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Originally published In Press as doi:10.1074/jbc.M300680200 on May 21, 2003

J. Biol. Chem., Vol. 278, Issue 31, 28553-28561, August 1, 2003
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Candida albicans Ssa1/2p Is the Cell Envelope Binding Protein for Human Salivary Histatin 5*

Xuewei S. Li {ddagger}, Molakala S. Reddy {ddagger}, Didi Baev {ddagger} and Mira Edgerton {ddagger} § 

From the Departments of {ddagger}Oral Biology and §Restorative Dentistry, School of Dental Medicine, State University of New York, Buffalo, New York 14214

Received for publication, January 21, 2003 , and in revised form, May 19, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Salivary histatins are a family of small histidine-rich peptides with potent antifungal activity. We previously identified a 70-kDa cell envelope protein in Candida albicans and Saccharomyces cerevisiae that mediates binding of histatin (Hst) 5. Isolation of Hst 5-binding protein followed by matrix-assisted laser desorption ionization mass spectrometry analysis identified this protein as the heat shock protein Ssa1p. Ssa protein and Hst 5-binding protein were found to be co-localized on immunoblots of yeast {beta}-mercaptoethanol cell wall extracts and cytosolic fractions. Yeast two-hybrid analysis showed strong interactions between Ssa1p and both Hst 3 and Hst 5. To assess functional roles of Ssa proteins in the Hst 5 antifungal mechanism in vivo, both binding and fungicidal assays were carried out using S. cerevisiae isogenic SSA1/SSA2 mutants. 125I-Hst 5 binding assays showed saturable binding (Kd = 2.57 x 10–6 M) with the wild-type SSA1/SSA2 strain; however, Hst 5 binding with the {Delta}ssa1ssa2 double mutant was reduced (Kd = 1.25 x 10–6 M). Cell wall HSP70 proteins were also diminished, but still detectable, in S. cerevisiae {Delta}ssa1ssa2 cells and are likely to be Ssa3p or Ssa4p. Hst 5 (31 µM) killed 80% of the wild-type cells in fungicidal assays at room temperature. However, only 50–60% killing of the single mutants ({Delta}ssa1 and {Delta}ssa2) was observed, and fungicidal activity was further reduced to 20–30% in the {Delta}ssa1ssa2 double mutant. Incubation of cells under heat shock conditions increased the sensitivity of cells to Hst 5, which correlated with increased Hst 5-binding activity in {Delta}ssa1ssa2 cells, but not in wild-type cells. This study provides evidence for a novel function for yeast Ssa1/2 proteins as cell envelope binding receptors for Hst 5 that mediate fungicidal activity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human saliva contains proteins with broad antibacterial and antifungal activities that protect oral tissues from pathogenic microorganisms (1). A major component of host nonimmune defense systems is salivary histatins, a family of small (3–4 kDa), histidine-rich, cationic proteins secreted by major salivary glands in humans and higher primates (2, 3). Histatin (Hst)1 5 is the most potent of the 12 histatin family members. It has fungicidal activity at physiological concentrations found in saliva (15–30 µM) against blastoconidial and filamentous forms of Candida albicans (4). Hst 5 possesses both fungistatic and fungicidal activities in vitro against a spectrum of other fungi, including Candida glabrata, Candida krusei, Saccharomyces cerevisiae, and Cryptococcus neoformans (5, 6). Importantly, Hst 5 is effective against azole- or amphotericin-resistant strains of these fungi (6), suggesting fundamental differences in their mechanisms of action. Thus, Hst 5 or its derivatives could potentially be used as alternative or complementary antifungal drug therapies for oral candidiasis.

Salivary Hst 5 does not function as a classical pore-forming antibiotic (7, 8). Rather, the fungicidal mechanism of Hst 5 is a multistep process initially characterized by binding with a yeast cell envelope protein, followed by intracellular translocation and efflux of ions including K+, Mg2+, and ATP (3, 9, 10). We found that binding of Hst 5 with cell-surface proteins of C. albicans was closely tied to its killing activity (7). Hst 5-specific binding was not detected in C. albicans spheroplasts following removal of the cell wall structure, and spheroplasts were 14-fold less susceptible to Hst 5 killing compared with the intact cells. Our in vivo binding experiments showed that whole fungal cells have ~106 saturable binding sites/cell with an affinity (Kd) for Hst 5 of ~1 x 10–6 M (7).

We identified a single component from whole cell lysates by SDS-PAGE, of ~67 kDa in C. albicans and 67–70 kDa in S. cerevisiae, that was responsible for binding Hst 5 (7). Upon cellular fractionation of yeast whole cell lysates, this Hst 5-binding protein was consistently found in yeast cell envelope fractions, but, unexpectedly, was also detected in the cytosolic fraction. This 70-kDa Hst 5-binding protein was detected only in Hst 5-sensitive C. albicans and S. cerevisiae, but not in human neutrophil membranes or human embryonic kidney epithelial cells. Thus, the selective toxicity of Hst 5 to eukaryotic fungal cells rather than host mammalian cells may be due to specific interactions between Hst 5 and this 70-kDa binding protein. In this regard, cell wall receptor-mediated binding and import of toxic proteins have been identified as a selective mechanism for the S. cerevisiae yeast K1 killer toxin system (11).

Hst 5 binding is followed by internalization and ultimately causes increased levels of reactive oxygen species, depletion of intracellular ATP, total cell volume reduction, and cell cycle arrest (12–14). Intracellular expression of Hst 5 alone results in substantial cell death, suggesting intracellular effector sites (15). Thus, Hst 5 binding with cell-surface proteins is precedent to its translocation to other cellular effector sites. Hst 5 binding and translocation within C. albicans cells are closely related and are temperature- and energy-dependent processes. The binding, cellular uptake, and fungicidal activity of Hst 5 were enhanced when cells were preincubated at 37 °C, whereas these processes were reduced when cells were maintained at 0–4 °C (9, 16). Pretreatment of C. albicans cells with sodium azide reduced internalization of Hst 5 (12) and provided substantial protection against Hst 5 killing (10). Thus, Hst 5 binding, translocation, and toxicity are closely tied processes and are optimal under conditions in which cells are transferred to higher temperatures (37 °C) and have ample intracellular ATP.

To isolate the Hst 5-binding protein, we used sequential column chromatography of whole cell lysates of C. albicans, followed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), and identified this protein as the heat shock protein Ssa1/2p. Here, we report the in vivo functional characterization of Ssa1p by co-localization of Hst 5-binding protein with Ssa protein, the positive interaction between these two proteins by yeast two-hybrid analysis, and the correlation of the binding and fungicidal activities of Hst 5 in isogenic SSA1 and/or SSA2 deletion mutants of S. cerevisiae. Consistently, we found that the SSA1 and SSA2 genes were required for binding and fungicidal activities of Hst 5.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Materials—The C. albicans strains used were SC5314 and DS1 (a clinical isolate) (7). Dr. E. A. Craig (University of Wisconsin, Madison, WI) generously provided the following S. cerevisiae isogenic SSA mutant strains with the indicated genotypes: JH27A (wild-type SSA, GAL2 his3-11,15 leu2-3,112 lys2 {Delta}trp1 ura3-52), JN114 ({Delta}ssa1, ssa1::HIS3), JN115 ({Delta}ssa2, ssa2::LEU2), and MW123 ({Delta}ssa1ssa2, ssa1::HIS3, ssa2::LEU2) (17). All strains were maintained on YPD (yeast extract, peptone, dextrose) agar plates and recultured monthly from–70 °C stock. YPD agar and media were from Difco. Na125I was purchased from Amersham Biosciences.

Isolation and Purification of Hst 5-binding Proteins—The histatin-binding protein (HstBP) was isolated and purified from large-scale whole cell lysates of C. albicans. C. albicans cells (~20–25 g) were washed twice with 10 mM sodium phosphate buffer (pH 7.4) and resuspended in 15 ml of cold lysis buffer supplemented with protease inhibitors and nonionic detergent (10 mM sodium phosphate buffer (pH 7.4), 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 1 µg/ml benzamidine, and 2.5% Triton X-100). Cells lysates were prepared in a 50-ml Bead Beater Chamber (BioSpec Products, Inc., Bartlesville, OK) with 15 ml of prechilled 0.5-mm glass beads by vortexing for five 1-min bursts at 4 °C. The efficiency of cell lysis was monitored by microscopic observation. The cell lysate was centrifuged at 15,000 x g for 15 min, and the clear supernatant was collected and subjected to gel filtration on a Sephadex G-200 column (1.6 x 95 cm, fine) that was pre-equilibrated with 10 mM phosphate buffer containing 1% Triton X-100. Fractions (1.5 ml) were collected, and aliquots (200 µl) were removed and subjected to 7.5% SDS-PAGE, followed by Western transfer to monitor for HstBP-positive fractions by overlay assay as previously described (7). HstBP-positive fractions (fractions 21–26) were pooled and fractionated by ion-exchange chromatography on a DEAE-Sepharose CL-6B column (1.0 x 15 cm) that was pre-equilibrated with 10 mM phosphate buffer containing 1% Triton X-100. The column was eluted with 0.25 M phosphate buffer (25 ml), followed by a linear elution gradient (0.25–0.5 M) of phosphate buffer over 50 ml. Fractions (1.2 ml) were collected, and aliquots were monitored for HstBP by overlay assay as described above. Two fractions (fractions 17 and 18) that eluted midway in the gradient were highly enriched for HstBP. Overlay assays showed that the HstBP-positive band was separated from other minor proteins in the fraction and permitted clear excision (see Fig. 1, lane 2). This 70-kDa HstBP-positive band was excised and in-gel digested with trypsin, and the peptides were subjected to MALDI-MS analysis at the Yale Cancer Center Mass Spectrometry Resource and the HHMI Biopolymer/W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University. Identified peptide masses were data base-searched using the ProFound algorithm, which relies upon the NCBI non-redundant data base, and the PeptideSearch algorithm, which is based on the EBI non-redundant data base.2 Both searches matched the same protein, the heat shock protein Ssa1p in C. albicans with a top score of 1.0e + 00.



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  		FIG. 1.
Isolation of Hst 5-binding protein in C. albicans. Isolation of the 70-kDa Hst 5-binding protein from C. albicans whole cell lysates was carried out by gel filtration, followed by ion-exchange chromatography. Solubilized proteins were subjected to 7.5% SDS-PAGE and stained or probed for the presence of Hst 5-binding proteins by overlay assay using biotinylated Hst 5 as the primary probe. Lane 1, C. albicans whole cell lysate (stain); lane 2, final isolation of Hst 5-binding protein (stain); lane 3, final isolation of Hst 5-binding protein (overlay detection). Molecular mass standards are indicated.

 

Cellular Fractionation and Western Blotting—Localization of Ssa proteins in C. albicans and S. cerevisiae isogenic SSA mutant strains was examined by three sequential cellular fractionation steps consisting of 1) {beta}-mercaptoethanol ({beta}-ME) cell wall extraction, 2) glucanidase cell wall extraction, and 3) cytosolic fractionation using published protocols (18, 19). Briefly, cells were washed twice with 10 mM sodium phosphate buffer (pH 7.4), and {beta}-ME-sensitive cell wall components were released by incubation of the cell suspension in ammonium carbonate buffer (pH 8.0) containing 1% (v/v) {beta}-ME for 30 min at 37 °C. The supernatant containing the {beta}-ME cell wall extract was collected following centrifugation at 500 x g. The {beta}-ME-treated cells were then washed with 1 M sorbitol and resuspended in spheroplast buffer (1 M sorbitol, 0.1 M sodium citrate (pH 5.8), 25 mM EDTA, and 100 µl {beta}-glucanidase) for 40–60 min at 30 °C until 90% of the cells were converted to spheroplasts. Cells were gently centrifuged at 500 x g, and cell wall material released from the {beta}-glucanidase treatment was collected in the supernatant. The cell pellet containing spheroplasts was lysed in ice-cold lysis buffer using glass bead disruption as described above, and the cytosolic fraction was collected following centrifugation at 10,000 x g for 10 min. The cell wall extracts ({beta}-ME- and glucanidase-extracted components) and cytosolic fractions were separated on a 7.5% SDS-polyacrylamide gel and probed following Western transfer. Ssa proteins were detected using mouse anti-HSP70 monoclonal antibody (Stressgen Biotech Corp., Victoria, British Columbia, Canada) and horseradish peroxidase-conjugated goat anti-mouse IgG (Pierce) as the secondary antibody. To verify that the detected Ssa proteins were identical to HstBP, blots probed with anti-HSP70 antibody were probed in parallel with biotinylated Hst 5 (20). Western blots of C. albicans cellular fractions were blocked by incubation with 1% skim milk for 2 h, and biotinylated Hst 5 (250 nM) was then added as the primary probe. Protein with bound biotinylated Hst 5 was visualized by colorimetric development following incubation with horseradish peroxidase-conjugated ExtrAvidin (Sigma) for 1 h.

The presence of HSP70 proteins in the cell wall of S. cerevisiae wild-type SSA or {Delta}ssa1ssa2 strains following room temperature or heat shock incubation was detected in {beta}-ME cell wall extracts as described above. For heat shock conditions, cells maintained at room temperature were transferred to prewarmed (37 °C) buffer and incubated at 37 °C for 60 min before cell wall extraction. Equal amounts of total cell wall proteins (15 µg) were subjected to SDS-PAGE and immunoblotted using anti-HSP70 antibody as described above.

Construction of a Yeast Two-hybrid System—Escherichia coli strain DH5{alpha} (Invitrogen) was used as a host for construction and maintenance of the plasmids used in this study and was cultivated in LB medium (Difco). Ampicillin was added at a final concentration of 100 µg/ml. The DupLEX-ATM yeast two-hybrid system kit was from OriGene Technologies Inc. (Rockville, MD). Standard conditions for plasmid DNA isolation, molecular cloning, transformation, and agarose gel electrophoresis were used (21). Restriction endonucleases and the GeneRuler 100-bp DNA Ladder Plus marker were from Fermentas Inc. (Hanover, MD). The Fast-LinkTM DNA ligation kit was from Epicentre Technologies Corp. (Madison, WI). Purification of restriction digests and PCR mixtures was done using a QIAquick PCR purification kit (QIAGEN Inc., Valencia, CA). SC5314 genomic DNA was isolated using a PUREGENE D-6000A DNA isolation kit (Gentra Systems, Inc., Minneapolis, MN) and was used as template for amplification of the SSA1 open reading frame.

Plasmid Construction—The open reading frame of the C. albicans SSA1 gene was amplified using the forward (f) primer nSSA1-f (5'-ATCccatggATGTCTAAAGCTGTTGGTATTGA-3') and the reverse (r) primer xSSA1-r (5'-ATCctcgagATCAACTTCTTCAACAGTTGGTC-3') as follows: initial denaturation for 3 min at 94 °C, followed by three-step cycling of 30-s denaturation at 94 °C, 30-s annealing at 57 °C, and 2-min extension at 72 °C and a 5-min final extension at 72 °C. The sequences encoding Hst 3 and Hst 5 were amplified using plasmid pCMVH3 (22) as template employing the following primers: for Hst 3, nHIS3/5-f (5'-ATCccatggGATTCACATGCAAAGAGACATC-3') and xHIS3-r (5'-ATCctcgagATTGTCATACAGATAATTTGATCTA-3'); and for Hst 5, nHIS3/5-f and xHIS5-r (ATCctcgagATAGCCTCGATGTGAATGATGC-3'). PCR conditions were as follows: initial denaturation for 3 min at 94 °C, followed by three-step cycling of 15-s denaturation at 94 °C, 15-s annealing at 57 °C, and 30-s extension at 72 °C and a 2-min final extension at 72 °C. The NcoI (forward primers) and XhoI (reverse primers) restriction sites introduced for in-frame cloning purposes into the bait and target plasmids are shown in lowercase in the above sequences. Thus amplified, the coding sequences were cloned as NcoI-XhoI restriction fragments into plasmid pEG202 to yield the LexA-bait fusion plasmids pB-SSA1 (LexA-Ssa1), pB-HIS3 (LexA-Hst 3), and pB-HIS5 (LexA-Hst 5), respectively. Likewise, the same fragments were cloned into the target plasmid pJG4-5, in which the unique EcoRI cloning site had been converted to an NcoI site since the C. albicans SSA1 open reading frame contains two EcoRI sites. The resulting fusion plasmids were designated pT-SSA1, pT-HIS3, and pT-HIS5, respectively. All constructs were verified for in-frame cloning by DNA sequencing.

Yeast Two-hybrid System Analysis—The yeast host strain EGY48 was first transformed with plasmids harboring the bait fusion genes and the reporter plasmid pSH18-34. All baits were assayed for autoinduction and nuclear localization. The plasmids containing the target fusion genes were then transformed into the strains that already contained the bait and reporter plasmids. Activation of the lacZ reporter gene was quantified by performing liquid culture {beta}-galactosidase assays (23). The resulting values are expressed as (A420 nm/culture A600 nm x (volume of culture) x (assay time in min)) x 1000 and were calculated from triplicate assays for five independent clones performed on four independent measurements for each bait-target combination.

Iodination of His 5 and Binding Assays—Hst 5 was iodinated using the chloramine-T method, following which Hst 5 retains full biological activity and binding specificity (7). Binding experiments were performed using wild-type and mutant SSA strains of S. cerevisiae grown under the same conditions as used for the fungicidal assays. The cells were washed and suspended at a density of 4 x 107 cells/ml in binding buffer (10 mM phosphate buffer (pH 7.4) and 2 mg/ml bovine serum albumin). Cell suspensions (25 µl, ~106 cells) were incubated with 125I-Hst 5 for 30 min at room temperature and washed twice with binding buffer, and cellular bound ligand was measured in a {gamma}-counter. Specific binding was calculated as total 125I-Hst 5 bound minus nonspecific bound, where nonspecific binding was determined in the presence of a 100-fold excess of unlabeled Hst 5. Cells were assayed for each experimental point in triplicate. Data were fitted to a single binding site equation to obtain binding curves and Kd values using Prism Version 3.03 software (GraphPAD, San Diego, CA). Binding of 125I-Hst 5 (6 µM) with S. cerevisiae wild-type SSA or {Delta}ssa1ssa2 mutant strains was examined using cells incubated at room temperature or following heat shock as described for fungicidal assays below. Differences in binding between cells following heat shock were calculated as the -fold change of bound 125I-Hst 5 relative to the binding activity of wild-type SSA at room temperature in three independent experiments. Unpaired Student's t test was used to compare sets of data.

Fungicidal Assays—The antifungal activities of Hst 5 with SSA mutants were examined by microdilution plate assays as described previously (7) with the following modifications. S. cerevisiae SSA strains (wild-type SSA, {Delta}ssa1, {Delta}ssa2, and {Delta}ssa1ssa2) were inoculated into 10 ml of YPD medium and grown overnight at 23 °C with rotary agitation until the cell density reached ~1.4–1.6 at 600 nm. Cells were washed twice with 10 mM phosphate buffer (pH 7.4) and resuspended at 2.5 x 105 cells/ml. Cell suspensions (20 µl) were mixed with 20 µlofHst 5 and incubated at room temperature (23 °C) with constant shaking for 1.5 h. For heat shock conditions, cells maintained at room temperature were transferred to prewarmed (37 °C) buffer and incubated at 37 °C for 30 min before addition of Hst 5. Cells were then incubated at 37 °C for another 1.5 h with Hst 5. Hst 5 was diluted (and the reaction stopped) by addition of 360 µl of 10 mM phosphate buffer. Cells were spread onto YPD agar plates and incubated for 36–48 h until colonies could be visualized. Percent cell killing was calculated as (1–(number of colonies from suspensions with Hst 5/number of colonies from control suspensions)) x 100.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
C. albicans HstBP Identified as the Heat Shock Protein Ssa1p—To identify the 70-kDa cell envelope Hst 5-binding protein, we developed a purification protocol using large-scale whole cell lysates of C. albicans and sequential column chromatography on Sephadex G-200, followed by DEAE-Sepharose CL-6B ion-exchange fractionation as described under "Experimental Procedures." Eluant fractions positive for Hst 5-binding activity were determined by overlay assay using biotinylated Hst 5 as a primary probe. Purified Hst 5-binding protein was eluted from the midpoint of a 0.25–0.5 M sodium phosphate buffer linear elution gradient of a DEAE-Sepharose CL-6B ion-exchange column. This fraction contained primarily a 67–70-kDa protein band as determined by SDS-PAGE (Fig. 1, lane 2), which strongly bound Hst 5 in overlay assays (lane 3). This Hst 5-binding protein was excised and in-gel digested with trypsin, and the fragments were analyzed by MALDI-MS. The peptide masses were data base-searched and identified as Ssa1p in C. albicans with the top score of 1.0e + 00. The predicted size of this protein is 70.31 kDa, a close match with the estimated size of the Hst 5-binding protein found in both C. albicans and S. cerevisiae.

Sequence Alignment of the SSA Family in C. albicans and S. cerevisiae—S. cerevisiae contains four SSA genes (SSA1–4), whereas only two SSA family members (SSA1 and SSA2) have been identified in C. albicans by screening a cDNA expression library (18, 24). However, the deduced amino acid sequence of C. albicans SSA2 from the cDNA clones showed only partial sequence information (193 amino acids) at the C terminus (24). To obtain full sequences of both Ssa1p and Ssa2p in C. albicans and to identify homologies among these proteins in C. albicans and S. cerevisiae, a BLAST similarity search (tBlastn) against the C. albicans genome data base3 was done using the S. cerevisiae Ssa1p sequence as query (Swiss-Prot accession number P10591 [GenBank] ). The results revealed two homologous genome sequences in C. albicans identified as contig 6–2136 and contig 6–2375. A tBlastn search using the S. cerevisiae Ssa2p sequence (Swiss-Prot accession number P10592 [GenBank] ) as query showed the same results. However, the tBlastn search using S. cerevisiae Ssa3p and Ssa4p sequences as query (Swiss-Prot accession numbers P09435 [GenBank] and P22202 [GenBank] , respectively) did not find out other homologous Ssa protein members in C. albicans, thus confirming that C. albicans contains only Ssa1p and Ssa2p.

Additional analyses using DNA-protein translation tools4 identified the 5'-3' frame 2 to contain the correct whole coding sequences for SSA1 and SSA2. Protein similarity search using the SIM alignment tool5 between the deduced amino acid sequences and the published amino acid sequences (18, 24) confirmed that the translated sequence from contig 6–2136 represents C. albicans SSA1 and that the translated sequence from contig 6–2375 represent C. albicans SSA2. The homology of deduced protein sequences among SSA1 and SSA2 family members in S. cerevisiae and C. albicans was analyzed using the ClustalW alignment tool6 and is shown in Fig. 2. The overall sequence identity of Ssa1p and Ssa2p between S. cerevisiae and C. albicans is >83%, showing high conservation of these proteins between the two species. The overall sequence identity between Ssa1p and Ssa2p in C. albicans is 87.2%. Both C. albicans Ssa1p (656 amino acids) and Ssa2p (645 amino acids) are highly conserved in the amino-terminal regions, whereas sequences are more varied in the most terminal portion of the carboxyl terminus.



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  		FIG. 2.
Alignment of deduced amino acid sequences of SSA1 and SSA2. The deduced whole amino acid sequences of SSA1 and SSA2 from C. albicans (Ca) and S. cerevisiae (Sc) are indicated. Dashes indicate gaps introduced during the alignment. On the consensus line, asterisks denote sequence identities, and decreased matching similarities are indicated by colons and periods. The alignment suggests that the amino-terminal sequences of SSA are highly conserved, whereas the carboxyl-terminal sequences are more varied. The overall sequence identity of SSA1 and SSA2 in the two species is >83%.

 

Comparison of peptide sequences from the trypsin-digested Hst 5-binding protein analyzed by MALDI-MS showed that these sequences have 97.6% homology to the deduced protein sequence of C. albicans Ssa2p (Fig. 2), and nearly 80% of these peptide fragments are identical in both C. albicans Ssa1p and C. albicans Ssa2p. Thus, although the initial data base search identified only C. albicans Ssa1p due to incomplete sequence information for C. albicans Ssa2p in the data base, our complete translated sequence data (Fig. 2) show that the identity of C. albicans Hst 5-binding protein is either Ssa1p or Ssa2p. Hst 5-binding Protein and Ssa Protein Co-localize on Immunoblots of C. albicans {beta}-ME Cell Wall Extracts and Cytosolic Compartments—Previously, we found Hst 5-binding proteins to be localized in the cell envelope of C. albicans (7); however, we could not explain why this binding protein was also detected in the cytosolic compartment. Ssa1p and Ssa2p are major cell wall-located immunogens in both S. cerevisiae and C. albicans (25, 26), although they are also found abundantly in the cytoplasm and intracellular compartments, where they participate in essential translocation functions (27–29). Both Ssa1p and Ssa2p are localized in the cell wall and cell membrane of C. albicans blastoconidia and germ tubes as assessed by {beta}-ME extraction and indirect immunofluorescence experiments (18). To further confirm the identity of Hst 5-binding protein as Ssa1/2p, we probed Western blots of cellular fractions prepared from C. albicans by three sequential cellular fractionation steps using anti-HSP70 antibody in parallel with biotinylated Hst 5. Localization of Ssa proteins with Hst 5-binding protein in C. albicans was assessed in cellular fractions obtained following sequential {beta}-ME cell wall extraction and glucanidase cell wall extraction and the remaining cytosolic materials. Cell wall proteins from C. albicans cells were initially released by treatment with {beta}-ME (Fig. 3A, lane 1). Cell wall glycoproteins were recovered after {beta}-glucanidase treatment (Fig. 3A, lane 2), followed by collection of total soluble cytosolic proteins (lane 3). Hst 5-binding protein (~70 kDa) was detected only in cell wall protein and cytosolic fractions, but not in {beta}-glucanidase extracts (Fig. 3B). An Ssa protein of very similar size to the Hst 5-binding protein was detected in the same cellular fractions using anti-HSP70 monoclonal antibody (Fig. 3C), consistent with its previously reported localization (18, 19). Thus, both Ssa protein and Hst 5-binding protein co-localized on immunoblots of the {beta}-ME cell wall extracts and cytosolic fractions, but were not present in the {beta}-glucanidase extracts. Similar results were found using S. cerevisiae cellular fractions (data not shown). These data are consistent with the known localization of Ssa proteins in both C. albicans and S. cerevisiae and further support the identification of Hst 5-binding protein as Ssa1/2p.



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  		FIG. 3.
Co-localization of Ssa protein (HSP70) and Hst 5-binding proteins in the C. albicans cell wall and cytoplasm. Cell wall proteins from C. albicans were prepared by sequential digestion with {beta}-ME and {beta}-glucanidase; cells were lysed using glass beads; and soluble cytosolic proteins were recovered. All cellular fractions were separated by 7.5% SDS-PAGE, followed by Western transfer (A). Ssa proteins were identified by immunoblotting using mouse anti-HSP70 monoclonal antibody (B). Hst 5-binding proteins were identified in parallel by overlay assay and probed with biotinylated Hst 5 (C). Lanes 1, {beta}-ME cell wall extract; lanes 2, {beta}-glucanidase extract; lanes 3, cytosolic fraction. Molecular mass standards are indicated.

 

Hst 5 Interacts with Ssa1p as Determined by Yeast Two-hybrid System Analysis—As our previous data show that Hst 5 and Hst 3 bind with a 70-kDa yeast protein identified here as Ssa1p (7), we wanted to systematically measure the potential in vivo interactions between these two proteins using the yeast two-hybrid system approach. A LexA (DNA-binding domain)-bait fusion construct was expressed together with the B42 (transcriptional activation domain)-target fusion in S. cerevisiae EGY48 cells, and interactions were analyzed by the activity of the {beta}-galactosidase reporter gene. To exclude the possibility of autoactivation induced by bait fusion proteins alone, the LexA-bait fusion constructs containing the SSA1, Hst 3, or Hst 5 gene were transformed into the EGY48 strain. No detectable {beta}-galactosidase activity was found compared with the positive control (data not shown). However, EGY48 strains coexpressing SSA1 and Hst 3 or SSA1 and Hst 5 as the target and bait pair both exhibited strong {beta}-galactosidase activity in several colonies on selective medium in the presence of 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside (X-gal). Quantification of these positive transformants in liquid medium showed interaction of SSA1 and Hst 3 to be 8.30 ± 0.83 {beta}-galactosidase units and that of SSA1 and Hst 5 to be 6.30 ± 0.75 units (Table I), demonstrating strong interactions between the heat shock protein Ssa1p of C. albicans with both Hst 3 and Hst 5.


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  		TABLE I
Protein-protein interactions between human salivary histatins 3 and 5 and the C. albicans heat shock protein Ssa1p

Values are means ± S.D. of four independent measurements.

 

To determine whether Hst 3, Hst 5, or Ssa1 proteins could interact with themselves to form homo-oligomeric structures in vivo, we used constructs with the same gene as both the bait and target in the yeast two-hybrid assay described above. Interestingly, these experiments showed that both Hst 3 and Hst 5 interacted with themselves. Hst 5-Hst 5 interactions were quite strong, with a score of 14.70 ± 5.41 {beta}-galactosidase units, whereas Hst 3-Hst 3 interactions were weaker, with a score of 4.87 ± 2.52 {beta}-galactosidase units (Table I). In contrast, Ssa1p demonstrated very little interaction with itself (1.22 ± 0.24), with values only slightly above control values. These results suggest that Hst 5 might have a strong tendency to form homo-oligomeric structures in vivo, which could be relevant for its functional interactions with Ssa1p or other target proteins.

S. cerevisiae ssa1ssa2 Mutants Have Reduced Binding of 125I-Hst 5—To determine the functional relevance of Ssa1/2p interactions with Hst 5 in yeast cells, we tested the effects of deletion of both Ssa1p and its closely related family member Ssa2p on Hst 5 binding and killing in mutant cells compared with wild-type cells. For these experiments, we chose to use SSA mutants of S. cerevisiae rather than C. albicans for several reasons. First, C. albicans and S. cerevisiae express Ssa1p and Ssa2p that have 83% primary sequence identity between the two species, and both are located at the cell surface (19), suggesting that Ssa1p and Ssa2p are likely to have very similar functions in both organisms. Second, since SSA genes are essential, S. cerevisiae mutants with inactivation of both the SSA1 and SSA2 genes survive only with a wild-type copy of SSA4. Thus, ssa1ssa2ssa4 triple mutant cells are inviable (30). Our concern with creation of SSA1 and SSA2 knockouts in C. albicans, which possesses only two SSA genes, is that inactivation of one or both genes could result in a lethal phenotype or in cells with significantly impaired growth properties not amenable to testing effects of Hst 5. However, S. cerevisiae SSA1 and SSA2 deletions are well tolerated (31) since the expression of SSA4 is elevated in these ssa1ssa2 double mutants (32). Conversely, it is also possible that the closely related proteins Ssa3p and Ssa4p have overlapping functions with Ssa1p or Ssa2p with respect to Hst 5 in S. cerevisiae. Thus, SSA1 and SSA2 deletion may potentially result in reduced Hst 5 effects, but not complete loss of function, in S. cerevisiae ssa1ssa2 mutants as a result of elevated expression of Ssa4 proteins. Thus, we chose S. cerevisiae ssa1ssa2 deletion mutants for these studies with the expectation that loss of Hst 5 function might be incomplete due to Ssa3p and Ssa4p effects.

Since intact C. albicans cells express a single class of saturable binding sites, we first assessed binding of 125I-Hst 5 with the S. cerevisiae wild-type SSA strain. Binding of 125I-Hst 5 to the S. cerevisiae wild-type SSA parental strain was found to be saturable (Fig. 4) and quantitatively similar to that of C. albicans. The calculated average equilibrium dissociation constant for S. cerevisiae wild-type SSA was Kd = 2.57 x 10–6 M (n = 3), whereas that for C. albicans is Kd = 1 x 10–6 M (7), and corresponds to 9.08 x 106 binding sites/cell compared with 8.6 x 105 binding sites/C. albicans cell. Thus, S. cerevisiae wild-type SSA cells have somewhat higher numbers of Hst 5-binding sites/cell compared with the C. albicans strain previously examined. This could be a result of cell wall Ssa3p or Ssa4p that may contribute to Hst 5-binding activity or higher numbers of Ssa1p or Ssa2p in the cell wall of S. cerevisiae. In contrast, binding of 125I-Hst 5 with S. cerevisiae ssa1ssa2 was significantly decreased (Fig. 4, inverted triangles), with Kd = 1.25 x 10–6 M (n = 3). Thus, S. cerevisiae ssa1ssa2 mutants retain ~1.84 x 106 Hst 5-binding sites/cell. As expected, loss of Hst 5 binding in ssa1ssa2 mutants was substantial, but not complete, suggesting that other S. cerevisiae proteins, perhaps Ssa3p or Ssa4p, may participate in Hst 5 binding. Nevertheless, the finding that Hst 5 binding is reduced by ~80% with S. cerevisiae ssa1ssa2 mutants compared with wild-type cells strongly supports the involvement of Ssa1p and Ssa2p as functional binding proteins that mediate association of Hst 5 with yeast cells.



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  		FIG. 4.
S. cerevisiae wild-type SSA contains saturable binding sites for 125I-Hst 5 that are reduced in the isogenic {Delta}ssa1ssa2 double mutant. S. cerevisiae wild-type (squares) and {Delta}ssa1ssa2 (inverted triangles) cells (1 x 106) were suspended in binding buffer and incubated with 125I-Hst 5 (10 nM to 10 µM) for 20 min at room temperature. Binding of 125I-Hst 5 was measured following washing to remove unbound Hst 5. Specific binding (picomoles/106 cells) is defined as the difference between total and nonspecific binding. Each data point is the mean ± S.E. of triplicate measurements from at least three independent experiments. Data were fitted to a single binding site equation to obtain binding curves and Kd values using Prism Version 3.03 software. The calculated average Kd for S. cerevisiae wild-type cells was Kd = 2.57 x 10–6 M, whereas that for {Delta}ssa1ssa2 was Kd = 1.25 x 10–6 M.

 

S. cerevisiae ssa1ssa2 Mutants Are Resistant to the Fungicidal Activity of Hst 5—Since Hst 5 binding is closely linked with fungicidal activity, we next examined whether reduction of binding in S. cerevisiae SSA mutants would also affect Hst 5 killing of these cells. The fungicidal profile of Hst 5 against S. cerevisiae wild-type cells was first assessed to determine the sensitivity of these cells to Hst 5. We found the concentration dependence of Hst 5-induced killing of S. cerevisiae JH27A (wild-type SSA) to be very similar to that of other S. cerevisiae strains characterized (7), with 65–81% killing corresponding to 7.5–62.5 µM Hst 5 (Fig. 5A, circles). Loss of the SSA1 gene function ({Delta}ssa1SSA2) reduced the fungicidal activity of Hst 5 to 41% at 7.5 µM Hst 5 and to 60% at 62.5 µM Hst 5 (Fig. 5A, triangles). Similarly, mutation of the SSA2 gene (SSA1{Delta}ssa2) reduced killing of Hst 5 to 54% at a low concentration of Hst 5 (7.5 µM) and to 59% at a high concentration of Hst 5 (62.5 µM) (Fig. 5A, squares). However, the effect of the SSA2 gene mutation was not as pronounced compared with the SSA1 gene mutation ({Delta}ssa1SSA2) at a lower Hst 5 concentration (<15.5 µM), showing that an intact SSA1 gene may contribute more to Hst 5 killing than the SSA2 gene. However, mutation of both SSA genes ({Delta}ssa1ssa2) resulted in substantial loss of Hst 5 fungicidal activity, as only 24–32% of cells were killed with the same concentrations of Hst 5 (Fig. 5A, diamonds). Thus, loss of function of either the SSA1 or SSA2 gene resulted in 25% resistance to Hst 5 in mutant cells, whereas loss of function of both the SSA1 and SSA2 genes resulted in 60% resistance of {Delta}ssa1ssa2 double mutant cells to Hst 5. As with the binding assay, we found a very substantial (but not complete) loss of Hst 5 toxicity with {Delta}ssa1ssa2 double mutant cells, also suggesting that Ssa3p or Ssa4p family members may interact with Hst 5.



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  		FIG. 5.
S. cerevisiae {Delta}ssa1ssa2 mutants are resistant to the fungicidal activity of Hst 5. S. cerevisiae wild-type SSA (circles), {Delta}ssa1SSA2 mutant (triangles), SSA1{Delta}ssa2 mutant (squares), and {Delta}ssa1ssa2 mutant (diamonds) cells were preincubated at room temperature (A) or under heat shock conditions (37 °C) (B) for 30 min before addition of Hst 5. The cells were then treated with Hst 5 (7.5–62.5 µM) for another 1.5 h at room temperature (A) or at 37 °C (B). The control groups were incubated with 10 mM phosphate buffer only. Loss of cell viability is expressed as (1–(colonies after Hst 5 addition/colonies after incubation with buffer only)) x 100. Each data point is the mean ± S.E. of triplicate independent experiments.

 

In S. cerevisiae wild-type cells, both Ssa1p and Ssa2p are expressed at moderate levels under normal growth conditions, whereas the expression of Ssa1p, Ssa3p, and Ssa4p is increased after heat shock induction (31). The expression of Ssa2p is primarily constitutive, with no obvious changes detected in cellular levels after temperature shift (18). To determine whether the fungicidal activity of Hst 5 could be altered as a result of an increase in Ssa protein following heat shock, the fungicidal activity of Hst 5 was measured under heat shock conditions by transfer of cell suspensions from room temperature to 37 °C before addition of Hst 5. Under heat shock conditions, sensitivity to the fungicidal activity of Hst 5 was increased in all four strains compared with cells incubated at room temperature (Fig. 5B). In S. cerevisiae {Delta}ssa1ssa2 double mutant cells, Hst 5 killing (31 µM) was more than doubled to 61 ± 4% (Fig. 5B, diamonds) compared with room temperature assays, suggesting that levels of Ssa3p and Ssa4p may be increased after heat shock induction and mediate Hst 5 fungicidal activity, in addition to Ssa1p and Ssa2p already identified. Loss of Ssa1p and Ssa2p in the {Delta}ssa1ssa2 double mutant cells still resulted in considerably reduced sensitivity to Hst 5 (31 µM) compared with wild-type cells with intact SSA1 or SSA2 genes following heat shock (91.3 ± 2% killing). As expected with the known responses of SSA genes to heat shock conditions, the sensitivity of the ssa2 mutant with an intact SSA1 gene to Hst 5 killing was identical to that of the wild-type cells (91 ± 2 and 91 ± 2%, respectively) (Fig. 5B, squares), whereas the sensitivity of the ssa1 mutant with an intact SSA2 gene (triangles) to Hst 5 (31 µM) was less increased by heat shock (78 ± 2%), in agreement with the known primarily constitutive expression of Ssa2p.

To determine whether the increased sensitivity of cells to Hst 5 following heat shock conditions was due to an increase in cell wall HSP70-binding activity, we probed wild-type and {Delta}ssa1ssa2 double mutant cell wall extracts with antibody to HSP70 in parallel with Hst 5 binding assays (Fig. 6). S. cerevisiae wild-type SSA cell wall extracts contained prominent amounts of HSP70 protein, which were not elevated in wild-type cells subjected to heat shock conditions (Fig. 6, upper panel, left two lanes). In contrast, HSP70 proteins were significantly reduced, but readily detectable, in {Delta}ssa1ssa2 double mutant cell wall extracts and appeared to be slightly increased in cell wall extracts from {Delta}ssa1ssa2 mutants following heat shock (Fig. 6, upper panel, right two lanes).



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  		FIG. 6.
S. cerevisiae {Delta}ssa1ssa2 mutants have reduced cell wall HSP70 protein and reduced Hst 5 binding that is modulated by heat shock. HSP70 proteins in the cell wall of S. cerevisiae wild-type SSA or {Delta}ssa1ssa2 strains following room temperature (RT) or heat shock (HS) incubation were immunodetected in {beta}-ME cell wall extracts (upper panel). HSP70 protein was significantly reduced in {Delta}ssa1ssa2 mutant cells and elevated in mutant (but not wild-type) cells under heat shock conditions (upper panel). Binding of 125I-Hst 5 (6 µM) with S. cerevisiae wild-type SSA cells was not significantly changed (n.s.) by heat shock (lower panel, left two bars), whereas binding of Hst 5 with {Delta}ssa1ssa2 cells was significantly increased (p = 0.01) following heat shock (right two bars). Differences in binding between cells following heat shock were calculated as the -fold change of bound 125I-Hst 5 relative to the binding activity of wild-type SSA at room temperature in three independent experiments. Unpaired Student's t test was used to compare sets of data.

 

We next examined binding of Hst 5 to wild-type and mutant cells at room temperature and under heat shock conditions. Binding of 125I-Hst 5 with S. cerevisiae wild-type SSA cells was not significantly increased (p > 0.05) following exposure of cells to heat shock conditions, in agreement with levels of cell wall HSP70 proteins detected by immunoblotting. Thus, although global expression of Ssa1p, Ssa3p, and Ssa4p in wild-type cells is increased by heat stress, the increase in Hst 5 fungicidal activity in cells subjected to heat shock conditions is not a result of increased HSP70-binding activity localized at the cell wall. However, binding of 125I-Hst 5 with S. cerevisiae ssa1ssa2 mutant cells was found to be significantly (p = 0.01) increased under heat shock conditions (Fig. 6, lower panel), suggesting that cell wall levels of Ssa3p or Ssa4p in the ssa1ssa2 background may be increased as a result of heat stress conditions. Although SSA4 transcription in {Delta}ssa1ssa2 cells was not found to be heat stress-inducible (32), our results suggest that heat stress may modulate the cellular distribution of Ssa4p in these cells. Thus, the observed increase in Hst 5 toxicity in {Delta}ssa1ssa2 cells may be due, in part, to elevated cell wall Ssa3/4 proteins.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Ssa proteins are conserved members of the HSP70 family in yeast that function in heat shock protection, protein folding assistance, and translocation across membranes (27, 28, 29). Ssa1p and Ssa2p are major cell wall-located immunogens of C. albicans that are capable of inducing cell-mediated immune responses in mice and humans colonized by C. albicans (25, 26). These prominent immunogenic characteristics of Ssa proteins demonstrate an outer cell-surface localization for these proteins and further suggest their potential to serve as receptor molecules. Our identification of C. albicans cell envelope Ssa1/2p as the Hst 5-binding protein and the requirement of SSA1 and SSA2 genes for in vivo binding and toxicity of Hst 5 provide evidence for a novel receptor function of Ssa proteins.

In S. cerevisiae, it is possible that two additional Ssa proteins, Ssa3p and Ssa4p, also participate in Hst 5 toxicity as suggested by the binding and fungicidal activities of Hst 5 in {Delta}ssa1ssa2 cells. Since the essential functions performed by Ssa proteins make their complete genetic deletion unfeasible, we cannot unequivocally ascribe all cell-surface Hst 5-binding functions to Ssa1/2 proteins. Rather, our results show that Ssa1/2 proteins play a significant role in Hst 5 binding and toxicity.

Similarly, S. cerevisiae Kre1p was recently identified as the cell-surface receptor for K1 toxin (11). K1 toxin action is initiated by binding to this receptor, although the subsequent mediating steps leading to cell death are not yet defined (33). However, like Hst 5 fungicidal activity, post-binding cellular events in K1 toxicity involve energy-dependent interactions that ultimately lead to disruption of transmembrane ion gradients. Thus, it appears that K1 toxin and Hst 5 share many features of a receptor-mediated multistep fungicidal mechanism.

Cytosolic Ssa proteins play an important role in the binding and transport of proteins across membranes and participate in protein folding or the assembly of oligomeric protein complexes (27, 34, 35). All Ssa proteins contain a highly conserved N-terminal ATPase domain and a more variable C-terminal peptide-binding domain that are coupled to allow ATP-dependent cyclical binding and release of peptide substrates (36). This energy-dependent binding of substrate proteins by Ssa proteins permits formation of intermediate protein complexes of the correct structure for transport across intracellular membranes. Our previous finding that Hst 5 killing is dependent on cellular ATP (10, 20) is consistent with the requirement of ATP hydrolysis for Ssa1 peptide-binding function. However, pretreatment of intact C. albicans cells with anti-HSP70 monoclonal antibody used in cell lysates to detect Ssa protein did not inhibit toxicity of Hst 5 (data not shown), suggesting that this epitope is inaccessible in whole cells in vivo. Additional in vitro experiments are needed to delineate the critical Hst 5 binding domain within Ssa1/2p and to assess whether nucleotide binding modulates substrate binding affinity.

It is not known whether cell surface-located Ssa proteins have any homologous role in oligomeric assembly or translocation of bound proteins as part of their function as Hst 5-binding proteins. As well as confirming Hst 5 and Ssa1p interactions by yeast two-hybrid analysis, we also found strong interactions between Hst 5 and Hst 5, suggesting that Hst 5 may form oligomeric structures in vivo. In this regard, it has recently been shown that the protective antigen for anthrax toxin formed oligomers rapidly after incubation with cells and that the oligomeric form was selectively internalized by a membrane-anchored receptor (37). Thus, specific multimer formation of protein is induced upon binding, whereas complex formation is required for translocation into the cell. Similar interactions between Hst 5 and Ssa1p may occur upon its binding at the cell surface and help oligomeric assembly of Hst 5 into a translocation-competent form. Although duplication of the functional domain of Hst 5 within the same molecule only slightly enhanced its candidacidal activity (38), others found no evidence of Hst 5 multimer formation in a hydrophobic environment (39). However, our evidence provided by yeast two-hybrid analysis strongly suggests the potential of Hst 5 to self-assemble in vivo. It may be speculated that Ssa1p binding of Hst 5 at the cell surface functions to stabilize interactions between Hst 5 molecules and to promote oligomer formation. Alternatively, Hst 5 may form inactive oligomeric structures in saliva that may be disassembled into active or transport-competent single proteins through binding to Ssa protein.

Binding of Hst 5 to Ssa proteins may also be required for intracellular transport and facilitation of transfer of Hst 5 across the plasma membrane. In C. albicans, extracellular binding of Hst 5 is invariably required for toxicity, although translocation and intracellular distribution of Hst 5 are also found (8, 12). Intracellular expression of Hst 5 in C. albicans without exogenous application results in substantial loss of cell viability (15). Thus, intracellular transport of Hst 5 is believed to be critical for delivery of Hst 5 to its ultimate cellular target. Cytosolic Ssa1p and Ssa2p are required for the transport of many proteins such as vacuolar hydrolase aminopeptidase-1 independently from the secretory pathway (40). Whether cell surface-localized Ssa1p and Ssa2p perform similar functions in transport of Hst 5 across the cell envelope remains an important question to be answered. Cytosolic Ssa proteins may play an additional role in Hst 5 transport once they reach the inner cell membrane. Our finding that the fungicidal activity of Hst 5 was substantially increased in wild-type cells under heat shock conditions that induce expression of Ssa1p, Ssa3p, and Ssa4p, but without elevated cell wall HSP70-binding activity, could be explained if these proteins have an additional role in Hst 5 transport or other functions within the cytosolic compartment. Thus, if cytosolic Ssa proteins are involved in intracellular transport of Hst 5 to its target site, then heat stress induction of cytosolic Ssa proteins may increase transport and thus elevate Hst 5 toxicity.

This study provides evidence for a novel function for yeast Ssa1/2 proteins in that they are cell envelope binding receptors for Hst 5 that are required for its fungicidal activity. Clarifying the additional functional roles of Ssa proteins in assembly or transport of Hst 5 in mediation of cytotoxic effects against pathogenic fungi will provide significant new understanding of the mechanisms of nonimmune protection by host defense systems.


   FOOTNOTES
 
* This work was supported by United States Public Health Service Grants DE10641 and DE00406 from NIDCR, National Institutes of Health (to M. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: SUNY at Buffalo Main Street Campus, 310 Foster Hall, 3435 Main St., Buffalo, NY 14214. Tel.: 716-829-3067; Fax: 716-829-3942; E-mail: edgerto{at}buffalo.edu.

1 The abbreviations used are: Hst, histatin; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry; HstBP, histatin-binding protein; {beta}-ME, {beta}-mercaptoethanol; contig, group of overlapping clones. Back

2 Available at info.med.yale.edu/wmkeck. Back

3 Available at www.ncbi.nlm.nih.gov/cgi-bin/Entrez/genom_table_cgi?organism=euk. Back

4 Available at us.expasy.org/tools/#translate. Back

5 Available at us.expasy.org/cgi-bin/sim.pl?prot. Back

6 Available at www.ebi.ac.uk/clustalw/. Back


   ACKNOWLEDGMENTS
 
We thank Dr. E. A. Craig for critical reading of the manuscript and helpful suggestions.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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J. R. Luque-Ortega, W. v. Hof, E. C. I. Veerman, J. M. Saugar, and L. Rivas
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FASEB J, June 1, 2008; 22(6): 1817 - 1828.
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N. L. van der Weerden, F. T. Lay, and M. A. Anderson
The Plant Defensin, NaD1, Enters the Cytoplasm of Fusarium Oxysporum Hyphae
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W. S. Jang, X. S. Li, J. N. Sun, and M. Edgerton
The P-113 Fragment of Histatin 5 Requires a Specific Peptide Sequence for Intracellular Translocation in Candida albicans, Which Is Independent of Cell Wall Binding
Antimicrob. Agents Chemother., February 1, 2008; 52(2): 497 - 504.
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Differentially Expressed Proteins in Derivatives of Candida albicans Displaying a Stable Histatin 3-Resistant Phenotype
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S. Vylkova, N. Nayyar, W. Li, and M. Edgerton
Human {beta}-Defensins Kill Candida albicans in an Energy-Dependent and Salt-Sensitive Manner without Causing Membrane Disruption
Antimicrob. Agents Chemother., January 1, 2007; 51(1): 154 - 161.
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X. S. Li, J. N. Sun, K. Okamoto-Shibayama, and M. Edgerton
Candida albicans Cell Wall Ssa Proteins Bind and Facilitate Import of Salivary Histatin 5 Required for Toxicity
J. Biol. Chem., August 11, 2006; 281(32): 22453 - 22463.
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J. Zhu, P. W. Luther, Q. Leng, and A. J. Mixson
Synthetic histidine-rich peptides inhibit Candida species and other fungi in vitro: role of endocytosis and treatment implications.
Antimicrob. Agents Chemother., August 1, 2006; 50(8): 2797 - 2805.
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S. Vylkova, X. S. Li, J. C. Berner, and M. Edgerton
Distinct Antifungal Mechanisms: {beta}-Defensins Require Candida albicans Ssa1 Protein, while Trk1p Mediates Activity of Cysteine-Free Cationic Peptides
Antimicrob. Agents Chemother., January 1, 2006; 50(1): 324 - 331.
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T. Tanida, T. Okamoto, E. Ueta, T. Yamamoto, and T. Osaki
Antimicrobial peptides enhance the candidacidal activity of antifungal drugs by promoting the efflux of ATP from Candida cells
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Z. Feng, B. Jiang, J. Chandra, M. Ghannoum, S. Nelson, and A. Weinberg
Human Beta-defensins: Differential Activity against Candidal Species and Regulation by Candida albicans
Journal of Dental Research, May 1, 2005; 84(5): 445 - 450.
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B. C. Monk, K. Niimi, S. Lin, A. Knight, T. B. Kardos, R. D. Cannon, R. Parshot, A. King, D. Lun, and D. R. K. Harding
Surface-Active Fungicidal D-Peptide Inhibitors of the Plasma Membrane Proton Pump That Block Azole Resistance
Antimicrob. Agents Chemother., January 1, 2005; 49(1): 57 - 70.
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D. Baev, A. Rivetta, S. Vylkova, J. N. Sun, G.-F. Zeng, C. L. Slayman, and M. Edgerton
The TRK1 Potassium Transporter Is the Critical Effector for Killing of Candida albicans by the Cationic Protein, Histatin 5
J. Biol. Chem., December 31, 2004; 279(53): 55060 - 55072.
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