence of S. pombe has been reported, which encodes a putative Pmt protein (GenBankTM accession number Z99126). Also, a PMT1 homologue appears to exist in the yeast Kluyveromycis lactis.5 Because we also obtained evidence for a homologue of the ScPMT2 gene in C. albicans, it appears that protein O-mannosylation by Pmt-type proteins is a reaction occurring generally in other fungi; this process may require two or more Pmt proteins. The sequence of the C. albicans PMT1 gene predicts a protein with a hydrophilic center and membrane-spanning termini, in agreement with the structures of the S. cerevisiae Pmt1 protein (17). Because we were unable to detect C. albicans homologues of the S. cerevisiae PMT3-7 genes, it is possible that C. albicans only contains two PMT genes. Conceivably, the two alleles of PMT1 and PMT2 provide sufficient genetic redundancy in the diploid yeast C. albicans, a function that is secured by multiple PMT loci in S. cerevisiae, which has a haploid or a diploid genome. The existence of only two PMT genes in C. albicans could also explain the drastic phenotypes of PMT1 deletion in C. albicans, as discussed below.
Although individual deletions of the S. cerevisiae PMT1 and PMT2 genes do not cause significant growth phenotypes, their simultaneous mutation significantly retards growth and causes cells to aggregate strongly (16). This phenotype is obtained in C. albicans by merely deleting both PMT1 alleles. Similarly, while S. cerevisiae pmt1 mutants do not show increased sensitivity toward hygromycin B, C. albicans pmt1 mutants are supersensitive, as is observed for S. cerevisiae pmt1 pmt2 double mutants.6 Unlike defects in outer chain addition to N-glycosyl chains in S. cerevisiae, which also lead to increased hygromycin B sensitivity (53), the pmt mutations did not cause resistance to vanadate. Surprisingly, deletion of a single PMT1 allele in C. albicans sufficed to increase hygromycin B sensitivity, providing evidence that the dosage of PMT1 expression determines the wild-type phenotype. Haplo-insufficiency has been observed for other genes in C. albicans (48).7 Supersensitivity for calcofluor white is another example of the strong consequence of PMT1 deletion in C. albicans, which compares to the phenotype of S. cerevisiae pmt1 pmt2 double mutants (19). The calcofluor white phenotype suggests that O-glycosylation of unknown proteins by Pmt1p stabilizes the C. albicans cell wall. In our study, we identified the Als1 protein (44), which recently has been recognized as a potential adhesion factor for C. albicans (45), as the first likely O-glycosylated protein in the cell wall of C. albicans. Recent results suggest that PMT1 activity is required to incorporate several proteins into the cell wall of S. cerevisiae (54). O-Glycosylated proteins may also be involved in an unknown system of resistance to various agents, such as aminoglycoside antibiotics, e.g. by promoting their export (39). However, a defect in Pmt1 function does not cause a general or an equal increase in the susceptibility toward damaging agents, since the sensitivity toward clotrimazole is increased only slightly and is absent for nystatin, amphotericin B, and methotrexate/sulfanilamide. Despite the multiple drastic phenotypes of the pmt1 mutation in C. albicans, a significant effect on osmotic sensitivity, as has been observed for multiple pmt mutations in S. cerevisiae (19), was not detected. Thus, although Pmt1 fulfills similar basic fungions in both fungal species, the resulting phenotypes differ significantly.
Depending on environmental conditions, C. albicans and S. cerevisiae are able to either grow as a budding yeast or in a multicellular filamentous form (reviewed in Ref. 2). Dimorphism of C. albicans is considered an important virulence trait. Hyphal formation of this pathogen can be induced either on certain depauperated solid media (spider media), a process requiring components of a conserved mitogen-activated protein kinase cascade (46); alternatively, positive stimuli, such as serum, are able to induce morphogenesis. Deletion of PMT1 completely abolished the ability of C. albicans to form hyphae on spider media. Surprisingly, even the heterozygous pmt1/PMT1 strain had a severe defect in morphogenesis, confirming the gene dosage effect observed for hygromycin B sensitivity and virulence in the animal model. Because serum still stimulated hyphae formation, we assume that not morphogenesis per se but rather sufficient levels of a component required for a signaling component operative on spider media requires O-glycosylation by Pmt1 for its function. A similar phenotype (block of hyphae formation on spider medium and normal hyphae formation on serum) has been reported for mutants defective in elements of a conserved mitogen-activated protein kinase pathway (34, 48). Therefore, it is possible that an unidentified O-glycosylated protein of this pathway, e.g. a membrane protein involved in signal transduction, is defective in pmt1 mutants. In contrast to the results obtained in C. albicans, pmt1 deletion in S. cerevisiae did not affect its ability to form pseudohyphae on low nitrogen media. Thus, even with regard to morphogenesis, PMT1 has a more important function in C. albicans compared with S. cerevisiae.
Despite the relatively minor growth defect in most media, pmt1 mutants were completely avirulent in the mouse model of systemic infection. Even the pmt1/PMT1 heterozygous strain showed a significant decline in virulence, although this strain grew identically to the wild-type strain and could be distinguished only by increased sensitivity to certain agents and by reduced morphogenesis (see above). Thus, even a 2-fold drop in Pmt1 activity (assuming equal contribution of each allele to overall PMT1 expression) interferes with pathogenicity. It remains to be determined experimentally which of the described various phenotypes of pmt1 mutants is responsible for attenuation of virulence. Conceivably, the alteration of cell surface properties and the morphogenesis defect of pmt1 strains cause defects in adhesion and penetration of epithelial and endothelial cell layers. We obtained experimental support for this possibility by demonstrating that pmt1 mutants adhere less strongly than wild-type cells to a Caco-2 epithelial cell layer. Thus, it appears that the ability to penetrate endothelial and epithelial layers, which is the precondition to infect organs (1), does not occur with pmt1 mutants. In agreement with this hypothesis, kidneys of infected animals were free of C. albicans pmt1 cells. Recently, the Als1 protein has been described as a likely adhesion factor for C. albicans (45). It appears possible that reduced O-glycosylation of this protein in pmt1 mutants, which is suggested here, reduces the adhesive properties of Als1p and thus affects virulence. Alternatively, the aggregation phenotype of pmt1 mutants could contribute to lower fungal dissemination in the infected animal. A third possibility is an increased host activity, e.g. phagocytosis, against pmt1 strains.
In conclusion, the characterization of the C. albicans PMT1 gene not only demonstrates that Pmt proteins exist and function in O-glycosylation in fungal species other than S. cerevisiae; the results also show that CaPmt1p assumes novel functions that are not observed (in hyphal morphogenesis and antifungal resistance) or are not considered relevant (in adherence and virulence) in S. cerevisiae. O-Glycosylation reactions mediated by Pmt proteins appear to be possible targets for antifungal agents for the following reasons. (i) The bulk of O-glycosylation occurs by different mechanisms in mammalian and fungal cells. (ii) Even a 2-fold reduction in PMT1 gene dosage causes defects in morphogenesis, as well as a significant drop in virulence. (iii) Reduced Pmt1p activity leads to an increased sensitivity to antifungal agents, thereby potentially amplifying any response to antifungals and offering the prospect for a combination therapy. (iv) Cells lacking Pmt1p activity show reduced adherence to host cells and reduced colonization of organs; this characteristic is in contrast to other avirulent C. albicans strains, which still show kidney colonization (55, 56). On the other hand, rare O-mannosylation of some human proteins has been reported (11, 12), and expressed sequence tags deposited at GenBankTM contain sequences encoding possible Pmt homologs derived from human (accession numbers AA670164, AA425494, AA496399, AA099791, N42494, and N35574), mouse (accession numbers AA274738, AA277592, AA274738, and Z31210), rat (accession number H33465), C. elegans (accession numbers C41029, C47075, C42623, C66447, and C69714), and rice (accession number C73075) cells. It is possible (although this remains to be established) that the corresponding genes perform essential functions in the respective cell types. In D. melanogaster, a gene encoding a Pmt homologue appears to be required for muscle development (20).
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ACKNOWLEDGEMENTS |
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We thank M. Spindler-Barth for advice on the chitinase assays, and we are grateful for the generous gifts of plasmids carrying PMT1-5 by W. Tanner and M. Gentzsch. We thank C. Marcireau (Rhone-Poulenc Rorer) and N. Gow for communication of unpublished results. We acknowledge A. Goffeau, M. Hostetter, L. Hoyer, L. Popolo, D. Sanglard, and R. Sentandreu for kindly supplying antisera. We thank U. Preuss for advice on the growth of the Caco-2 cells and Christian Herder for the construction of the pCH1,2-plasmids. We thank S. Badock for excellent technical assistance.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF000232 (for CaPMT1).
To whom correspondence should be addressed: Institut für
Mikrobiologie, Heinrich-Heine Universität, Universitätsstr.
1/26.12, D-40225 Düsseldorf, Germany. Tel./Fax: 49-211-8115176;
E-mail: joachim.ernst{at}uni-duesseldorf.de.
The abbreviations used are: Pmt, dolichyl-phosphate-D-mannose:protein O-D-mannosyltransferase (EC 2.4.1.190)PAGE, polyacrylamide gel electrophoresisSD, yeast synthetic minimal medium with dextrosekb, kilobase pair(s)bp, base pair(s)PCR, polymerase chain reactionDol, dolichyl.
2 A. Sonneborn and J. Ernst, unpublished results.
3 F. Rademacher and J. F. Ernst, manuscript in preparation.
4 Available on the World Wide Web (http://ulrec3.unil.ch/software/ TMPRED_form.html).
5 C. C. Marcireau, personal communication.
6 S. Wickert and J. F. Ernst, unpublished results.
7 F. Rademacher and J. F. Ernst, unpublished results.
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REFERENCES |
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