(Received for publication, August 25, 1995; and in revised form, January 5, 1996)
From the
cDNA sequences encoding a cell wall protein have been isolated from the opportunistic pathogen, Candida albicans, an organism that can cause serious disease in immunocompromised patients such as those with AIDS. The cDNA encodes a peptide that is largely composed of an acidic, repeated motif 10 amino acids in length that is rich in proline and glutamine residues. The cDNA gene product was found to be present on hyphal surfaces by immunofluorescence assays using monospecific antisera raised to the recombinant protein produced in Pichia pastoris. The hyphae-specific surface location was also seen on organisms colonizing the gastrointestinal mucosa of mice, indicating that the antigen is produced and developmentally regulated during growth in host tissues. The cDNA clone hybridized to an abundant messenger RNA 2.3 kilobases in size that was present in hyphal but not yeast forms. These studies demonstrate that the bud-hypha transition is accompanied by the de novo synthesis of proteins that are targeted to hyphal surfaces. The primary sequence of the unique amino acid motif shares features with surface proteins of other lower eukaryotic microorganisms and with host acidic salivary proline-rich proteins.
The cell surface structures of Candida albicans mediate several important processes in the molecular interactions that occur between host and parasite. To establish a commensal host parasite relationship, C. albicans must be adherent enough to persist on mucosal surfaces where cell-mediated immune responses are induced that serve to counteract overgrowth. When risk factors such as immunosuppression due to AIDS or chemotherapy are present, fungal surface structures mediate attachment to and invasion of host tissues. Thus surface structures of C. albicans are of central importance in the complex balancing mechanisms that serve to maintain the host-parasite equilibrium and are also involved in pathogenesis.
The facile transition between yeast and hyphal growth and the presence of intermediate pseudohyphal forms are hallmarks of C. albicans that are accompanied by molecular surface changes that confer differing antigenic, chemical, and physical surface properties to the growth forms(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) . Thus, the existence of yeast and hyphal forms increases the repertoire of surface structures of C. albicans that are available for survival within the host environment. The differences between growth forms in interactions with phagocytic cells(12, 13, 14, 15) , adherence to epithelial cells(16) , and binding to host proteins (17, 18, 19, 20, 21) probably result from the differing surface biochemical compositions of the growth forms.
Although the numerous binding activities that have been attributed to hyphae involve a variety of host cells and host proteins, a frequent finding is that surface proteins of hyphae are involved in binding(8, 9, 15, 17) . Therefore it is surprising that primary structures of candidal hyphal wall proteins have not been described. We have shown that unique antigens exist on hyphal surfaces (8) and propose that identification of the primary sequences of these antigens will provide insight into the structure and function of hyphal surfaces. In this report, we describe a novel proline- and glutamine-rich amino acid segment that is exposed on surfaces of hyphae grown in mammalian hosts and in laboratory cultures. Synthesis of this protein occurred exclusively during hyphal growth, showing that the bud-hypha transition controls the antigenic surface composition of hyphae by production of de novo proteins. The predicted protein sequence provided new insights into possible mechanisms of interaction between C. albicans and host cells.
The 5` region of HWP1 mRNA was amplified using the
5` RACE System kit (Life Technologies, Inc.) according to the
manufacturer's instructions. First strand cDNA synthesis was
performed on total RNA isolated from yeast and hyphal forms using a
gene-specific oligo (5`-GGGTAATCATCACATGG) complementary to nucleotides
199-214. A second nested gene-specific oligo
(5`-GATAGTAATCATAAGATCTC) complementary to nucleotides 174-193
was used as a primer to amplify the 5` end using PCR. The resulting PCR ()product was cloned directly into pBluescript SK-
(Stratagene, La Jolla, CA) that had been digested with EcoRV
and treated with TaqPlus DNA polymerase (Stratagene) in the
presence of dTTP to generate a T-vector (24) . The nucleotide
sequence of four independent clones was determined (U. S. Biochemical
Corp.).
Purified clones
were further tested for their ability to selectively adsorb antibodies
that cross-reacted with hyphal surfaces from the anti-C. albicans antiserum. Each of the purified clones was used to produce nearly
confluent isopropyl-1-thio--D-galactopyranoside-induced
plaques on 100-mm NZY agar plates that were then transferred to
nitrocellulose. Filters were soaked overnight in TBST (20 mM Tris, pH 7.5, 50 mM NaCl, and 0.05% Tween 20) and washed
three times in TBST prior to blocking with TBST containing 1% skim
milk, 1 mM EDTA, and 0.02% sodium azide. Filters were washed
and incubated in the anti-C. albicans antiserum described
above in blocking solution for 2 h at 37 °C. Unattached antibodies
were removed with five washes in TBST followed by one wash in 0.15 M NaCl containing 0.05% Tween 20. Bound antibodies were eluted
with a solution containing 0.2 M glycine, pH 2.8, 0.15 M NaCl, 1 mM EDTA, and 0.05% Tween 20. Antibodies were
immediately neutralized with Tris base and concentrated using Centricon
membranes (Amicon, Beverly, MA). The concentrated antibodies were
assayed for their ability to bind hyphal surfaces in the
immunofluorescence assay(8) .
Procedures for electrophoresis, transfer, and
hybridization conditions and radiolabeling of probes were similar to
those previously described (26, 32) except that 2
10
cpm of gel-purified HWP1 and cENO were
used and the incubation temperature during hybridization was 37 °C.
Molecular sizes of mRNAs were determined using a standard curve based
on gel migrations of RNA standards (0.24-9.5 kilobases) (Life
Technologies, Inc.).
Recombinant
hwp1 was purified by standard column chromatography. P. pastoris GS115-13 culture supernatant (30 ml) was fractionated by
(NH)
SO
precipitation; the fraction
enriched for rhwp1 (40-60% saturation) was dialyzed against
column buffer (50 mM Tris-Cl, pH 7.5, 1 mM EDTA, 10%
glycerol, 2 mM 2-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride) and applied to a 2.5
18-cm
DEAE-Sephacel (Pharmacia Biotech Inc.) column at 4 °C. The bound
proteins were eluted with a linear gradient of KCl from 0.0 to 0.5 M. Aliquots of column fractions were tested for rhwp1 by
SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting,
followed by immunostaining. Fractions containing rhwp1 were pooled and
concentrated. A single protein adjacent to the anode was seen following
two-dimensional SDS-PAGE and Western blotting as described above
showing that rhwp1 was pure. Attempts to quantitate rhwp1 with
Coomassie-based protein assays and derivative methods (Lowry and biuret
enhanced with bicinchoninic acid; Pierce) were unsuccessful. Therefore
the absorbance at 205 nm was used to approximate the protein
concentration(37) . The molecular size of rhwp1 was determined
using a standard curve based on gel migrations of purchased protein
standards ranging from 14.3 to 200 kDa (Rainbow Markers, Amersham
Corp.). The amino acid composition of rhwp1 was determined at the Ohio
State Biochemical Instrument Center.
Monospecific antiserum to rhwp1 was prepared by immunizing New Zealand White rabbits with rhwp1 that had been excised from one-dimensional SDS-PAGE as described above, using conventional methods(38) . Antibodies to P. pastoris were removed by adsorptions with P. pastoris GS115 untransformed cells. The immune serum (1:200 dilution in phosphate-buffered saline (170 mM NaCl in 10 mM potassium phosphate buffer, pH 7.4)) was used for binding to surfaces of C. albicans using the immunofluorescence assay(8) . Immunofluorescence blocking experiments were performed by incubating chromatographically purified rhwp1 (see above) at concentrations of 4.25 or 8.5 µg/ml at 37 °C for 15 min with diluted primary antibody before applying to C. albicans cells. In control experiments, irrelevant proteins enolase and lactate dehydrogenase (Boehringer Mannheim) were used at concentrations of 1.25 mg/ml.
Preliminary results showed that two clones were able to select
antibodies from the screening antiserum that cross-reacted with hyphal
surfaces of C. albicans in an immunofluorescence assay that
employs germ tube forms as antigens (not shown). Yeast surfaces were
negative or weakly fluorescent. In control experiments, antibodies
selected by a clone containing cDNA encoding the cytoplasmic enzyme
enolase did not stain C. albicans surfaces, indicating that
the reactivity with hyphal surfaces of antibodies selected by the
unknown clones was not a result of nonspecific binding of
immunoglobulins. One of the positive clones, 13, was selected for
further study. The cDNA from this clone was termed HWP1 for
hyphal wall protein 1, and the protein product, hwp1.
Figure 1:
A, composite 5` rapid amplification of
cDNA ends product and 13 partial cDNA clone DNA sequence. Clone 13
DNA sequence begins at nucleotide 156. The deduced amino acid sequence
for ORF1 is shown below the nucleotide sequence. B,
alignment of the repetitive amino acid sequences of hwp1. Gaps have
been introduced to maximize the similarities of the
repeats.
The deduced amino acid sequence of the partial
cDNA revealed a series of tandem repeats 10 amino acids in length that
comprised the majority of the open reading frame. An alignment of the
repeats starting with amino acid 14 is shown in Fig. 1B. Common features included proline residues at
positions 2, 6, and 9 in all but two of the repeats. Cysteine residues
predominated at position 3 and aspartate was found at position 4. The
first six repeats had a tyrosine in the fifth position and glutamate in
the tenth position, whereas the more carboxy proximal repeats had
asparagine and aspartate, at the fifth and tenth positions,
respectively. The amino acid composition of the entire ORF was notable
in having 27% proline, 16% glutamine, and 12% aspartate residues (mole
percents). The C terminus of hwp1 was threonine- and serine-rich
providing abundant potential sites for O-glycosylation. The
deduced amino acid composition was hydrophilic throughout the entire
sequence with a net negative charge of 32 at neutral pH and a
calculated M of 22,750. No consensus sequence N-glycosylation sites were found. Chou-Fasman predictions of
secondary structure (44) showed the sequence to be primarily
composed of turns with two helical sections in locations containing
five and six consecutive glutamine residues. The surface probabilities (45) were also highest for the sites with consecutive glutamine
residues and for glutamines bounded by prolines. Searches of computer
data bases showed that hwp1 was similar to a wide variety of proteins
containing amino acid repeats having periodic proline residues and/or
glutamine residues; however, no identical sequences were found.
Figure 2: Northern blot analysis of HWP1 expression in different growth forms of C. albicans. Total RNA (5 µg) was electrophoresed in formaldehyde gels and transferred to nitrocellulose membranes (see under ``Experimental Procedures''). The membranes were hybridized with radiolabeled HWP1 and 1.5 kilobases of enolase cDNA(25) . Growth conditions and cell morphologies are indicated above each lane.
The finding that antiserum that
had been raised to whole C. albicans organisms bearing germ
tubes and adsorbed to yeast forms also recognized Pichia-produced rhwp1 of transformant GS115-13 (Fig. 3)
provided strong confirmatory evidence that a hyphal surface antigen
cDNA had been cloned. The M of the rhwp1 was
determined to be 55,000, much larger than the calculated 27,215 (see
``Discussion''). The position of rhwp1 at the anode following
isoelectrofocusing was consistent with the calculated pI of 3.39 (data
not shown). Coomassie Blue did not stain the recombinant protein in
SDS-PAGE. Preimmune serum was negative.
Figure 3: Western blot and immunodetection of rhwp1 produced in Pichia pastoris. Samples of P. pastoris GS115-13 culture supernatant were subjected to SDS-PAGE, and the separated proteins transferred to an Immobilon-P membrane (Millipore Corporation, Bedford, MA). The membrane was cut and each portion was treated with anti-C. albicans hyphae-specific polyclonal antiserum (I) or preimmune serum (P) followed by goat anti-rabbit IgG conjugated to horseradish peroxidase. The membranes were developed with ECL reagents (Amersham Corp.) and exposed to x-ray film (Eastman Kodak's X-OMAT). Protein mass standards (Rainbow Markers, Amersham Corp.) are shown at left.
A further prediction of the conclusion that HWP1 cDNA encodes a hyphal surface protein is that antiserum raised to a recombinant protein encoded by HWP1 cDNA should cross-react with hyphal surfaces. Serum from rabbits immunized with rhwp1 was tested for the ability to recognize antigens on C. albicans hyphal surfaces in immunofluorescence assays (Fig. 4). Immune serum, at dilutions of 1:200, which was determined to be optimal by titration experiments, stained hyphal surfaces but not the parent blastoconidia of C. albicans. Thus the antiserum was specific for rhwp1 and hyphal surfaces of C. albicans. In addition, the hyphae-specific staining could be blocked by incubation of chromatographically purified rhwp1 with anti-rhwp1 antiserum used in immunofluorescence assays, and blocking was concentration-dependent (Fig. 4, C and D). Control incubations with higher concentrations of irrelevant proteins (enolase and lactate dehydrogenase) did not affect antibody detection of native hwp1 (Fig. 4E). The blocking experiments showed that native hwp1 on hyphal surfaces and rhwp1 shared the same antigenic epitopes. Blastoconidia in logarithmic growth at temperatures of 25 °C or 37 °C were also negative for hwp1 (not shown). These results confirmed that the partial HWP1 cDNA encoded an antigen that is exposed on hyphal surfaces and not on yeast surfaces of C. albicans and that the rhwp1 antiserum was specific for a hyphal antigen encoded in part by the HWP1 cDNA clone.
Figure 4: Detection of hwp1 on hyphal surfaces of C. albicans grown in laboratory cultures or in host tissues. Formalin-fixed C. albicans yeasts bearing germ tubes (A-E) were treated with various primary antisera followed by fluoresceinated goat anti rabbit IgG in indirect immunofluorescence assays. A, preimmune serum. B, monospecific antiserum to rhwp1. C and D, monospecific antiserum to rhwp1 incubated with rhwp1 at concentrations of 8.5 µg/ml (C) or 4.25 µg/ml (D) or with enolase 1.25 mg/ml (E). F-J, sections from an 11-month-old female bg/bg-nu/+ mouse that had been colonized for 9 months with C. albicans were treated with various antisera (G-J) or histologically stained with the periodic acid-Schiff reagent (F). G, preimmune serum. H, anti-C. albicans serum I, preimmune serum. J, monospecific antiserum to rhwp1. The large arrowhead points to positive-staining hyphae, and the small arrowhead points to negative-staining yeast. The bars represent 5 µm.
To determine if hwp1 was the sole antigen recognized by the screening hyphae-specific antiserum, the blocking experiments were performed with this antiserum as well. Recombinant hwp1 did not block the fluorescence exhibited by the hyphae-specific antiserum, indicating that antigens other than hwp1 are present on hyphal surfaces (data not shown).
To ensure that hwp1 expression was not limited to strain SC5314, five additional strains including B311, ATCC 28367, ATCC 38696, and 15 recent clinical isolates were tested for the presence of hwp1 on hyphae induced by growth in M199 at 37 °C (not shown). All C. albicans isolates tested expressed hwp1 on hyphal surfaces. In contrast, four strains of Candida tropicalis did not produce hwp1. These results demonstrated that hwp1 is not specific to SC5314 but is a feature of C. albicans strains in general. Although hwp1 was not detected on four strains of C. tropicalis, more experiments are needed to determine if hwp1 is present in other species of Candida.
To gain further information about native hwp1,
Western blots of cell wall digests using monospecific antiserum from
rhwp1 were performed. The Western blots showed a polydisperse pattern
of antibody binding that was concentrated at a M of approximately 34,000 (Fig. 5A, arrow). Because the enzyme used to prepare cell wall digests,
Zymolyase, contains a protease and because cell wall proteins are
highly processed, the M
of the material detected
on the Western blot does not represent that of the intact native
protein. The same pattern of antibody binding was seen in antisera from
two rabbits that and been immunized with rhwp1 (Fig. 5A, lanes 4 and 8). Although
the pattern of background bands differed, the polydisperse pattern was
similar for both sera and was absent in preimmune sera. The specificity
for hwp1 was demonstrated by the ability of rhwp1 to block antibody
binding, whereas higher concentrations of an irrelevant protein,
enolase, had no effect (Fig. 5A, lanes 5 and 9 versus lanes 6, 7, 10, and 11).
Native hwp1 was not found in yeast cell wall digests (Fig. 5B), consistent with the developmental expression
seen by immunofluorescence and by Northern blotting. Polydisperse
patterns of binding are typical of glycoproteins and may reflect O-glycosylation of hwp1.
Figure 5: Western blot and immunodetection of hwp1 in the hyphal wall of C. albicans. Cell wall proteins were subjected to SDS-PAGE, and the separated proteins were transferred to an Immobilon-P membrane. Immunodetection of hwp1 was performed in an Immunetics manifold (see under ``Experimental Procedures''). Rabbit anti-rhwp1 sera (321 and 326) were used alone, preincubated with of rhwp1 (7.08 µg/ml, lanes 6 and 10 and 14.2 µg/ml, lanes 7 and 11) or preincubated with enolase (31.3 µg/ml, lanes 5 and 9) as indicated above each lane. Lanes 1 and 2 were incubated with preimmune sera 321 and 326, respectively. Lane 3 was incubated with rabbit antiserum to whole C. albicans organisms bearing germ tubes; the antiserum was not adsorbed to yeast forms. The membrane was subsequently incubated with goat anti-rabbit horse radish peroxidase conjugate and developed with ECL reagents (Amersham Corp.). A, hyphal wall proteins. B, yeast wall proteins. Protein mass standards (Rainbow Markers, Amersham Corp.) are shown on the left. The arrows indicate the position of the immunodominant hwp1 protein fragment recognized by anti-rhwp1 antiserum that is present in cell wall digests of hyphae but not yeast.
Our use of recombinant techniques to identify surface antigens of C. albicans hyphae has led to the discovery of a novel amino acid segment that is both developmentally regulated and expressed on hyphal surfaces. Given the high antigen density required for visualization by immunofluorescence assays, the positive immunofluorescence of hyphae when tested with monospecific antiserum to rhwp1 indicates that hwp1 is an abundant antigen. The same monospecific antiserum was also able to detect ``native'' hwp1 in Western blots of hyphal walls digested with Zymolyase. Coupled with the strong signal intensity on Northern blots probed with HWP1 cDNA, the results suggest that HWP1 encodes a major, immunodominant hyphal surface protein.
Messenger RNA levels of HWP1 were clearly correlated with C. albicans morphology and not with a particular medium component or temperature. This pattern of expression is similar to that found for ECE1(46) but is in contrast to other genes that have been correlated with hyphal growth(47, 48) . The presence of HWP1 mRNA correlated with hwp1 protein expression as well. The absence of hwp1 mRNA in yeast forms under three different environmental conditions compared with the abundant message in hyphal forms suggests that putative cis-acting gene elements play a role in maintaining transcription control until hyphae-specific signals allow for HWP1 expression. Similar regulatory mechanisms have been demonstrated for gene expression during switching between white and opaque forms of C. albicans(49) , although the relationship between expression of genes controlled by switching and genes controlled by the bud-hypha transition is unknown. The presence of hwp1 on hyphal and not yeast surfaces in murine tissues indicated that the developmental regulation of hwp1 is also operative in vivo and thus hwp1 could be an important determinant of pathogenicity.
Several mechanisms have been proposed to contribute to the changes in surface composition of C. albicans during morphogenesis. Ultrastructural studies support the occurrence of rearrangements or losses of wall components during morphogenesis(50, 51) , whereas unmasking of cryptic antigenic determinants has been suggested using monoclonal antibodies to localize specific antigens(6, 52, 53) . Although we showed in previous work that hyphal surfaces contained unique surface proteins(8, 39) , the mechanisms contributing to the expression of these proteins were not determined. The present finding that control of expression of hwp1 was mediated by mRNA levels proves that synthesis of new proteins is an important process in controlling hyphal surface gene expression. However, other mechanisms cannot be ruled out and are supported by the changes in carbohydrate structures on hyphal forms compared with yeast forms(10) .
The amino acid sequence provided plausible explanations for the unusual physical characteristics of rhwp1. The lack of agreement between the predicted molecular weight and the SDS-polycrylamide gel-determined molecular weight is typical for proteins with high proline content (54, 55, 56, 57) , although O-glycosylation of serine residues located near the C terminus might also contribute to the discrepancy. A feature in common with acidic proline-rich salivary proteins was the poor binding of Coomassie Blue(57) . Recombinant hwp1 stained blue with a cationic carbocyanine dye (58) and migrated to the anode following isoelectric focusing (data not shown), features that were correlated with the low isoelectric point of the amino acid sequence.
The
deduced amino acid sequence of hwp1 suggested several potential
functional properties in addition to antigenicity. First, the high
percentage of proline residues and the length of the repeat (10 amino
acids) place hwp1 within a varied group of proline-rich proteins in
which the proline residues are proposed to function in maintaining the
polypeptide chains in extended conformations and to mediate noncovalent
interactions between protein chains or, in the case of salivary
proteins, to bind toxic plant polyphenols(59) . Of particular
interest is the presence of acidic salivary proline-rich proteins
(aprp) in this group because of the findings by Bradway et
al.(60, 61) that aprp are substrates for buccal
epithelial cell transglutaminase and that an aprp-like,
transglutaminase substrate might also be present on surfaces of C.
albicans. Like hwp1, aprp contain glutamine residues within
proline-rich repeats, a conformational arrangement that may be
favorable for formation of (
glutamyl) lysine cross-links by
transglutaminase, given the properties of other known substrates for
epithelial cell transglutaminases(62, 63) . The
similarity of hwp1 to aprp could be important for interactions between C. albicans and the oral mucosa. The presence of a
polyphenol-binding extracellular proline-rich wall protein of the plant
fungal pathogen Colletotrichum graminicola(64) lends
support to the idea that the presence of proteins with aprp-like
properties might be a common feature of fungal cell walls. A cell wall
protein containing abundant proline residues has also been reported
from S. cerevisiae(65) . The primary sequences of
these proteins are unknown.
Other features of the hwp1 repeats are also likely to be functionally important. The presence of a cysteine residue in each repeat probably leads to the formation of regularly spaced extracellular disulfide bonds that may be important for the surface conformation of hwp1 and possibly for intermolecular cross-linking of proteins on the cell surface. Given the specificity of hwp1 for hyphal surfaces, the presence of cysteines may be related to the enhanced sensitivity and increased protein release from hyphal forms following treatment with dithiothreitol(4, 66, 67) . An additional feature of the repeats is the presence of acidic amino acids that would confer a negative charge to hyphal surfaces at physiological pH. The presence of anionic proteins on hyphal surfaces has been demonstrated by others(68) . The serine and threonines near the C-terminal end of hwp1 are likely to be sites for O-glycosylation and serve as wall spanning domains as has been proposed for other yeast surface proteins(69, 70, 71) . Finally, the presence of amino acid repeats on hyphal surfaces is not surprising given that tandem amino acid repeats are widely distributed on surfaces of broad groups of microorganisms including fungi, parasites, viruses, and bacteria. The functions of repetitive surface proteins have frequently been found to involve the host and include attachment sites to host cells, evasion of phagocytosis, invasion of host cells, and neutralization epitopes(54, 72, 73, 74, 75, 76, 77, 78, 79, 80) . Thus it is likely that hwp1 plays a significant role in interaction with the oral mucosa and with components of the host immune system. Future molecular and biochemical characterization of hwp1 will provide insights into the pathogenesis of candidiasis and will be important for developing new strategies for the medical management of candidiasis.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U29369[GenBank].