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Antimicrobial Agents and Chemotherapy, February 2008, p. 497-504, Vol. 52, No. 2
0066-4804/08/$08.00+0     doi:10.1128/AAC.01199-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Woong Sik Jang,¹ Xuewei Serene Li,¹ Jianing N. Sun,¹ and Mira Edgerton^1,2*

Department of Oral Biology,¹ Restorative Dentistry, School of Dental Medicine, State University of New York at Buffalo, Buffalo, New York 14214²

Received 11 September 2007/ Returned for modification 22 October 2007/ Accepted 1 November 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The activity of histatin 5 (Hst 5) against Candida albicans is initiated through cell wall binding, followed by translocation and intracellular targeting. The C. albicans cell wall protein Ssa2 is involved in the transport of Hst 5 into cells as part of cell killing. P-113 (a 12-amino-acid candidacidal active fragment of Hst 5) and P-113Q2.10 (which is inactivated by a glutamine substitution of the Lys residues at positions 2 and 10) were compared for their levels of cell wall binding and intracellular translocation in Candida wild-type (wt) and ssa2 strains. Both P-113 and P-113Q2.10 bound to the walls of C. albicans wt and ssa2 cells, although the quantity of P-113Q2.10 in cell wall extracts was higher than that of P-113 in both strains. Increasing the extracellular NaCl concentration to 100 mM completely inhibited the cell wall association of both peptides, suggesting that these interactions are primarily ionic. The accumulation of P-113 in the cytosol of wt cells reached maximal levels within 15 min (0.26 µg/10⁷ cells), while ssa2 mutant cells had maximal cytosolic levels of less than 0.2 µg/10⁷ cells even after 30 min of incubation. Furthermore, P-113 but not P-113Q2.10 showed specific binding with a peptide array of C. albicans Ssa2p. P-113Q2.10 was not transported into the cytosol of either C. albicans wt or ssa2 cells, despite the high levels of cell wall binding, showing that the two cationic lysine residues at positions 2 and 10 in the P-113 peptide are important for transport into the cytosol and that binding and transport are independent functional events.

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Candida albicans is an opportunistic pathogen that causes oropharyngeal candidiasis in compromised hosts (9). Oral mucosal infections by Candida species are also frequent in patients receiving radiation therapy for head and neck cancer (25, 26, 30) or in individuals with compromised salivary function (15, 19). There are three main classes of clinically useful antifungal agents: polyene antibiotics, the fluoropyrimidine flucytosine, and azoles (36). However, the toxicities of the currently used polyene antifungal drugs and the emergence of candidal species resistant to fluoropyrimidine flucytosine and azole-based agents have resulted in the initiation of a search for innate peptide antibiotics as alternative drug therapies (32).

Histatins are a group of small, histidine-rich cationic antimicrobial peptides secreted into the saliva by human parotid and submandibular-sublingual glands (1). These proteins exhibit fungicidal activity against several Candida species, Saccharomyces cerevisiae, and Cryptococcus neoformans (11, 20). Among at least 50 histatin peptides derived from posttranslational proteolytic processing (7, 18), histatin 5 (Hst 5), which contains 24 amino acids, has the highest level of activity against C. albicans (38).

The fungicidal activity of Hst 5 is a distinctive multistep mechanism involved in the binding of Hst 5 to Candida cell wall proteins, followed by translocation to intracellular compartments (24). When it reaches the cytotosol, Hst 5 causes the nonlytic efflux of cellular ATP and the loss of other small nucleotides and ions from the cell through mechanisms that involve the Trk1p potassium transporter (3, 4). The initial binding of histatins to the candidal cell wall is followed by rapid internalization, which can be slowed by low temperature conditions or azide pretreatment, which depletes the cell of energy (17, 39). Thus, specific energy-dependent processes are involved in the binding and/or uptake of the peptide. Hst 5 was shown to have weak interactions with liposome membranes compared with those of the histatin variant dhvar4 (8), implying that translocation across biological membranes requires additional proteins. In this regard, we identified the C. albicans Ssa2 protein to be a binding partner for Hst 5 (23), and ssa2 mutants are defective in Hst 5 translocation and killing (24). Ssa proteins are conserved members of the heat shock protein 70 family in yeast, of which Candida only has two members: Ssa1p and Ssa2p. The major localization of Ssa proteins in Candida is the cytoplasm, where they are involved in heat shock protection and assistance in protein folding and translocation across the membranes. The C. albicans Ssa1 and Ssa2 proteins are both exported to the cell wall, where the Ssa2 protein but not the Ssa1p protein is required for Hst 5 binding and intracellular uptake.

On the basis of the structure of Hst 5, several synthetic congeners have been designed and evaluated for their activities against a variety of antibiotic-resistant bacteria and fungi (16, 27). One histatin variant with a higher amphipathicity (dhvar4) was found to have enhanced non-energy-dependent fungicidal activity (28), while cyclized histatin 3 was 100-fold more active against Saccharomyces cerevisiae than Hst 5 (6). Both of these structural derivatives appear to have more disruptive interactions with fungal membranes than the parent Hst 5 protein (6, 28). To evaluate whether histidine residues are crucial for the activity of Hst 5, amino acid substitutions were made in two adjacent histidine residues without altering the conformation of the protein, and these substitutions resulted in 8- to 20-fold reductions in candidacidal activity (33). Among other derivatives, P-113 (amino acid residues 4 to 16 of Hst 5) is as active as full-length Hst 5 in terms of its in vitro candidacidal activity (27). However, the replacement of the Lys residue at position 2 (Lys₂) and Lys₁₀ of P-113 with glutamine (P-113Q2.10) resulted in the nearly complete loss of killing function, while the substitution of histidine residues within this peptide did not alter the killing function (27). Interestingly, the replacement of four cationic residues (Lys₂ Arg₃, Arg₉, Lys₁₀) of P-113 with glutamine resulted in the complete loss of candidacidal activity; however, increasing the amphipathicity of P-113 by amino acid substitution had no effect on its killing activity (27). Thus, that study identified a minimum of two nonhistidine cationic residues that are crucial for the toxicity of the peptide and further showed that the degree of the amphipathic molecular moment of the peptide is unrelated to its killing activity.

While the overall process of binding and uptake required for the antifungal activity of Hst 5 has been well studied, the individual cellular components involved in translocation into the cell remain largely unknown. Therefore, P-113 and the inactive substituted peptide P-113Q2.10 were chosen to probe for the competence of the intracellular translocation in a C. albicans wild-type (wt) strain and an ssa2 mutant strain. We examined translocation by measuring the time-dependent levels of each peptide in the cell wall and cytosolic compartments and found that P-113Q2.10 is competent in cell wall binding but is defective in cytosolic translocation. Thus, we show for the first time that the cell wall binding and intracellular transport of Hst 5 in C. albicans are independent events and that translocation is dependent on the primary sequence of the imported peptide.

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Materials. C. albicans wt strain CAF4-2 is the parental strain for the ssa1 and ssa2 mutant strains used in these experiments. Cells were cultured for assays in yeast nitrogen base (YNB; Qbiogene, Morgan Irvine, CA), maintained on yeast extract-peptone-dextrose (Qbiogene) agar plates, and recultured monthly from –78°C stocks. The peptides (P-113 and P-113Q2.10) and N-terminal biotin-labeled peptides were synthesized by using standard solid-phase synthesis protocols and were purified by reversed-phase high-performance liquid chromatography by Genemed Synthesis Inc. (San Francisco, CA). The primary structures of these peptides are shown in Table 1.

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TABLE 1. Primary structures of antimicrobial peptides used in this study

Candidacidal assay. The activities of the peptides against the Candida strains were tested by using standard microdilution plate candidacidal assays (37) and radial diffusion assays (21). For the candidacidal assays, C. albicans cells were grown in YNB medium and washed twice with 10 mM sodium phosphate buffer (NaPB; Na₂HPO₄, NaH₂PO₄, pH 7.4), and then the cells (1 x 10⁶) were mixed with different concentrations of the peptides and incubated at room temperature with constant shaking for 1 h. The cell suspensions were diluted in 10 mM NaPB, and aliquots of 500 cells were spread onto YNB agar plates and incubated for 48 h at room temperature to visualize the surviving colonies. Cell survival was expressed as a percentage of that for the control, and the loss of viability was calculated as [1 – (number of colonies from peptide-treated cells/number of colonies from control cells)] x 100. Candidacidal assays were performed in triplicate. For the radial diffusion assay, C. albicans cells were grown in YNB medium overnight and washed twice with 10 mM NaPB. Washed yeast-phase C. albicans cells (1 x 10⁶ CFU) were trapped in thin underlay gels, which contained 9 mM sodium phosphate, 1 mM sodium citrate buffer, 1% (wt/vol) agarose (A 6013; Sigma), and 0.3 mg of Sabouraud dextrose broth (Difco)/ml. In some experiments, the underlay agars were supplemented with 99 mM sodium phosphate. Stock peptide solutions and serial twofold dilutions with concentrations ranging from 7.81 µM to 250 µM were prepared in distilled water. Peptide samples (5 µl) were loaded into 3-mm-diameter wells that had been punched in the underlay gels. After incubation at 37°C for 3 h, a 10-ml overlay gel of 1% agarose and 6% Sabouraud dextrose broth was poured on the underlay gel. After the plates were incubated overnight at 37°C, the clear-zone diameters were measured to the nearest 0.1 mm and graphed against the peptide concentration. Zone diameters are expressed in units (0.1 mm = 1 unit).

Interaction of P-113 with the component of the Candida cell wall and cytoplasmic transport of P-113. The localization of the peptides in the C. albicans wt and ssa2 mutant strains was examined by two sequential cellular fractionation steps consisting of β-mercaptoethanol (β-ME) cell wall extraction, followed by cytosolic fractionation, as described previously (24). Briefly, early-log-phase cells (1 x 10⁸) were washed twice with 10 mM NaPB and suspended in 1 ml of NaPB, and biotin-labeled peptides were added to a final concentration of 31.25 µM. The cell mixtures were incubated with constant shaking for 0, 5, 15, 30, 60, and 90 min. After centrifugation at 6,000 rpm and 4°C, the supernatant of each sample was collected in a fresh 1.5-ml tube to measure the level of remaining peptides, and the cells were washed with 10 mM NaPB. β-ME-extractable cell wall components were released by incubation of the cell suspension in ammonium carbonate buffer (pH 8.0) containing 1% (vol/vol) β-ME for 30 min at 37°C. The supernatant containing the cell wall extract obtained by extraction with β-ME was collected following centrifugation at 500 x g. The β-ME-treated cells were then washed twice with 10 mM NaPB, and the cell pellet was disrupted in 1 volume of 0.5-mm glass beads and 1 volume of cold lysis buffer supplemented with protease inhibitors (10 mM NaPB, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, and 1 µg/ml benzamidine). Cell lysates were prepared by using a Fastprep apparatus at 4°C. The cytosolic fraction was collected following centrifugation at 13,000 rpm for 10 min. The protein concentrations of the cell wall extracts obtained by extraction with β-ME and the cytosolic proteins were measured by a bicinchoninic acid assay. Subsequently, the cell wall proteins and cytosolic proteins were electrophoresed by 16% Tricine sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (29) and transferred to polyvinylidene difluoride (PVDF) membranes. Supernatant samples were loaded onto PVDF membranes by using a slot blot apparatus (Hoefer) that had been equilibrated in Tris-buffered saline (TBS; 20 mM Tris, 500 mM NaCl, pH 7.5) for 10 min. The membrane was then incubated for 60 min in blocking solution (3% skim milk in TBS containing 0.05% Tween 20 [TBST]). After a brief wash in TBST, the membrane was incubated for 1 h in blocking solution containing 1,000-fold-diluted streptavidin conjugated with horseradish peroxidase (HRP; Pierce). Finally, the membrane was washed twice in TBST and once in TBS and was finally submerged in HRP color development solution (50 ml of TBS containing 30 µl of H₂O₂ mixed with 0.3% of chloronaphthol in 10 ml of ice-cold methanol). The concentrations of P-113 and P-113Q2.10 in the cell wall extracts and the cytosolic extracts (10 µg/lane) were determined by comparison with P-113 and P-113Q2.10 concentration standards (0.5, 0.25, 0.125, and 0.06125 µg) and analysis with Quantity One software (version 4.2).

Mapping of P-113 and P-113Q2 binding motifs on Ssa2 peptide arrays. The primary sequence of C. albicans Ssa2p was obtained from the Universal Protein Resource (http://www.ebi.uniprot.org/index.shtml), and a peptide array of the Ssa2 protein was designed so that the peptide fragment consisted of sequential 13-amino-acid fragments with an overlap of 2 amino acids between each peptide. Overlapping peptides comprising the entire sequence of Ssa2p (645 amino acids) were synthesized by JPT Peptide Technologies GmbH (Berlin, Germany) and were printed on cellulose-β-alanine membranes. The preparation of the membrane, as well as control experiments for the detection of the false-positive binding of streptavidin-HRP to the membrane, was performed by following the manufacturer's protocol. Spot array cellulose-β-alanine membranes were rinsed with methanol for 5 min, followed by three washes with 20 ml TBS (50 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl, pH 8.0) for 10 min each. The membrane was then blocked with Starting Block (Pierce) blocking buffer (an albumin and endogenous biotin-free buffer compatible with a streptavidin system) for 2 h at room temperature. P-113 or P-113Q2.10 (2 µM) was added to the blocking buffer, and the mixture was incubated with the membrane for 3 h at room temperature. After an extensive wash with TBS buffer to remove the unbound peptides, the membrane was incubated with streptavidin-HRP (1:20,000) for 1 h at room temperature. After the membranes were washed six times with TBS buffer, they were developed with the SuperSignal West Pico chemiluminescent substrate (150 µl/cm²; Pierce) for 5 min and scanned by using a Fuji LAS-1000 Plus chemiluminescence imager. The repeatabilities of the binding signals were confirmed by reprobing each membrane following regeneration with regeneration buffer (62.5 mM Tris-HCl, 2% SDS, 0.7% 2-mercaptoethanol, pH 6.7) for 30 min at 50°C.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Candidacidal activities. We have found that Hst 5 binds to Candida albicans cell wall Ssa proteins and is involved in the transport of Hst 5 into cells as part of its mechanism of cell killing (23), as evidenced by the results for Candida ssa2 mutants, which have a reduced uptake of Hst 5 and a subsequently reduced toxicity compared with the levels of uptake and toxicity for wt cells (24). It is not known if P-113 (the smaller active fragment of Hst 5) also utilizes this mechanism of binding and uptake. Therefore, the antifungal activities of P-113 and P-113Q2.10 were examined by using C. albicans ssa1 and ssa2 mutant strains by two independent assays: our standard candidacidal assay, in which cells are exposed to peptides for 1 h and then removed and placed in growth medium, and a radial diffusion assay, in which growing cells are continuously exposed to the peptide. These assays were compared, since the levels of expression of Ssa proteins may differ depending on the growth conditions. The treatment of C. albicans wt or ssa1 cells with P-113 (31.5 µM) under standard assay conditions (Fig. 1 A) resulted in 90% cell killing, while the ssa2 strain had reduced susceptibility (76% killing). At lower P-113 concentrations, both the ssa1 and the ssa2 strains had reduced sensitivities. In contrast, P-113Q2.10 (in which the Lys₂ and Lys₁₀ of P-113 are replaced with glutamine) had no activity against any of the Candida strains at concentrations below 31.5 µM and less than 10% killing at 31.5 µM (Fig. 1A). When the activities of P-113 and P-113Q2.10 against the C. albicans wt and the ssa1 and ssa2 mutant strains were visualized by radial diffusion assays, they were found to be nearly identical to those obtained by conventional candidacidal assays (Fig. 1B). P-113 had potent activity against the C. albicans wt and ssa1 strains, while the ssa2 strain had substantial resistance to this peptide (Fig. 1B, left). P-113Q2.10 had very little activity against any of Candida strains (Fig. 1B, right). Thus, P-113 has similar effects when its activities were measured by either conventional candidacidal assays or the radial diffusion assay; and as for Hst 5, Ssa2p is involved in the toxicity of P-113, while Ssa1p makes little contribution to the toxicity.

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FIG. 1. Candidacidal activities of P-113 and P-113Q2.10. The susceptibilities of cells to P-113 and P-113Q2.10 killing was examined with cells of the C. albicans wt (closed circles and open circles, respectively), ssa1 mutant (closed squares and open squares, respectively), and ssa2 mutant (closed triangles and open triangles, respectively) by microdilution plate (A) and radial diffusion (B) assays. In the standard microdilution plate candidacidal assays, the cells (1 x 106) were incubated with peptides for 1 h, diluted to quench their activity, and then spread onto YNB agar plates to assess cell survival. Cell death (loss of viability) as a result of peptide exposure was calculated as [1 – (number of colonies from peptide-treated cells/number of colonies from control cells)] x 100. For the radial diffusion assays, C. albicans cells were incorporated into underlay gels and the antifungal activities of P-113 and P-113Q2.10 were determined following overnight incubation of peptides loaded into wells that had been punched in the underlay gels (B, bottom panel). Cell death was calculated from the clear-zone diameters (in which cells were unable to grow) and graphed against the peptide concentration (B, upper panel). Values are the means and standard deviations from three independent experiments. The candidacidal activity measured for each peptide was equivalent whether either short incubation periods in the microdilution plate assay or continuous incubation in the radial diffusion assay was used. Both assays showed that P-113 had a high level of potency against C. albicans wt cells and that the ssa2 mutant (but not the ssa1 mutant) had reduced susceptibility to P-113. P-113Q2.10 showed greatly reduced killing activity against wt cells, while the ssa2 mutants had virtually no sensitivity to this peptide.

Extracellular salt prevents cell wall binding and toxicity of P-113. Because divalent cations and NaCl reduce the activity of P-113 (27), we questioned whether salts inhibit the initial interactions between P-113 and the cell wall or if subsequent translocation and/or cytosolic functions are affected. Since 100 mM NaPB completely inhibits the candidacidal activity of Hst 5 (22), we examined the effect of this salt concentration on the activities of P-113 and P-113Q2.10 by the radial diffusion assay. P-113 had potent activity against C. albicans cells in 10 mM NaPB (Fig. 2, left), but it was completely inactivated in the presence of 100 mM NaPB (Fig. 2, right). The low level of toxicity elicited by P-113Q2.10 at high concentrations (>62 µM) was also lost when it was incubated with 100 mM NaPB. Previous studies with Hst 3 have suggested that high external salt conditions prevent the efficient import of the protein into the cytosolic compartment, resulting in impaired toxicity (39). Therefore, we examined the cytosolic levels of translocated P-113 in C. albicans over a time course (5, 15, and 30 min) under conditions with low (10 mM NaPB) or high (100 mM NaPB) salt concentrations (Fig. 3). Total cytosolic proteins were extracted after incubation with biotin-labeled P-113 under each condition, and equal amounts of total protein were immunoblotted to detect the levels of P-113 (Fig. 3A, middle panel). Under the conditions of the low salt concentration used for the candidacidal assays, P-113 was present in the C. albicans cytosol within 5 min, while P-113 was absent in cytosolic extracts from cells incubated under conditions with a high salt concentration (Fig. 3A, middle panel). This result could be due to the loss of the transport function of bound P-113 or the inability of P-113 to bind to the cell wall under conditions with a high salt concentration. To differentiate between these two possibilities, cell wall proteins were stripped from whole C. albicans cells immediately following 5, 15, and 30 min of incubation of the cells with P-113 under conditions with low and high salt concentrations and immunoblotted as described above for the cytosolic extracts. P-113 was detected in the cell wall extracts within 5 min in cells suspended in 10 mM NaPB (Fig. 3B, middle panel), but little to no P-113 was found in the cell walls of C. albicans cells incubated in 100 mM NaPB (Fig. 3B, middle panel). These results show that the high-salt-concentration condition inhibits the initial binding of P-113 with the cell wall, so that subsequent cytosolic transport does not occur. Furthermore, the interruption of binding of P-113 to the cell wall by salt suggests that initial contacts between this protein and C. albicans cells are largely nonspecific ionic interactions that are disrupted under conditions with high salt concentrations.

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FIG. 2. Increasing salinity inhibits candidacidal activities of P-113 and P-113Q2.10. C. albicans wt cells were tested for their susceptibilities to P-113 and P-113Q2.10 in the presence or the absence of 100 mM NaPB by the radial diffusion assay. The antifungal activities of P-113 (closed circles) and P-113Q2.10 (open circles) in 10 mM NaPB are shown on the left, while the antifungal activities of these peptides incubated in 100 mM NaPB are shown on the right. The diameters of the clear zones (lower panels) are displayed in units (10 units = 1 mm) and are graphed against the peptide concentration (upper panels). Incubation in the presence of 100 mM NaPB completely inhibited the fungicidal activities of both the P-113 and the P-113Q2.10 peptides.

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FIG. 3. Conditions with high salt concentrations prevent the cell wall binding of P-113. CAF4-2 wt cells were incubated with biotin-labeled P-113 (31.5 µM) in 10 mM NaPB (closed circles) or 100 mM NaPB (closed diamonds) buffer for 1 to 30 min at 37°C. The cells were washed, and then cell wall extraction and cytosolic fractionation were done sequentially to recover P-113 from each cellular compartment. The cell wall and cytosolic fractions for each time point (10 µg/lane) were subjected to 16% Tricine SDS-PAGE (upper panels) and were immunoblotted with streptavidin-HRP to detect P-113 (middle boxes). P-113 immunoblots were quantitated by using P-113 standards, and the cytosolic (A) and cell wall (B) levels of P-113 were plotted as a function of time (lower graphs). P-113 was detected in the cell wall and cytosol within 5 min of peptide incubation in 10 mM NaPB. However, incubation in 100 mM NaPB prevented cell wall binding of P-113 (B), so that no cytosolic localization of P-113 was detected (A). Lanes M, molecular mass markers; lanes C, controls (cells treated with buffer alone).

P-113 but not P-113Q2.10 binds to a peptide array of Candida Ssa2p. Since Hst 5 binds to Ssa2p as part of the mechanism of peptide uptake into the cell (24), we wanted to determine whether P-113 was also capable of direct interactions with Ssa2p. For this purpose, a peptide array of Ssa2p comprising the entire 645-amino-acid sequence was constructed so that each peptide spot consisted of sequential 13-amino-acid fragments from Ssa2p with an overlap of 2 amino acids between each peptide. Biotin-labeled P-113 or P-113Q2.10 was incubated with each membrane and extensively washed to remove unbound peptide, and bound P-113 or P-113Q2.10 was visualized by chemiluminescence. Strong binding of P-113 was detected in two regions of Ssa2p, spot 7 (AKRLIGRKFDDHE) and spot 23 (KRKNKKDISTNQR), while weaker binding was visualized with three other peptide fragments (Fig. 4, upper membrane). In sharp contrast, no binding with any region of Ssa2p was detected when P-113Q2.10 was used as a probe (Fig. 4, bottom membrane). These results show that P-113 is capable of direct binding to selective peptide regions of Ssa2p; however, the mutation of two amino acids in P-113Q2.10 results in the loss of this binding capability. This suggests that the reduced sensitivity of the ssa2 mutant to P-113 and the complete loss of antifungal activity of P-113Q2.10 could be a result of differences in the abilities of the peptides to bind with the Ssa2 protein and subsequent intracellular transport. Therefore, we next investigated the properties of the transport of P-113 or P-113Q2.10 into the cytosol in wt and ssa2 mutant cells.

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FIG. 4. P-113 binds to selective peptides in an Ssa2 peptide array, while P-113Q2.10 does not bind to the Ssa2 peptide array. Peptide arrays containing the complete library of the Candida albicans Ssa2 protein (JPT Peptide Technologies GmbH) were used to probe for binding sites for P-113 and P-113Q2.10. Each spot (0.37 cm by 0.37 cm) contains 5 nmol of a 13-mer peptide (more than 70% purity) encoding the Ssa2 protein sequence from the N terminus to the C terminus with a 2-amino-acid overlap between consecutive peptides. The arrays were first incubated with streptavidin-HRP antibody to verify the absence of nonspecific binding. The Ssa2 arrays were then incubated with biotin-labeled P-113 or P-113Q2.10 (2 µM) and extensively washed, and the binding was visualized with streptavidin-HRP and enhanced chemiluminescence development. P-113 strongly bound to two peptides of the Ssa2 protein and more weakly to three other peptides. In sharp contrast, P-113Q2.10 did not bind to any region of the peptide array.

P-113 import is facilitated, but not limited, by Ssa2p. Cells were treated with 31.5 µM each peptide under low-salt-concentration conditions, as described above for the candidacidal assays, and three compartments (unbound peptide in the supernatant, cell wall-associated peptide, and cytosolic peptide) were collected for each cell type at selected time points. Biotin-labeled P-113 or P-113Q2.10 was added to C. albicans wt and ssa2 mutant cells; and then the unassociated peptide not taken up by the cells was collected from the supernatant over 5, 15, 30, 60, and 90 min and measured by slot blot analysis (Fig. 5). P-113 rapidly disappeared from the supernatant samples of wt cells, so that by 15 min no further free peptide was detected in the cell supernatants. The level of P-113 was also reduced in the ssa2 supernatant samples, but the time required for P-113 to be substantially depleted from the supernatant was increased to 30 min. Even after 90 min of incubation with ssa2 cells, some P-113 could be detected in the cell supernatant. In contrast, the levels of P-113Q2.10 were slightly reduced in the supernatants of both the C. albicans wt and ssa2 strains after 15 min of incubation, but they were not further reduced even after 90 min of incubation (Fig. 5). Thus, the cells took up P-113Q2.10 to a threshold level beyond which no further peptide could be adsorbed. This threshold level appeared to be equivalent in wt and ssa2 cells, suggesting that Ssa2p is not involved in the uptake of P-113Q2.10. In contrast, C. albicans ssa2 cells had a reduced velocity of uptake of P-113 as well as incomplete total uptake from the supernatant, showing the involvement of Ssa2p in the uptake of P-113, perhaps as a facilitator protein.

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FIG. 5. P-113 is rapidly depleted from cell supernatants following incubation with C. albicans wt and ssa2 cells, while P-113Q2.10 remains in the cell supernatant. C. albicans wt and ssa2 mutant cells were each incubated with biotin-labeled P-113 or P-113Q2.10 (31.5 µM) for 5, 15, 30, 60, and 90 min. After centrifugation to remove the cells, the cell supernatants (50 µl) were loaded onto a slot blot apparatus and the membrane was probed with streptavidin-HRP and visualized by enhanced chemiluminescence. C, control (cells treated with buffer only). P-113 was completely depleted from the supernatants of wt cells following 15 min of incubation, while depletion from the ssa2 mutant cell supernatants was slower, requiring 90 min before no further P-113 could be detected in the supernatant. In contrast, the P-113Q2.10 levels were somewhat reduced at 15 min, but no further depletion from the supernatant was detected even after 90 min of incubation.

P-113Q2.10 binds to the Candida cell wall but is defective in translocation. To differentiate between cell wall binding and the intracellular transport of P-113 and P-113Q2.10, cell wall proteins were first extracted by β-ME, and then the cells were disrupted and the cytosol was collected. The protein content was normalized for each cell extract, and the content of P-113 or P-113Q2.10 at each time point was analyzed by immunoblotting. P-113 was detected in cell wall extracts of both wt and ssa2 strains within 5 min after addition of the peptide (Fig. 6 A). The P-113 levels in the cell wall reached a maximum by 15 min and then remained relatively constant over 90 min of treatment (Fig. 6C). Cell wall extracts from both the wt and the ssa2 strains contained similar levels of P-113 (0.1 µg/10⁷ cells). Surprisingly, cell wall extracts from both the wt and the ssa2 strains had significantly higher levels of P-113Q2.10 within 5 min of incubation (0.15 µg/10⁷ cells) compared to the levels of P-113, and within 15 min the maximal level of P-113Q2.10 was nearly double (0.2 µg/10⁷ cells) that of P-113 (Fig. 6B and C). As for P-113, no difference in the quantity of P-113Q2.10 in the cell wall extracts was observed between the wt and the ssa2 mutant. These results suggest that Ssa2p has no influence on the total level of P-113 or P-113Q2.10 in the cell wall but that its effect may be in the subsequent transport. In addition, the higher levels of P-113Q2.10 in the cell wall may reflect the fact that the peptide is accumulated within the wall but is unable to be transported intracellularly. Therefore, we next examined the cytosolic levels of P-113 and P-113Q2.10.

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FIG. 6. The cell wall association of P-113Q2.10 is greater than that of P-113 in both wt and ssa2 cells. Strain CAF4-2 wt and ssa2 cells were incubated with 31.5 µM biotin-labeled P-113 or P-113Q2.10 in 10 mM NaPB for 5 to 90 min at 37°C. The cell wall proteins were then extracted with ammonium carbonate buffer containing β-ME, and the extracts (10 µg/lane) were subjected to Tricine SDS-PAGE and immunoblotted with streptavidin-HRP to detect P-113 (A, lower box) or P-113Q2.20 (B, lower box). The immunoblots were quantitated by using respective peptide standards, and the cell wall levels of P-113 and P113Q2.10 were plotted as a function of time (C). CAF4-2 (closed circles) and ssa2 (closed triangles) cells had equal levels of cell wall-associated P-113; however, cells treated with P-113Q2.10 had significantly higher levels of P-113Q2.10 in cell wall extracts from both CAF4-2 (open circles) and ssa2 (open triangles) cells at all times assayed.

Cytosolic P-113 was detected in the wt strain within 5 min of incubation and reached maximal levels within 15 min (0.26 µg/10⁷ cells) (Fig. 7A and C). Subsequently, the cytosolic levels of P-113 were reduced in a linear manner to about 0.2 µg/10⁷ cells after 90 min. In contrast, in the ssa2 mutant P-113 did not reach a maximal cytosolic concentration until 30 min of incubation with the peptide, and this level was less than 0.2 µg/10⁷ cells. Thus, the ssa2 mutant had a reduced rate of cytosolic transport and reached a maximal cytosolic level that was slightly less than the level of P-113 peptide found in wt cells after 90 min. Strikingly, no amount of P-113Q2.10 peptide was detected in the cytosol of either C. albicans wt or ssa2 mutant cells (Fig. 7B and C). Preincubation of C. albicans wt cells with P-113Q2.10 inhibited the subsequent binding and uptake of P-113 (data not shown), showing that both peptides utilize the same binding sites on the cell wall. However, the mutation of two lysine residues of P-113, which created P-113Q2.10, resulted in the complete loss of intracellular transport of P-113Q2.10, although it is efficiently bound to the cell wall. Thus, the higher cell wall levels of P-113Q2.10 compared to those of P-113 likely reflect the inability of the peptide to be transported into the cell and the lack of turnover of peptide as a result of intracellular transport. Furthermore, Ssa2p appears to be involved in the efficiency of transport of P-113 into the cytosol.

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FIG. 7. P-113, but not P-113Q2.10, undergoes Ssa2p-dependent cytosolic translocation. C. albicans cells (wt or ssa2) were incubated with 32 µM P-113 and P-113Q2.10 for 5 to 90 min in 10 mM NaPB at 37°C, and then the cells were fractionated to recover cytosolic proteins (250 µg/ml). The cytosolic fractions were subjected to Tricine SDS-PAGE (10 µg/lane) (A and B, upper panels) and Western blot analysis (A and B, lower panels), and the cytosolic levels of P-113 and P113Q2.10 were quantitated as a function of time (C). Cytosolic P-113 was detected in wt strain CAF4-2 cells (closed circles) within 5 min of incubation, while the P-113 levels in ssa2 cells (closed triangles) were substantially reduced at all observation times (C). Neither CAF4-2 wt cells (open circles) nor ssa2 cells (open triangles) treated with P-113Q2.10 had any detectable P-113Q2.10 in the cytosolic extracts, showing that P-113Q2.10 is defective in intracellular translocation.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Peptide import into cells has attracted increased attention not only as a basis for understanding the basic mechanisms of intracellular trafficking of peptides but also for the generation of peptide-mediated drug delivery systems or targeted antimicrobial peptides (12). Because of the positive charge of cationic antifungal peptides at physiological pH, the prevailing view is that the initial binding to the cell surface is through electrostatic interactions with anionic surface mannans and glycans. Indeed, we found this to be the case, as an increase in the extracellular salt concentration to 100 mM completely inhibited the binding of P-113 as well as that of P-113Q2.10, showing that initial binding with the cell wall is predominantly through electrostatic interactions rather than by binding with specific amino acid sequences or motifs. However, the charge interactions between cationic antimicrobial peptides and the anionic moieties of cell surfaces can be important determinants for organism specificity as well as salt sensitivity (31). Cationic charge clustering of dibasic amino acids such as Arg or Lys is commonly found at the N termini of many open-chain antimicrobial peptides. Thus, the number, proximity, and topology of charged amino acids may confer the specificity of binding interactions with cell surface molecules. For Candida, outer cell wall galactomannans, mannans, or beta-1,3-glucans may possess specific determinants of binding for Hst 5. Such interactions are supported by the findings from our previous studies, which found reduced killing by Hst 5 upon digestion of the Candida cell wall, producing spheroplasts (13), and reduction of the total level of cell-associated Hst 5 upon incubation in higher-ionic-strength buffers (10).

P-113 binding with the cell wall is transitory, since the peptide is detected in the cytosol within 5 min. Furthermore, the lower levels of P-113 associated with the cell wall compared with those of the translocation-incompetent peptide P-113Q2.10 indicate that the association of the peptide with the cell wall is a transitional step prior to internalization. Thus, ongoing functional import reduces the cell wall levels of the peptide as the peptide is cycled into the cell cytosol, while translocation-incompetent peptides remain bound at saturated levels with the cell wall. The mechanism by which candidal cells rapidly import Hst 5 peptides is yet unknown; however, this work has shown that import is dependent upon a specific amino acid sequence found in P-113 but not in P-113Q2.10 and that additional candidal proteins, including Ssa2p, also facilitate import. One possibility is that the Ssa2 protein serves as a chaperone that transfers cell wall-bound peptides to a membrane permease which specifically transports cationic peptides. S. cerevisiae uses at least four plasma membrane permeases, DUR3 and SAM3 (34), AGP2 (2), and GAP1 (35), that catalyze the uptake of extracellular polyamines in an energy-dependent manner. Candidal homologues of these permeases are potential candidates as intracellular transporters of Hst 5. We are investigating this possibility by evaluation of the translocation of Hst 5 in C. albicans permease null mutants. Alternatively, Hst 5 could be taken up by Ssa2-mediated endocytosis and then released into the cytosol by retrograde transport, as has been described for yeast killer toxins in S. cerevisiae (5, 14). Evidence for the involvement of endocytotic processes has also been reported for branched Hst 5 analogues (40). However, the time required for endocytotic trafficking does not seem to coincide with the rapid (<5 min) appearance of Hst 5 in the cytosol, nor does Hst 5 possess a classical HDEL endoplasmic retention signal essential for retrograde transport in S. cerevisiae. However, endocytotic processes in Candida are not well understood, leaving this possibility open to further study.

This work shows that intracellular transport is the specific and rate-limiting step for the delivery of cationic peptides to the cytosol of C. albicans. Similarities in the hydrophobic molecular moments and net charges between P-113 and P-113Q2.10 (27) suggest that the transport process depends on the recognition of specific amino acid sequences or motifs of the transported substrate rather than helical structural features. It will be important to identify the substrate requirements for intracellular transport as well as the translocation mechanism in order to best design peptides for therapeutic use for candidiasis.

 
This work was supported by U.S. PHS R01 grant DE010641 from the National Institute of Dental and Craniofacial Research (to M.E.) and a Korea Research Foundation grant funded by the Korean Government (MOEHRD) (KRF-2006-352-C00061) (to W.S.J.).

 
* Corresponding author. Mailing address: Department of Oral Microbiology, 310 Foster Hall, SUNY at Buffalo Main Street Campus, 3435 Main Street, Buffalo, NY 14214. Phone: (716) 829-3067. Fax: (716) 829-3942. E-mail: edgerto{at}buffalo.edu

Published ahead of print on 12 November 2007.

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Antimicrobial Agents and Chemotherapy, February 2008, p. 497-504, Vol. 52, No. 2
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