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• W2023647231 abstract "Histatin 5 is a 24-residue peptide from human saliva with antifungal properties. We recently demonstrated that histatin 5 translocates across the yeast membrane and targets to the mitochondria, suggesting an unusual antifungal mechanism (Helmerhorst, E. J., Breeuwer, P., van't Hof, W., Walgreen-Weterings, E., Oomen, L. C. J. M., Veerman, E. C. I., Nieuw Amerongen, A. V., and Abee, T. (1999) J. Biol. Chem. 274, 7286–7291). The present study used specifically designed synthetic analogs of histatin 5 to elucidate the role of peptide amphipathicity, hydrophobicity, and the propensity to adopt α-helical structures in relation to membrane permeabilization and fungicidal activity. Studies included circular dichroism measurements, evaluation of the effects on the cytoplasmic transmembrane potential and on the respiration of isolated mitochondria, and analysis of the peptide hydrophobicity/amphipathicity relationship (Eisenberg, D. (1984) Annu. Rev. Biochem. 53, 595–623). The 14-residue synthetic peptides used were dh-5, comprising the functional domain of histatin 5, and dhvar1 and dhvar4, both designed to maximize amphipathic characteristics. The results obtained show that the amphipathic analogs exhibited a high fungicidal activity, a high propensity to form an α-helix, dissipated the cytoplasmic transmembrane potential, and uncoupled the respiration of isolated mitochondria, similar to the pore-forming peptide PGLa (Peptide with N-terminal Glycine and C-terminal Leucine-amide). In contrast, histatin 5 and dh-5 showed fewer or none of these features. The difference in these functional characteristics between histatin 5 and dh-5 on the one hand and dhvar1, dhvar4, and PGLa on the other hand correlated well with their predicted affinity for membranes based on hydrophobicity/amphipathicity analysis. These data indicate that the salivary protein histatin 5 exerts its antifungal function through a mechanism other than pore formation. Histatin 5 is a 24-residue peptide from human saliva with antifungal properties. We recently demonstrated that histatin 5 translocates across the yeast membrane and targets to the mitochondria, suggesting an unusual antifungal mechanism (Helmerhorst, E. J., Breeuwer, P., van't Hof, W., Walgreen-Weterings, E., Oomen, L. C. J. M., Veerman, E. C. I., Nieuw Amerongen, A. V., and Abee, T. (1999) J. Biol. Chem. 274, 7286–7291). The present study used specifically designed synthetic analogs of histatin 5 to elucidate the role of peptide amphipathicity, hydrophobicity, and the propensity to adopt α-helical structures in relation to membrane permeabilization and fungicidal activity. Studies included circular dichroism measurements, evaluation of the effects on the cytoplasmic transmembrane potential and on the respiration of isolated mitochondria, and analysis of the peptide hydrophobicity/amphipathicity relationship (Eisenberg, D. (1984) Annu. Rev. Biochem. 53, 595–623). The 14-residue synthetic peptides used were dh-5, comprising the functional domain of histatin 5, and dhvar1 and dhvar4, both designed to maximize amphipathic characteristics. The results obtained show that the amphipathic analogs exhibited a high fungicidal activity, a high propensity to form an α-helix, dissipated the cytoplasmic transmembrane potential, and uncoupled the respiration of isolated mitochondria, similar to the pore-forming peptide PGLa (Peptide with N-terminal Glycine and C-terminal Leucine-amide). In contrast, histatin 5 and dh-5 showed fewer or none of these features. The difference in these functional characteristics between histatin 5 and dh-5 on the one hand and dhvar1, dhvar4, and PGLa on the other hand correlated well with their predicted affinity for membranes based on hydrophobicity/amphipathicity analysis. These data indicate that the salivary protein histatin 5 exerts its antifungal function through a mechanism other than pore formation. Peptide with N-terminal Glycine and C-terminal Leucine-amide 3,3′-dipropyl-2,2′-thiadicarbocyanine iodide trifluoroethanol carbonyl cyanidep-chlorophenylhydrazone Throughout living nature, distinct groups of cationic peptides have been identified that display antimicrobial activity in vitro. Best characterized with respect to their structure-function relationship are magainins from the skin of the frog Xenopus laevis (1Zasloff M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5449-5453Crossref PubMed Scopus (1984) Google Scholar, 2Soravia E. Martini G. Zasloff M. FEBS Lett. 1988; 228: 337-340Crossref PubMed Scopus (176) Google Scholar), cecropins from the giant silk moth Hyalophora cecropia (3Steiner H. Hultmark D. Engstrom A. Bennich H. Boman H.G. Nature. 1981; 292: 246-248Crossref PubMed Scopus (1077) Google Scholar), and defensins (α and β) from human leukocytes and various epithelial sources, respectively (4Ganz T. Selsted M.E. Szklarek D. Harwig S.S. Daher K. Bainton D.F. Lehrer R.I. J. Clin. Invest. 1985; 76: 1427-1435Crossref PubMed Scopus (1110) Google Scholar, 5Bensch K.W. Raida M. Magert H.J. Schulz-Knappe P. Forssmann W.G. FEBS Lett. 1995; 368: 331-335Crossref PubMed Scopus (491) Google Scholar, 6Harder J. Bartels J. Christophers E. Schroder J-M. Nature. 1997; 387: 861-862Crossref PubMed Scopus (1178) Google Scholar). These peptides show considerable variation in chain length, hydrophobicity, and charge distribution; however, they share the common feature of being cationic in nature and able to adopt 70 ordered amphipathic α-helix or β-sheet conformations in structure-promoting solvents, such as trifluoroethanol, and in membrane-mimicking liposome vesicles. Their antimicrobial mode of action is believed to arise from the attraction to negatively charged surface molecules on the target cell and the subsequent formation of a membrane-spanning porelike structure, thereby altering membrane permeability leading to cell lysis. The regulated and constitutive expression of such cationic peptides provides an immediate protection to tissues that are continuously subjected to microbial challenges (7Mathews M. Jia H.P. Guthmiller J.M. Losh G. Graham S. Johnson G.K. Tack B.F. McCray Jr., P.B. Infect. Immun. 1999; 67: 2740-2745Crossref PubMed Google Scholar, 8Weinberg A. Krisanaprakornkit S. Dale B.A. Crit. Rev. Oral Biol. Med. 1998; 9: 399-414Crossref PubMed Scopus (203) Google Scholar). In the oral cavity, the hard and soft tissues are constantly exposed to a variety of microorganisms that can lead to caries and periodontal disease. This condition requires that antimicrobial components are continually secreted to provide a permanent line of defense against microbial invasion or at least to maintain the harmonious relationship between the commensal microflora and the host (9Marsh P.D. J. Dent. Res. 1988; 68: 1567-1575Google Scholar). Examples of such salivary antimicrobial systems are histatins. Histatins are a group of histidine-rich cationic peptides that are secreted by the parotid and the submandibular/sublingual human salivary glands (10Oppenheim F.G. Yang Y. Diamond R.D. Hyslop D. Offner G.D. Troxler R.F. J. Biol. Chem. 1986; 261: 1177-1182Abstract Full Text PDF PubMed Google Scholar, 11Oppenheim F.G. Xu T. McMillian F.M. Levitz S.M. Diamond R.D. Offner G.D. Troxler R.F. J. Biol. Chem. 1988; 263: 7472-7477Abstract Full Text PDF PubMed Google Scholar). At least 12 fragments have been identified (12Troxler R.F. Offner G.D. Xu T. Vanderspek J.C. Oppenheim F.G. J. Dent. Res. 1990; 69: 2-6Crossref PubMed Scopus (139) Google Scholar) that derive from the proteolytic degradation of two gene products, histatin 1 and histatin 3 (13Sabatini L.M. Azen E.A. Biochem. Biophys. Res. Commun. 1989; 160: 495-502Crossref PubMed Scopus (69) Google Scholar). Histatin 5 has been shown to be the most potent member with respect to its fungicidal activity against the oral opportunistic pathogenCandida albicans, and the domain responsible for this activity has been located in the C-terminal 14 residues (14Xu T. Telser E. Troxler R.F. Oppenheim F.G. J. Dent. Res. 1990; 69: 1717-1723Crossref PubMed Scopus (31) Google Scholar,15Raj P.A. Edgerton M. Levine M.J. J. Biol. Chem. 1990; 265: 3898-3905Abstract Full Text PDF PubMed Google Scholar). Despite a great interest in understanding the mechanism of histatin function, the molecular events leading to cell death have so far been largely elusive (16Helmerhorst E.J. van't Hof W. Veerman E.C.I. Somoons-Smit I. Nieuw Amerongen A.V. Biochem. J. 1997; 326: 39-45Crossref PubMed Scopus (142) Google Scholar, 17Edgerton M Koshlukova S.E. Lo T.E. Chrzan B.G. Straubinger R.M. Raj P.A. J. Biol. Chem. 1998; 273: 20438-20447Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 18Helmerhorst E.J. Breeuwer P. van't Hof W. Walgreen-Weterings E. Oomen L.C.J.M. Veerman E.C.I. Nieuw Amerongen A.V. Abee T. J. Biol. Chem. 1999; 274: 7286-7291Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 19Koshlukova S.E. Lloyd T.L. Araujo M.W.B. Edgerton M. J. Biol. Chem. 1999; 274: 18872-18879Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 20Gyurko C. Lendenmann U. Troxler R.F. Oppenheim F.G. Antimicrob. Agents Chemother. 2000; 44: 348-354Crossref PubMed Scopus (77) Google Scholar). Salivary histatins differ from other natural antimicrobial peptides in a number of respects. First, histatins are enriched in the amino acid histidine. Histidine has an isoelectric point of 6.5 and, therefore, modulates the cationicity of the peptide considerably at lower pH values. In addition to this, histidine side chains are known participants in metal chelation. Indeed, histatin 5 is able to form complexes with various metal ions (21Gusman H. Lendenmann U. Troxler R.F. Oppenheim F.G. J. Dent. Res. 1999; 78: 179Google Scholar), and this adds potential biological properties to this peptide compared with other basic peptides. Secondly, although histatins like other antimicrobial peptides adopt helical conformations in hydrophobic environments (15Raj P.A. Edgerton M. Levine M.J. J. Biol. Chem. 1990; 265: 3898-3905Abstract Full Text PDF PubMed Google Scholar,22Raj P.A. Soni S. Levine M.J. J. Biol. Chem. 1994; 269: 9610-9619Abstract Full Text PDF PubMed Google Scholar), the amphipathicity of this helix quantified by the hydrophobic moment is rather low compared with other antimicrobial peptides, such as the magainins (16Helmerhorst E.J. van't Hof W. Veerman E.C.I. Somoons-Smit I. Nieuw Amerongen A.V. Biochem. J. 1997; 326: 39-45Crossref PubMed Scopus (142) Google Scholar). Because peptide amphipathicity is believed to be a key factor in governing the cytolytic activity of pore-forming basic antimicrobial peptides (23Lee Maloy W. Prasad Kari U. Biopolymers. 1995; 37: 105-122Crossref PubMed Scopus (455) Google Scholar), it is questionable whether histatin 5 operates by the same mode of action. We recently demonstrated that histatin 5 translocates across the yeast membrane and shows intracellular targeting to the mitochondria, suggesting an unusual antifungal mechanism (18Helmerhorst E.J. Breeuwer P. van't Hof W. Walgreen-Weterings E. Oomen L.C.J.M. Veerman E.C.I. Nieuw Amerongen A.V. Abee T. J. Biol. Chem. 1999; 274: 7286-7291Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). The present investigation focuses on physical peptide parameters, such as amphipathicity, hydrophobicity, and the propensity to adopt helices in relation to these unique functional characteristics of histatin 5. The biophysical and biological results obtained show that histatin 5, in contrast to more amphipathic peptides employed for comparison, is not a classic pore former, and these aspects are discussed with respect to its capacity to translocate across the yeast membrane. Histatin 5, the C-terminal domain dh-5, and peptides derived from this domain, dhvar1 and dhvar4, were chemically synthesized as previously described (16Helmerhorst E.J. van't Hof W. Veerman E.C.I. Somoons-Smit I. Nieuw Amerongen A.V. Biochem. J. 1997; 326: 39-45Crossref PubMed Scopus (142) Google Scholar, 24van't Hof W. Driedijk P.C. Van den Berg M. Beck-Sickinger A.G. Jung G. Aalberse R.C. Mol. Immunol. 1991; 28: 1225-1232Crossref PubMed Scopus (99) Google Scholar). Synthetic PGLa1(Peptide with N-terminal Glycine and C-terminal Leucine-amide) was a kind gift from H. V. Westerhoff (Dept. of Molecular Cell Physiology, Vrije Universiteit, Amsterdam). CD spectra were recorded under computer control on an AVIV 62DS CD spectropolarimeter (AVIV Associates, Inc., Lakewood, NJ) from 250 to 183 nm at 25 ± 0.5 °C using a 0.1-cm quartz cuvette. Peptides were dissolved in pure water, in pure trifluoroethanol (TFE), or in a mixture of both solvents to a final concentration of 0.04–0.14 mg/ml. Spectra (1-nm intervals, 1 s/interval with 5–15 repeats) were averaged and corrected for base-line contribution. Molar ellipticity values [θ] were calculated according to Equation 1, [θ](deg·cm2·decimol−1)=(θ×MRW)/(10×l×c)Equation 1 where θ is the displacement from the base-line value for the full range in degrees, MRW is the mean residue weight of the amino acids in the protein structure, l is the path length of the cuvette in cm, and c is the concentration of the protein in g/ml. C. albicans 315 (ATCC 10231) was cultured from a glycerol stock on Sabouraud dextrose agar (Difco, Detroit, MI) and maintained at most for 1 week. Several yeast colonies were picked from the plate and suspended in 1, 10, 20, or 50 mm potassium phosphate buffer, pH 7.0, to yield a suspension of 5.2 × 106 cells/ml. From this suspension, 250 μl was added to 250 μl of a dilution of histatin 5, dhvar1, or dhvar4 in the same buffer. After 0, 6, 12, 18, 24, 30, 40, 60, and 90 min of incubation in a 37 °C water bath, 50-μl samples were diluted in 9 ml of phosphate-buffered saline and mixed. From this suspension, 25 μl was plated on Sabouraud dextrose agar and incubated for 48 h at 30 °C to determine the percentage reduction in viable counts. Log count reductions were determined by plating 25 μl from a 10-fold serial dilution series of the samples in phosphate-buffered saline. Relative changes in the cytoplasmic transmembrane potential of C. albicans were measured using the fluorescent probe 3,3′-dipropyl-2,2′-thiadicarbocyanine iodide (diS-C3-(5)) (Molecular Probes, Inc., Eugene, OR) (25Eddy A.A. Hopkins P.G. Biochem. J. 1985; 231: 291-297Crossref PubMed Scopus (26) Google Scholar, 26Shapiro H.M. Darzynkiewicz Z. Crissman H.A. Methods in Cellular Biology. 33. Academic Press Inc. London, United Kingdom1990: 25-35Google Scholar). The measurement was carried out in a 3-ml fluorescence cuvette connected to a 30 °C thermostated water bath. Cells were picked from a Sabouraud dextrose agar plate, suspended in 1 mm potassium phosphate buffer, pH 7.0, and added to the thermostated cuvette containing 1 mm potassium phosphate buffer to a final cell density of 2.6 × 106 cells/ml. Subsequently, diS-C3-(5) was added from a stock solution of 0.5 mm in ethanol to a final concentration of 3.3 μm. Changes in the fluorescence intensity were measured at λex = 643 ± 10 nm and λem = 666 ± 10 nm with a PerkinElmer LS 50 B spectrofluorimeter supplied with a pulsed xenon light source. Mitochondria were isolated from C. albicans spheroplasts essentially as described previously (27Jault J-M. Comte J. Gautheron D.C. Di Pietro A. J. Bioenerg. Biomembr. 1994; 26: 447-456Crossref PubMed Scopus (10) Google Scholar). C. albicans (ATCC 10231) were grown to late log phase in 1 liter of Sabouraud dextrose broth (Difco), washed once in 1.2 m sorbitol (Sigma), and suspended in 1.2m sorbitol supplemented with 50 mm potassium phosphate, pH 7.4. For every milliliter of yeast suspension with anA 620 of 10, 2 μl of β-mercaptoethanol (Sigma) and 10 units of yeast lytic enzyme (ICN Biomedicals, Costa Mesa, CA) were added, and the suspension was incubated for 1 h at 37 °C. Spheroplasts were collected by centrifugation in a Sorvall tabletop centrifuge with a H-1000B rotor at 2100 ×g at 4 °C, washed 4 times in 1.2 m sorbitol, suspended in 5–10 ml of 1.2 m sorbitol, and stored at 4 °C for up to 16 h. After centrifugation for 5 min at 2100 × g, spheroplasts were suspended in 15 ml of 0.4m sorbitol, 0.2% (w/v) bovine serum albumin (fraction V, Sigma), and 10 mm imidazole (Fisher), pH 6.4, and homogenized on ice for 10 min using a manual Potter-Elvehjem homogenizer. The homogenate was mixed 1:1 with buffer containing 1m sorbitol, 25 mmKH2PO4, 4 mm EGTA (Sigma), 0.2% (w/v) bovine serum albumin, and 10 mm imidazole, pH 6.4, and centrifuged in a Sorvall RC-5B centrifuge with a SS-34 rotor for 5 min at 1000 × g at 4 °C. The supernatant was carefully removed and centrifuged using the same rotor for 10 min at 12,000 × g at 4 °C. The reddish pellet containing the mitochondria was suspended in a small volume (typically 1 ml) of 0.6 m mannitol, 2 mm EGTA, 0.2% bovine serum albumin, and 10 mm imidazole, pH 6.4, and kept on ice. In each experiment the same volume of the mitochondrial suspension was used for polarographic measurements (final A 620of 0.15–0.25). Mitochondrial oxygen consumption was measured using a biological oxygen monitor model 5300 equipped with a 5331 standard oxygen probe (YSI, Inc., Yellow Springs, OH). Measurements were performed in air-saturated buffer containing 0.65 mmannitol, 2 mm MgCl2, 16 mmKH2PO4, 10 mm imidazole, pH 6.4. State 2 or basal rate respiration was measured after the addition of NADH (Sigma) to a final concentration of 1 mm, a substrate that can be directly oxidized by yeast mitochondria (28Averet N. Fitton V. Bunoust O. Rigoulet M. Guerin B. Mol. Cell. Biochem. 1998; 184: 67-79Crossref PubMed Google Scholar). State 3 respiration was determined after the addition of ADP (Sigma) to a final concentration of 0.33 mm. State 4 respiration was determined after all the ADP was used and the respiration had returned to the basal rate. The amino acid sequences, the isoelectric point, the mean hydrophobicity (H), and the mean hydrophobic moment (μ) of histatin 5, the fungicidal domain dh-5, the two substitution analogs dhvar1 and dhvar4, and the two magainin peptides, magainin 2 and PGLa, are summarized in TableI. The design of dhvar1 and dhvar4 was based on a helical wheel projection of dh-5 (16Helmerhorst E.J. van't Hof W. Veerman E.C.I. Somoons-Smit I. Nieuw Amerongen A.V. Biochem. J. 1997; 326: 39-45Crossref PubMed Scopus (142) Google Scholar). The peptides are all basic with isoelectric points ranging from 10 to 12. Major differences were observed in the mean hydrophobicities and mean hydrophobic moments. Histatin 5 and dh-5 have a relatively low hydrophobic moment and, therefore, are weakly amphipathic in contrast to dhvar1, dhvar4, magainin 2, and PGLa, which have a high hydrophobic moment and are highly amphipathic. These amphipathicities were calculated assuming pure helix conformation. Previous studies with histatin 5 and a number of recombinant histatin analogs have shown that these peptides all adopted helical conformations in pure TFE (29Tsai H. Raj P.A. Bobek L.A. Infect. Immun. 1996; 64: 5000-5007Crossref PubMed Google Scholar, 30Tsai H. Bobek L.A. Antimicrob. Agents Chemother. 1997; 41: 2224-2228Crossref PubMed Google Scholar). To assess the inducibility of α-helix formation and to detect potential differences between histatin 5, dh-5, dhvar1, and dhvar4, the CD spectra were recorded in aqueous solvents with a stepwise increase of the TFE content (Fig. 1). In pure water (water/TFE ratio of 100/0), the spectra of all peptides showed a maximum molar ellipticity between 215 and 220 nm and a minimum molar ellipticity below 200 nm, which is typical for random coil conformation. At increasing TFE content of the solvent, α-helical conformations were inducible in all peptides, characterized by two negative CD bands between 218–222 and 206–208 nm and a maximum molar ellipticity below 200 nm. The peptides differed in their propensity to adopt helix conformations. Dh-5 was most reluctant to adopt helices being only helical in pure TFE, whereas dhvar4 most rapidly adopted helices being only random coil in pure water. Histatin 5 and dhvar1 showed the spectra consistent with mixtures of random coil and α-helix conformations in mixtures of water and TFE. Although the tendency to adopt helices at increasing hydrophobicity was predominant, it is interesting to note that it was repeatedly found that peptide dhvar1 did not assume the highest degree of α-helix in pure TFE but did in a 50/50 water/TFE mixture.Table IPrimary structure and physical properties of peptidesPeptideSequenceCharge at pH 7.0PIaThe net peptide charge and the isoelectric point (PI) were calculated using the software program “Peptool.” In PGLa, the positive charge at the N terminus was not compensated by a negative charge at the C terminus, thereby increasing the net peptide charge by 1 unit.HbH, mean hydrophobicity of the most amphipathic 11-residue sequence calculated from the sum of the normalized consensus hydrophobicity values (36). In histatin 5, this sequence started from Ala4. In dh-5, dhvar1, and dhvar4, this sequence started from Arg2. In magainin 2, this sequence started from Lys4, and in PGLa, this sequence started from Gly11.μcμ, mean hydrophobic moment of the most amphipathic 11-residue sequence in the peptide in pure helix conformation.Histatin 5D S H A K R H H G Y K R K F H E K H H S H R G Y5+10.3−0.7040.377dh-5K R K F H E K H H S H R G Y4+10.5−0.8470.294dhvar1K R L F K E L K F S L R K Y5+10.6−0.2930.710dhvar4K R L F K K L L F S L R K Y6+11.2−0.1250.726Magainin 2G I G K F L H S A K K F G K A F V G E I M N S3+10.0−0.1800.689PGLaG M A S K A G A I A G K I A K V A L K A La5+12.00.2310.6001-a The net peptide charge and the isoelectric point (PI) were calculated using the software program “Peptool.” In PGLa, the positive charge at the N terminus was not compensated by a negative charge at the C terminus, thereby increasing the net peptide charge by 1 unit.1-b H, mean hydrophobicity of the most amphipathic 11-residue sequence calculated from the sum of the normalized consensus hydrophobicity values (36Eisenberg D. Annu. Rev. Biochem. 1984; 53: 595-623Crossref PubMed Scopus (730) Google Scholar). In histatin 5, this sequence started from Ala4. In dh-5, dhvar1, and dhvar4, this sequence started from Arg2. In magainin 2, this sequence started from Lys4, and in PGLa, this sequence started from Gly11.1-c μ, mean hydrophobic moment of the most amphipathic 11-residue sequence in the peptide in pure helix conformation. Open table in a new tab To compare the kinetics of the killing of C. albicans, cells were exposed to histatin 5, dh-5, dhvar1, and dhvar4 for different time intervals after which the viability of the cells was determined in a colony-forming assay (Fig. 2). At 8.3 μm, histatin 5, dhvar1, and dhvar4 caused a 100% reduction in the viability of the yeast inoculum, which was equivalent to a reduction by 3 log units in viable counts (data not shown). With histatin 5, almost 100% reduction in viability was observed after 20 min of incubation. Incubation for 90 min with 8.3, 3.3, 1.6, and 0.8 μm histatin 5 resulted in a 100, 90, 35, and 15% reduction in viable counts, respectively. With dh-5 comprising residues 11–24 of histatin 5, very comparable results were obtained. On the other hand, exposure of the cells to only 1.6 μm dhvar1 or only 0.8 μm dhvar4 resulted in a 100% reduction in viable counts within 6 min of incubation, demonstrating the higher activity of these analogs. It is well known that the presence of ions affects the antimicrobial activity of several antimicrobial peptides including histatins (31Xu T. Levitz S.M. Diamond R.D. Oppenheim F.G. Infect. Immun. 1991; 59: 2549-2554Crossref PubMed Google Scholar). Sensitivity studies to ions with histatin 5, dh-5, dhvar1, and dhvar4 were carried out with equally effective rather than equimolar concentrations to compensate for the higher molar potencies of dhvar1 and dhvar4 compared with histatin 5 and dh-5. Such concentrations were derived from experiments as shown in Fig. 2. Under these conditions, histatin 5, dhvar1, and dhvar4 showed comparable dependence on the ionic strength (Table II). Because the peptides are well soluble in all buffers used, these data suggest that ions interfere with the peptide-cell interaction, either by preventing electrostatic interactions or by modifying groups on the yeast cell envelope.Table IIPercent viability of C. albicans cells after 1.5-h exposure to equally effective concentrations of histatin 5, dh-5, dhvar1, and dhvar4BufferPeptideHistatin 5dh-5dhvar1dhvar41 mm PPB0.5 ± 0.52.9 ± 0.30.5 ± 0.50.5 ± 0.510 mmPPB29.5 ± 0.540.4 ± 2.732.0 ± 3.030.5 ± 3.520 mm PPB57.5 ± 0.573.6 ± 4.758.0 ± 1.066.5 ± 7.550 mmPPB93.4 ± 0.193.6 ± 6.491.1 ± 1.899.7 ± 0.3The concentrations that were equally effective in killing C. albicans were 8.3 μm histatin 5, 16.6 μm dh-5, 1.6 μm dhvar1, and 1.6 μm dhvar4.PPB, potassium phosphate buffer. Open table in a new tab The concentrations that were equally effective in killing C. albicans were 8.3 μm histatin 5, 16.6 μm dh-5, 1.6 μm dhvar1, and 1.6 μm dhvar4. PPB, potassium phosphate buffer. Although the initial interaction for histatin 5, dh-5, dhvar1, and dhvar4 is presumably electrostatic in nature, the ability of these peptides to interact with lipophilic portions of the bilayer and to insert into the microbial membrane might be significantly different. Such interactions, which may lead to the disruption of membrane integrity, have often been studied using liposome vesicles as a model system for microbiological membranes (32Matsuzaki K. Murase O. Fujii N. Miyajima K. Biochemistry. 1995; 34: 6521-6526Crossref PubMed Scopus (307) Google Scholar, 33Dathe M. Schümann M. Wieprecht T. Winkler A. Beyermann M. Krause E. Matsuzaki K. Murase O. Bienert M. Biochemistry. 1996; 35: 12612-12622Crossref PubMed Scopus (342) Google Scholar). In the present study, the effect of peptide on aspects of the integrity of wholeC. albicans cells was studied by the measurement of changes in the cytoplasmic transmembrane potential. For this purpose, diS-C3-(5), a fluorescent dye with membrane potential-dependent distributional properties, was used. This probe is fluorescent in solution but autoquenches when it accumulates intracellularly (26Shapiro H.M. Darzynkiewicz Z. Crissman H.A. Methods in Cellular Biology. 33. Academic Press Inc. London, United Kingdom1990: 25-35Google Scholar). It should be pointed out that the accumulation of the dye is driven by the potential across the cytoplasmic membrane rather than the potential across the mitochondrial membrane because sodium azide, which dissipates the mitochondrial transmembrane potential (18Helmerhorst E.J. Breeuwer P. van't Hof W. Walgreen-Weterings E. Oomen L.C.J.M. Veerman E.C.I. Nieuw Amerongen A.V. Abee T. J. Biol. Chem. 1999; 274: 7286-7291Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar), does not prevent the intracellular accumulation of diS-C3-(5) (data not shown). For this experiment, relatively high doses of peptide were chosen so that the dissipating effect could be determined immediately after the addition of the peptide. Fig. 3 shows that the exposure of C. albicans cells, loaded with diS-C3-(5), to 18 μm dhvar4 results in an increase in the fluorescence signal to the level just after adding the probe to the cells, indicating the complete release of the dye into the extracellular environment due to cell membrane permeation. At this concentration, histatin 5 and dh-5 had no effect, and testing at a 6-fold higher concentration to compensate for their lower molar potency only caused a small increase in fluorescence intensity. Mitochondria were isolated from C. albicans for two purposes. The first purpose was to investigate the effect of histatin 5 on the respiratory activity of isolated yeast mitochondria. Interest in this stems from our previous observation that fluorescein isothiocyanate-labeled histatin 5 associates with C. albicans mitochondria in situ (18Helmerhorst E.J. Breeuwer P. van't Hof W. Walgreen-Weterings E. Oomen L.C.J.M. Veerman E.C.I. Nieuw Amerongen A.V. Abee T. J. Biol. Chem. 1999; 274: 7286-7291Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). The second purpose was to assess whether dhvar4, which was shown in the previous experiment to alter the membrane integrity in whole cells, would in analogy of magainin peptides (34Westerhoff H.V. Hendler R.W. Zasloff M. Juretic D. Biochim. Biophys. Acta. 1989; 975: 361-369Crossref PubMed Scopus (69) Google Scholar, 35De Waal A. Vaz Gomes A. Mensink A. Grootegoed J.A. Westerhoff H.V. FEBS Lett. 1991; 293: 219-223Crossref PubMed Scopus (38) Google Scholar) act as an uncoupler of the respiration of isolated mitochondria. In Fig.4 A, the respiratory activity of the mitochondria is shown in the presence of the substrate NADH (State 2 respiration), after the addition of ADP (State 3), and after the depletion of ADP (State 4). The respiratory control ratio defined as the ratio of the respiratory rates in States 3 and 4 was 2.6, indicating that the respiration was coupled to ATP formation. The increase in respiration upon the addition of ADP is a measure for the integrity of the mitochondrial inner membrane, which was a prerequisite to study the uncoupling potential of the peptides. The peptides were added to mitochondria respiring in State 2. Histatin 5 at a final concentration of 33 μm inhibited mitochondrial respiration by 63%. Inhibition of the respiratory chain was verified by the observation that the addition of the uncoupler CCCP after histatin 5 did not increase respiration. dh-5 at the same concentration had no inhibitory effect on respiration, and as expected, the addition of CCCP after dh-5 increased respiration. In contrast to histatin 5 and d" @default.
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