is the most common human fungal pathogen that can be isolated from the oral and vaginal mucosa and the gastrointestinal tract [1
]. In healthy individuals, the fungus is commensal and does not cause significant disease. However, in the absence of proper immunity, such as in patients with immunodeficiency, the fungus can cause mucosal and even life-threatening systemic infections [3
]. With the significant growth in the population exhibiting oral and systemic candidiasis, there is a great need for the development of novel antifungal agents.
P-113 (AKRHHGYKRKFH), a 12-amino-acid peptide derived from histatin 5, retains antifungal activity comparable to that of the parent molecule [4
]. It is active against clinically important microorganisms such as Pseudomonas
spp., and C. albicans
]. Recently, a clinical study on human immunodeficiency virus (HIV) patients showed that P-113 has a positive result for oral candidiasis therapy [6
]. Another study on the application of P-113 to gingivitis showed its safety and efficacy in a clinical study [7
]. The proposed mechanism of the candidacidal activity of P-113 is similar to that of histatin 5. Initially, the positively charged residues of P-113 bind to the negatively charged C. albicans
surface through electrostatic interactions, followed by binding to the cell-wall protein Ssa2 and translocation to the cytoplasm [8
]. Ssa proteins belong to the heat-shock protein 70 (HSP70) family with roles in heat shock protection, protein folding assistance, and translocation across membranes [9
]. In addition, Ssa1p and Ssa2p play important roles in cell-mediated immune responses in mice and humans infected by C. albicans
]. The two cationic amino acids Lys2 and Lys10 of P-113 play important roles in transport into the cytosol [8
]. The efficacy of P-113 is greatly reduced at high salt concentrations [11
Despite the promising results of P-113 as antifungal, Candida
can become resistant to antimicrobial peptides by producing antimicrobial peptide (AMP)-degrading proteases. Specifically, Candida
produces secreted aspartic proteinases (Saps), which are also suggested to function as virulence factors [12
]. There are 10 Sap proteinases, encoded by a family of 10 SAP
genes, which account for all of the extracellular proteolytic proteins produced by C. albicans
. The size of the Sap1–10 proteins is between 35 and 50 kDa, and they contain Lys/Arg (KR) or Lys/Lys (KK) processing sites along with four conserved cysteine residues [13
]. Sap1 to Sap8 are secreted to the extracellular environment, while Sap9 and Sap10, which play roles in cell surface integrity and cell separation, are located in the cell membrane/wall via a glycosylphosphatidylinositol (GPI) anchor [14
]. It was reported that Saps are able to inactivate a vast number of host defense proteins such as salivary lactoferrin, immunoglobulins [15
], LL-37 cathelicidin [16
], and kininogen-derived NAT26 and HKH20 [17
]. Recently, degradation of histatin 5 with Saps derived from C. albicans
was shown. Sap9 is mainly responsible for the degradation of histatin 5 at physiological pH [18
]. In addition, at optimal pH conditions, histatin 5 can be cleaved by other Saps [19
]. The C-terminal end of dibasic (KR, KK) or monobasic (K, R) residues of histatin 5 seemed to be the preferred cleavage sites of Sap9 and Sap10 [13
]. Despite the extensive information on the interactions between Saps and histatin 5 in vitro, the in vivo interaction between C. albicans
and AMPs, such as P-113 with potent antifungal activity, is not fully understood.
To improve the resistance of antimicrobial peptides to hydrolysis, several studies developed antimicrobial peptides with modifications that can reduce their sensitivity to proteases; these include adding N-terminal acetylation and C-terminal amidation, replacing d
-amino acids at specific positions, and introducing peptidomimetics to increase half-lives [4
]. Furthermore, increasing the hydrophobicity of peptides by conjugating with an acyl chain at their termini and aromatic amino acid end-tags were effective in conferring them stability against proteolytic degradation. Recently, we found that histidine residues in P-113 substituted with bulky unnatural amino acids, such as Nal (β-naphthylalanine), β-diphenylalanines (Dip), and β-(4,4′-biphenyl)alanines (Bip), boost their salt resistance and serum proteolytic stability [11
Here, we used solution nuclear magnetic resonance (NMR) methods to elucidate the molecular mechanism of interactions between P-113 and living C. albicans cells. We also characterized the functional roles of the amino-acid residues of P-113 in this interaction. Furthermore, we investigated the anti-Candida activity and mechanism of these bulky amino acids replaced peptides to identify whether they could be translocated to cytosol or localized into membranes.
long adapted to human hosts and commonly colonizes the human mucosal surfaces. However, under conditions of immune dysfunction such as those found in HIV+ individuals, C. albicans
can frequently cause superficial mucosal infections and life-threatening disseminated infections [23
]. The virulence of C. albicans
pathogens appears to correlate with the level of Saps activity that not only facilitates the availability of nutrients for fungal growth, but can also inactivate complement components [24
]. In addition, Saps are reported to neutralize and cleave some human antimicrobial peptides (AMPs) such as LL-37 and histatin 5 [16
]. In-depth investigations of the degradation of P-113, a histidine-rich cationic salivary peptide with strong anticandidal activity, by C. albicans
are reported in our present study. Here, we demonstrated for the first time the interactions between P-113 and living C. albicans
cells over time using NMR. In the 15
H HSQC spectra, the chemical shifts of the P-113 peptide dramatically moved after cell titration (Figure 1
b). Initially, we expected the chemical shifts to be due to the peptides targeting cell membranes and being imported into cells. Unfortunately, due to low sensitivity and the complexity of living matter, we were not able to demonstrate that the chemical shift perturbations were due to intracellular translocation. Instead, chemical shift movements were caused by hydrolytic cleavage by C. albicans
proteases (Figure 1
). Comparing 15
C CON experiments and backbone assignment of P-113 after degradation, we found that the cleavage sites were located at Tyr7-Lys8, Lys8-Arg9, and Lys10-Phe11, thus producing four fragments (Figure 3
Intracellular translocation and accumulation to a threshold concentration in cells are necessary for P-113 to exert candidacidal activity [8
]. In a previous study, C. albicans
cell concentrations of 106
cells/mL and below were shown to be unable to degrade histatin 5 at concentrations of 50–200 μg/mL [18
]. However, the hydrolytic level of histatin 5 was proportional to the cell density (>107
cells/mL) and time of exposure. This phenomenon was also observed with P-113 by NMR spectroscopy in the present study. The degradation of P-113 by C. albicans
resulted in chemical shift perturbations that were recorded in 1
N HSQC spectra. We found that a cell density of 107
cells/mL could degrade 0.25 mM P-113 in five hours. However, chemical shifts did not show any noticeable changes at cell densities below 107
cells/mL (data not shown), indicating lack of insufficient C. albicans
enzyme levels to fully cleave P-113. That is, complete hydrolysis occurs at the cell-to-peptide ratio of 1:1.5 × 1013
(one mole of peptide contains 6.02 × 1023
number of peptide molecules). Candida
concentrations of 600 colony-forming units (CFU)/mL were reported in concentrated rinse samples for healthy commensal carriage [27
], with higher levels (above 2–3 × 103
CFU/mL) in individuals predisposed to oral candidiasis [28
]. These results suggested that maintaining a concentration of P-113 higher than 0.075 μM (3 × 103
× 1.5 × 1013
/6 × 1023
) in the oral cavity is important for controlling the proliferation of commensal C. albicans
strains. Our findings also explain the clinical trial report that 0.01% P-113 (0.07 μM) in a mouthrinse treatment reduced the development of gingivitis and plaque [29
The interactions between P-113 and the Ssa proteins were studied by Jang et al. [8
]. Lysine residues of P-113 were shown to have specific role in antifungal activity. The substitution of lysine residues at positions 2 and 10 by glutamine results in loss of activity against C. albicans
, and lack of transport to the cytosol, although it could efficiently bind to the cell wall [8
]. Furthermore, the three amino acids Lys-Phe-His of P-113 at the C-terminus of P-113 were suggested to contribute to peptide translocation [30
]. However, lysine residues in the histatin 5 sequence are important for its recognition and are prone to be cleaved by the Saps secreted from C. albicans
cells. In our study, we found that the peptide bond between Lys10 and Phe11 was cleaved by proteases. In contrast, Lys2, at the N-terminus, was able to resist hydrolysis. Thus, we designed N-terminal Ala-Lys-truncated P-113 (RHHGYKRKFH) and C-terminal Phe-His-truncated P-113 (AKRHHGYKRK) peptides to identify the functional role of the N-terminus and the C-terminus of P-113. Although both truncated peptides lost their anti-Candida
activity, the lower fluorescent intensity level of C-terminal truncated P-113 in the cells suggests that the C-terminal Phe-His residues are an important sequence for active P-113 translocation (Figure 9
). This may be the reason why the enzymes from C. albicans
specifically cut off the C-terminal Phe-His residues of P-113 to prevent peptide entry into the cells via Ssa proteins. This observation can be used to design better antimicrobial peptides to fight against C. albicans
In our previous studies, we found that replacing histidine residues with non-natural amino acids, such as β-naphthylalanine (Nal), β-diphenylalanine (Dip), and β-(4,4′-biphenyl)alanine (Bip), protects Nal-P-113, Dip-P-113, and Bip-P-113 from degradation in serum [31
]. To investigate whether Dip-P-113 and Bip-P-113 peptides target to the cell surface instead of translocating into cells, we performed fluorescence microscopic studies to monitor FITC-labeled Dip-P-113 and Bip-P-113 interacting with living C. albicans
cells (Figure 10
a). Our results showed that both Dip-P-113 and Bip-P-113 FITC-labeled peptides localized to the cell surface in 5 min, indicating that Dip-P-113 and Bip-P-113 may possess membrane lytic action. These results are in accordance with the effects observed by SEM, which showed that cells treated with Dip-P-113 or Bip-P-113 had wrinkly surfaces, indicating the loss of membrane integrity leading to pore formation and cell death (Figure 10
b). Thus, it seems that introduction of bulky hydrophobic side chains enables the peptide to attack cell membranes. The localization and changes in cell morphology observed with Dip-P-113 or Bip-P-113 suggest that they employ different mechanisms for killing C. albicans
compared with P-113 (Figure 11
). The rapid antimicrobial activity resulting from increased hydrophobicity is suggested to prevent the peptide from being hydrolyzed. Interestingly, Dip-P-113 and Bip-P-113, with the same net charge and hydrophobicity showed differences in killing kinetics (Figure 7
b). This could be due to the longer hydrophobic side chain of Bip-P-113 resulting in more efficient insertion and disruption of C. albicans
membranes compared to Dip-P-113.
In conclusion, we observed the interactions between the salivary anticandidal peptide P-113 and living C. albicans cells by NMR spectroscopy. We found that Phe-His residues at the C-terminus of P-113 play a pivotal role in peptide translocation, while the N-terminal Ala-Lys residues are key for anticandidal activity. The resistance strategy of C. albicans relies on the proteolytic defensive enzymes that specifically cleaved Phe–His amino-acid sequences, preventing peptides from importing into the cytosol. These findings provide further in-depth information for the design of effective anti-Candida agents. Furthermore, elongating the length of the hydrophobic alkyl tails at the position of histidine alters the C. albicans killing mechanism of P-113. With its high anti-Candida activity, Bip-P-113 deserves further attention in the development of antifungal therapeutics in the future.
4. Materials and Methods
15N-labeled ammonium chloride and 13C-labeled glucose were purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). Thermanox substrate-coated cover slips were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Sabouraud dextrose (SD) broth and SD agar were purchased from Becton, Dickinson, and Company (Franklin Lakes, NJ, USA). All other chemicals used in this study were obtained from Sigma-Aldrich (St. Louis, MO, USA).
P-113, FITC-conjugated P-113, and P-113 derivatives (>95% purity) were purchased from Kelowna International Scientific Inc. (Taipei, Taiwan).
4.3. Yeast Strain
C. albicans (ATCC 10231) from the American Type Culture Collection (Manassas, VA, USA) was cultured at 28 °C on SD agar plates.
4.4. Nuclear Magnetic Resonance Spectroscopy
To obtain 15
C-labeled peptide for three-dimensional NMR analysis, P-113 was expressed in Escherichia coli
BL21 (DE3) cells grown in M9 minimal media containing 15
N-labeled ammonium chloride (1 g/L) and 13
C-labeled glucose (2 g/L). Peptides were purified according to our previous study [32
]. All NMR samples were prepared in SD broth containing 10% D2
O. Sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) was used as an internal chemical shift standard.
For living cell NMR experiments, the interaction between 15N-labeled P-113 peptides and C. albicans was monitored by 2D heteronuclear single quantum coherence (HSQC) spectroscopy. The peptides were dissolved in SD broth for a total volume of 500 μL at a 0.275 mM concentration. After centrifugation, 107 CFU/mL of cells were resuspended in 50 μL of SD broth and then titrated into the peptide solution. The 15N-1H HSQC spectra were recorded on a Bruker Avance 600-MHz NMR spectrometer in the States-TPPI mode for quadrature detection at 301 K. The 1H and 15N carriers were placed at 4.699 and 120 ppm, respectively. After titration, 2D HSQC spectra were collected every hour during 24 h of incubation at 301 K. All spectra were processed with NMRPipe and analyzed using the Sparky program (T.D. Goddard and D.G. Kneller, SPARKY3, University of California, San Francisco, CA, USA).
To obtain sequence assignment information, 3D spectra, including those from HNCA, HN(CO)CA, HNCACB, HN(CO)CACB, HN(CA)CO, and HNCO experiments, were recorded on a Bruker Avance 600-MHz NMR spectrometer at 301 K. The 13C carriers were placed at 48 ppm for Cα, at 38 ppm for 13Cα/β and at 174 ppm for C′. All spectra were processed with NMRPipe and analyzed using the Sparky program.
To observe the correlation of amide nitrogen (15Ni) to the carbon (13C′) spin in the proceeding residue (13C′i−1), 2D CON experiments were performed on Bruker Avance 850-MHz NMR spectrometer equipped with a triple-resonance cryogenic probe. The spectra comparing 15N-, 13C-labeled P-113 in SD broth and in a C. albicans titration were recorded with the center frequencies at 120 ppm (15N) and 174 ppm (13C), respectively. All spectra were processed with NMRPipe and analyzed using the Sparky program.
4.5. Inhibition of Degradation by Aspartyl Protease Inhibitors
The inhibition of P-113 degradation by C. albicans was determined using living cell NMR experiments as described above in the presence of 0.5 mM of the aspartic protease inhibitor pepstatin A.
4.6. Expression and Purification of Amino-Acid-Selective 15N-Unlabeled P-113
To conveniently observe the change in the chemical shift of each amino acid during the time course experiments, we expressed the selectively unlabeled hG31P-P-113 protein in E. coli. The procedures for expressing selectively 15N-unlabeled protein were the same as those for the uniformly 15N-labeled protein expression. The only difference is the composition of M9 minimal medium, which contains the desired amino acid to be selectively unlabeled at a concentration of 1.0 g/L and together with 15NH4Cl (0.5 g/L). In the present study, we chose three amino acids for selective unlabeling: Lys (K), Arg (R), and His (H).
4.7. Scanning Electron Microscopy Analysis
Time-course morphological studies of C. albicans were performed by scanning electron microscopy (SEM). Overnight cultures of C. albicans were sub-cultured and grown in SD broth. Then, 107 CFU/mL of C. albicans was treated with 0.25 mM P-113 in SD broth at 28 °C. Samples were collected following 1, 2, 3, 4, 5, and 6 h of incubation. Another experiment was performed to observe the morphological changes caused over in a short time caused by Bip-P-113 and Dip-P-113. Briefly, 107 CFU/mL of C. albicans in SD broth was treated for 5 min at 28 °C with 50 μM P-113, Bip-P-113, and Dip-P-113. After harvest, cells were resuspended in fixation solution (2.5% glutaraldehyde in 20 mM phosphate buffer pH 7.4) and then placed on Thermanox substrate-coated cover slips at 4 °C for 12 h. The cover slips were carefully washed three times with 20 mM phosphate buffer (pH 7.4), followed by incubation in post-fixation solution (1% OsO4 in 20 mM phosphate buffer pH 7.4) at room temperature for an hour. After being washed three times with 20 mM phosphate buffer (pH 7.4), the cover slips were air-dried for an hour by dehydration with a graded series of ethanol. Samples were gold-coated and observed under an S-4700 field-emission (FE)-SEM (Hitachi, Ltd., Tokyo, Japan) at an acceleration voltage of 5 kV.
4.8. Confocal Laser Microscopy
Firstly, 107 CFU/mL of C. albicans was incubated with 0.25 mM FITC P-113 and 0.25 mM FITC alone (as a negative control) in SD broth at 28 °C for 1, 2, 3, 4, 5, and 6 h. Another experiment was performed to observe the short-term localization of Bip-P-113 and Dip-P-113 peptides. Briefly, log-phase C. albicans cultures were resuspended to a cell density of 107 CFU/mL in SD broth and treated for 5 min at 28 °C with 50 μM FITC-conjugated P-113, Bip-P-113, and Dip-P-113. After centrifugation, the cells were washed three times with 20 mM phosphate buffer (pH 7.4) to remove nonbinding peptides and fixed with 4% (w/v) paraformaldehyde in 20 mM phosphate buffer (pH 7.4) at room temperature for 20 min. Following fixation, the cells were centrifuged at 3500× g for 5 min, resuspended in phosphate buffer, and loaded onto a glass slide with a 1.4% solid agarose bed. The images were taken with a confocal laser scanning microscope (LSM 510 META, Carl Zeiss, Jena, Germany) equipped with a 40× C-Apochromat water-immersion objective lens (Carl Zeiss, Jena, Germany).
4.9. Anticandidal Activity Assay
To determine the minimum inhibitory concentration (MIC) values of the FITC conjugated-P-113 peptides and their derivatives, a broth microdilution assay was used. Overnight-grown cultures of C. albicans in SD broth were sub-cultured for several hours and subsequently diluted to a final concentration of 5 × 104 CFU/mL in Mueller Hinton (MH) broth and LYM broth. A total of 100 μL of diluted microbe was transferred into each well of a 96-well plate, into which a 1 μL dilution series of peptides in sterile water (ranging from 50 to 0.78 μg/mL) was previously added. After incubation for 24 h at 28 ℃, the MIC value of P-113 peptides was determined as the lowest concentration at which no change in optical density was observed. The experiment was conducted three times, and average values were reported.
4.10. Time Killing Assay
C. albicans were cultivated onto Sabouraud dextrose agar plates overnight and sub-cultured twice before time killing assays. A total of 5 × 104 CFU/mL of C. albicans cells were treated with 1× MIC of FITC-conjugated P-113 peptides and their derivatives in SD broth for 10, 20, 30, 60, and 120 min at 28 °C. After that time, serial dilutions (1:10) of the incubation mixtures were added onto SD agar. The numbers of CFU were counted after 24 h of incubation at 28 °C. The percentage of killed cells was calculated by comparison with the number of cells in a control sample incubated without P-113 peptide. The assays were performed in triplicate.
4.11. Fluorescence Microscopy
Firstly, 107 CFU/mL of C. albicans cells were incubated with 50 µM FITC N-terminal truncated P-113, FITC C-terminal truncated P-113 peptides, and FITC alone for 30 min at 28 °C in SD broth. After incubation, the cells were centrifuged at 3500× g for 5 min and then washed three times with 20 mM phosphate buffer (pH 7.4) to remove unbound peptides. The resulting pellets were resuspended and immobilized on the glass slips with a 1.4% solid agarose bed. Fluorescence images were captured using an inverted fluorescence microscope (ECLIPSE E400, Nikon, Tokyo, Japan) with a camera (AxioCam ICc5, ZEISS, Jena, Germany).
4.12. Flow Cytometry
Firstly, 5 × 106 CFU/mL of C. albicans cells were incubated with 50 µM FITC P-113, FITC N-terminal truncated P-113, and FITC C-terminal truncated P-113 peptides for 30 min at 28 °C in SD broth. The resuspended cell pellets described above were also used for flow cytometry experiments. Flow cytometric cell sorting was performed (10,000 events/sample) by using flow cytometry (BD Accuri™ C6 Plus Flow Cytomete, BD Biosciences, NJ, USA). The quantitative data were analyzed by Prism software.
4.13. Statistical Analysis
All statistical results are expressed as the means ± standard deviation and were analyzed using one-way ANOVA. Statistical analysis was performed using GraphPad Prism version 5.0, where p < 0.05 was considered to indicate a statistically significant difference.