oles of Cellular Respiration, CgCDR1, and CgCDR2 in Candida glabrata Resistance to Histatin
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《抗菌试剂及化学方法》
ABSTRACT
Histatin 5, a human salivary protein with broad-spectrum antifungal activity, is remarkably ineffective against Candida glabrata. Fluconazole resistance in this fungus is due in most cases to upregulation of CgCDR efflux pumps. We investigated whether the distinct resistance of C. glabrata to histatin 5 is related to similar mechanisms.
TEXT
In the past decade, Candida glabrata has emerged as the second most common fungal pathogen in opportunistic fungal infections in humans. This increase is likely related to the widespread use of the antimycotic drug fluconazole, to which C. glabrata is inherently less sensitive than C. albicans, C. parapsilosis, and C. tropicalis (18). Resistance to azoles has been associated with the upregulation of the genes CgCDR1 and CgCDR2 (also designated PDH1) which encode proteins belonging to the ATP binding cassette family of multidrug resistance membrane-associated efflux pumps (1, 2, 12, 19, 20, 23). Other reported mechanisms of azole resistance in C. glabrata which may actually be related to CDR upregulation involve the generation or selection of respiratory-deficient petite mutants (3, 4). In the quest to develop new antimycotics with possibly different cellular targets, histatins, a group of salivary cationic proteins, have received special attention (5, 8, 16, 17, 21). While histatins are highly effective against diverse classes of fungi, they are significantly less effective against C. glabrata (9, 24). Furthermore, other, structurally unrelated, cationic antifungal peptides, belonging to the magainin and defensin family, also show a selectively reduced activity against this fungus (6, 9, 11). The cellular basis for the apparent insensitivity of C. glabrata to various antifungal agents as different as azoles and cationic antifungal proteins is as yet unknown. In the current study we evaluated the role of petite mutation, fermentation, and the activity of the efflux pumps CgCDR1 and CgCDR2 in the resistance of C. glabrata to histatin 5.
C. albicans (ATCC 10231) and C. glabrata (ATCC 90030) were cultured in 1:10 diluted Sabouraud dextrose broth (SDB; Difco, Becton Dickinson, Franklin Lakes, NJ) (9) and containing either 0, 56, 112, or 225 μg/ml of histatin 5. Assessment of the optical density at 620 nm (OD620) after 48 h of incubation at 30°C showed that C. glabrata was able to grow even in the presence of the highest concentration of histatin 5, while the growth of C. albicans was inhibited by all three histatin concentrations (Fig. 1).
Since C. glabrata resistance to fluconazole in some cases is explained by induction or selection of petite mutants (3, 4) and petite mutation in C. albicans is known to abolish sensitivity to histatin 5 (7), the respiratory competence of C. glabrata cells grown in the presence of histatin 5 was evaluated. Two experimental approaches were followed. First, 10-μl aliquots of cells grown for 48 h in the presence of the indicated three concentrations of histatin 5 were plated on glucose- or glycerol-limited agar (7), the latter strictly supporting cellular respiration. Second, since the C. glabrata petite phenotype may be a transient cellular characteristic caused by mitochondria that switch between states of respiratory competence and incompetence (12), we also measured directly the respiration of C. glabrata cells in the histatin 5-containing cell cultures. Plated C. albicans cell suspensions containing histatin 5 did not grow on agar (Fig. 2). In contrast, the histatin 5-grown C. glabrata cells grew well on glucose- and glycerol-limited agar, the latter indicating that the presence of histatin 5 during growth in diluted SDB broth had not induced "petite" characteristics. In addition, respiratory measurements using a biological oxygen monitor (Yellow Springs Instruments, Yellow Springs, Ohio) (7) of cells grown in the presence of 56, 112, or 225 μg/ml of histatin 5 indicated that the oxygen consumption of these cells in their respective supernatants was certainly not lower than that from cells that had been cultured in the absence of histatin 5 (10.6, 10.6, 10.2, and 9.0 nmol O2/min/OD620, respectively).
In previous studies it was demonstrated that cellular respiration is important in C. albicans sensitivity to histatin 5 (8). It was hypothesized that C. glabrata might escape histatin 5 activity by utilizing fermentative pathways, since theoretically dextrose (glucose) can either be fermented or assimilated. Furthermore, C. glabrata, but not C. albicans, is a Crabtree-positive fungus (22), and its respiration may be negatively affected by certain levels of glucose (15). To exclude the possibility of substrate fermentation by C. glabrata, growth inhibition assays were also conducted in broth containing only glycerol as the carbon source. C. glabrata was insensitive in either glycerol- or glucose-limited broth, exhibiting 50% inhibitory concentrations (IC50s) higher than 225 μg/ml (Fig. 3). This result shows that the resistance of this fungus to histatin 5 is not explained by mitochondrial dysfunction or lack of respiration. In contrast, C. albicans was sensitive to histatin 5 in glycerol- and glucose-containing broths, exhibiting IC50s of 13.8 ± 4.8 and 21.2 ± 2.5 μg/ml, respectively. The sensitivity of C. albicans in glucose broth is consistent with the fact that respiration of C. albicans is not suppressed by glucose (10).
Previous studies have shown that the site-directed mutation in C. glabrata of CgCDR1, CgCDR2, or both affects sensitivity to fluconazole, in particular when both genes were disrupted simultaneously (20). Using the standardized antifungal susceptibility protocol M27-A2 of the National Committee for Clinical Laboratory Standards (NCCLS) (14), we confirmed published results (20) that strains in which either CgCDR1 or both CgCDR genes were abolished showed a marked increased sensitivity to fluconazole (Table 1). Since the intracellular accumulation of histatins is required for their activity toward C. albicans (8, 13, 25), it was hypothesized that C. glabrata resistance could be related to the fact that histatin 5 fails to accumulate intracellularly as a result of the activity of efflux pumps. While it is unknown whether histatin 5 could be transported by the CgCDR1 and CgCDR2 pumps that are so effective in exporting fluconazole, it was assessed whether the mutants lacking one or both of these genes would show an increased sensitivity to histatin 5. Growth inhibition assays with histatin 5 were conducted in diluted SDB (9), since salts in RPMI medium interfere with histatin activity (9). As indicated in Table 1, the efficacy of fluconazole in RPMI medium and in diluted SDB was in most cases comparable, allowing the comparison of C. glabrata sensitivities to fluconazole and histatin 5 in diluted SDB. The results indicated that while the various C. glabrata mutants showed differential sensitivities to fluconazole, all C. glabrata strains were resistant to histatin 5 growth inhibition (IC50 > 225 μg/ml). Notably, the observed histatin 5 resistance was relative to the histatin 5 sensitivity of C. albicans, which displayed IC50 values of 9.5 ± 0.5 μg/ml. In killing assays conducted in 5 mM potassium phosphate buffer, pH 7.0 (9), similar results of C. glabrata resistance were obtained, except that the ura3 C. glabrata strain (DSY 1029) was somewhat sensitive (Table 1). These results combined indicated that C. glabrata resistance to histatin 5 is independent of mutations in CgCDR1 or CgCDR2.
The data obtained here and in our previous study (9) reveal a seemingly fundamental and widespread resistance of C. glabrata to histatin 5. While the resistance of C. glabrata to fluconazole is known to occur in cells exhibiting petite mutations and/or overexpressing specific efflux pumps, the resistance to histatin 5 is not related to either of these two mechanisms. The absence of a correlation between the responses to fluconazole and histatin 5 of the various C. glabrata mutants evaluated suggests that the cellular characteristics providing resistance against azoles and cationic antifungal proteins in this fungus are not identical.
ACKNOWLEDGMENTS
We thank Nerline Grand-Pierre and Ana S. Fraga for technical assistance.
This study is supported by NIH/NIDCR grants DE05672, DE07652, and DE14950.
EFERENCES
Bennett, J. E., K. Izumikawa, and K. A. Marr. 2004. Mechanism of increased fluconazole resistance in Candida glabrata during prophylaxis. Antimicrob. Agents Chemother. 48:1773-1777.
Borst, A., M. T. Raimer, D. W. Warnock, C. J. Morisson, and B. A. Arthington-Skaggs. 2005. Rapid acquisition of stable azole resistance by Candida glabrata isolates obtained before the clinical introduction of fluconazole. Antimicrob. Agents Chemother. 49:783-787.
Brun, S., C. Aubry, O. Lima, R. Filmon, T. Bergès, D. Chabasse, and J. P. Bouchara. 2003. Relationship between respiration and susceptibility to azole antifungals in Candida glabrata. Antimicrob. Agents Chemother. 47:847-853.
Defontaine, A., J. P. Bouchara, P. Declerk, C. Planchenault, D. Chabasse, and J. N. Hallet. 1999. In vitro resistance to azoles associated with mitochondrial DNA deficiency in Candida glabrata. J. Med. Microbiol. 48:663-670.
Edgerton, M., and S. E. Koshlukova. 2000. Salivary histatin 5 and its similarities to the other antimicrobial proteins in human saliva. Adv. Dent. Res. 14:16-21.
Feng, Z., B. Jiang, J. Chandra, M. Ghannoum, S. Nelson, and A. Weinberg. 2005. Human beta-defensins: differential activity against candidal species and regulation by Candida albicans. J. Dent. Res. 84:445-450.
Gyurko, C., U. Lendenmann, R. F. Troxler, and F. G. Oppenheim. 2000. Candida albicans mutants deficient in respiration are resistant to the small cationic salivary antimicrobial peptide histatin 5. Antimicrob. Agents Chemother. 44:348-354.
Helmerhorst, E. J., and F. G. Oppenheim. 2004. The antifungal mechanisms of antimicrobial proteins, p. 245-277. In R. E. W. Hancock and D. Devine (ed.), Mammalian antimicrobial proteins, 1st ed. Cambridge University Press, Cambridge, United Kingdom.
Helmerhorst, E. J., C. Venuleo, A. Beri, and F. G. Oppenheim. 2005. Candida glabrata is unusual with respect to its resistance to cationic antifungal proteins. Yeast 22:705-714.
Helmerhorst, E. J., M. Stan, M. P. Murphy, F. Sherman, and F. G. Oppenheim. 2005. The concomitant expression and availability of conventional and alternative, cyanide-insensitive, respiratory pathways in Candida albicans. Mitochondrion 5:200-211.
Joly, S., C. Maze, P. B. McCray Juniorperiod, and J. M. Guthmiller. 2004. Human -defensins 2 and 3 demonstrate strain-selective activity against oral microorganisms. J. Clin. Microbiol. 42:1024-1029.
Kaur, R., I. Castano, and B. P. Cormack. 2004. Functional genomic analysis of fluconazole susceptibility in the pathogenic yeast Candida glabrata: roles of calcium signaling and mitochondria. Antimicrob. Agents Chemother. 48:1600-1613.
Koshlukova, S. E., M. W. Araujo, D. Baev, and M. Edgerton. 2000. Released ATP is an extracellular cytotoxic mediator in salivary histatin 5-induced killing of Candida albicans. Infect. Immun. 68:6848-6856.
National Committee for Clinical Laboratory Standards. 2002. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard, 2nd ed. M27-A2. National Committee for Clinical Laboratory Standards, Wayne, Pa.
Niimi, M., A. Kamiyama, and M. Tokunaga. 1988. Respiration of medically important Candida species and Saccharomyces cerevisiae in relation to glucose effect. J. Med. Vet. Mycol. 26:195-198.
Oppenheim, F. G., T. Xu, F. M. McMillian, S. M. Levitz, R. D. Diamond, G. D. Offner, and R. F. Troxler. 1988. Histatins, a novel family of histidine-rich proteins in human parotid secretion. Isolation, characterization, primary structure, and fungistatic effects on Candida albicans. J. Biol. Chem. 263:7472-7477.
Oppenheim, F. G. 1989. Salivary histidine-rich proteins, p. 151-160. In J. O. Tenovuo (ed.), Human saliva: clinical chemistry and microbiology. CRC Press, Boca Raton, Fla.
Pfaller, M. A., S. A. Messer, R. J. Hollis, R. N. Jones, and D. J. Diekema. 2002. In vitro activities of ravuconazole and voriconazole compared with those of four approved systemic antifungal agents against 6,970 clinical isolates of Candida spp. Antimicrob. Agents Chemother. 46:1723-1727.
Sanglard, D., F. Ischer, D. Calabrese, P. A. Majcherczyk, and J. Bille. 1999. The ATP binding cassette transporter gene CgCDR1 from Candida glabrata is involved in the resistance of clinical isolates to azole antifungal agents. Antimicrob. Agents Chemother. 43:2753-2765.
Sanglard, D., F. Ischer, and J. Bille. 2001. Role of ATP-binding cassette transporter genes in high frequency acquisition of resistance to azole antifungals in Candida glabrata. Antimicrob. Agents Chemother. 45:1174-1183.
Tsai, H., and L. A. Bobek. 1998. Human salivary histatins: promising anti-fungal therapeutic agents. Crit. Rev. Oral Biol. Med. 9:480-497.
Van Urk, H., W. S. Leopold Voll, W. A. Scheffers, and J. P. van Dijken. 1990. Transient-state analysis of metabolic fluxes in Crabtree-positive and Crabtree-negative yeasts. Appl. Environ. Microbiol. 56:281-287.
Vermitsky, J.-P., and T. D. Edlind. 2004. Azole resistance in Candida glabrata: coordinate upregulation of multidrug transporters and evidence for a Pdr1-like transcription factor. Antimicrob. Agents Chemother. 48:3773-3781.
Wei, G. X., and L. A. Bobek. 2004. In vitro synergistic antifungal effect of MUC7 12-mer with histatin 5 12-mer or miconazole. J. Antimicrob. Chemother. 53:750-758.
Xu, Y., I. Ambudkar, H. Yamagishi, W. Swaim, T. J. Walsh, and B. C. O'Connell. 1999. Histatin 3-mediated killing of Candida albicans: effect of extracellular salt concentration on binding and internalization. Antimicrob. Agents Chemother. 43:2256-2262.
Department of Periodontology and Oral Biology, Goldman School of Dental Medicine, Boston University, 700 Albany Street, Boston, Massachusetts 02118,1 Institute of Microbiology, University Hospital Lausanne (CHUV), Rue de Bugnon 48, CH-1011 Lausanne, Switzerland2(Eva J. Helmerhorst, Cater)
Histatin 5, a human salivary protein with broad-spectrum antifungal activity, is remarkably ineffective against Candida glabrata. Fluconazole resistance in this fungus is due in most cases to upregulation of CgCDR efflux pumps. We investigated whether the distinct resistance of C. glabrata to histatin 5 is related to similar mechanisms.
TEXT
In the past decade, Candida glabrata has emerged as the second most common fungal pathogen in opportunistic fungal infections in humans. This increase is likely related to the widespread use of the antimycotic drug fluconazole, to which C. glabrata is inherently less sensitive than C. albicans, C. parapsilosis, and C. tropicalis (18). Resistance to azoles has been associated with the upregulation of the genes CgCDR1 and CgCDR2 (also designated PDH1) which encode proteins belonging to the ATP binding cassette family of multidrug resistance membrane-associated efflux pumps (1, 2, 12, 19, 20, 23). Other reported mechanisms of azole resistance in C. glabrata which may actually be related to CDR upregulation involve the generation or selection of respiratory-deficient petite mutants (3, 4). In the quest to develop new antimycotics with possibly different cellular targets, histatins, a group of salivary cationic proteins, have received special attention (5, 8, 16, 17, 21). While histatins are highly effective against diverse classes of fungi, they are significantly less effective against C. glabrata (9, 24). Furthermore, other, structurally unrelated, cationic antifungal peptides, belonging to the magainin and defensin family, also show a selectively reduced activity against this fungus (6, 9, 11). The cellular basis for the apparent insensitivity of C. glabrata to various antifungal agents as different as azoles and cationic antifungal proteins is as yet unknown. In the current study we evaluated the role of petite mutation, fermentation, and the activity of the efflux pumps CgCDR1 and CgCDR2 in the resistance of C. glabrata to histatin 5.
C. albicans (ATCC 10231) and C. glabrata (ATCC 90030) were cultured in 1:10 diluted Sabouraud dextrose broth (SDB; Difco, Becton Dickinson, Franklin Lakes, NJ) (9) and containing either 0, 56, 112, or 225 μg/ml of histatin 5. Assessment of the optical density at 620 nm (OD620) after 48 h of incubation at 30°C showed that C. glabrata was able to grow even in the presence of the highest concentration of histatin 5, while the growth of C. albicans was inhibited by all three histatin concentrations (Fig. 1).
Since C. glabrata resistance to fluconazole in some cases is explained by induction or selection of petite mutants (3, 4) and petite mutation in C. albicans is known to abolish sensitivity to histatin 5 (7), the respiratory competence of C. glabrata cells grown in the presence of histatin 5 was evaluated. Two experimental approaches were followed. First, 10-μl aliquots of cells grown for 48 h in the presence of the indicated three concentrations of histatin 5 were plated on glucose- or glycerol-limited agar (7), the latter strictly supporting cellular respiration. Second, since the C. glabrata petite phenotype may be a transient cellular characteristic caused by mitochondria that switch between states of respiratory competence and incompetence (12), we also measured directly the respiration of C. glabrata cells in the histatin 5-containing cell cultures. Plated C. albicans cell suspensions containing histatin 5 did not grow on agar (Fig. 2). In contrast, the histatin 5-grown C. glabrata cells grew well on glucose- and glycerol-limited agar, the latter indicating that the presence of histatin 5 during growth in diluted SDB broth had not induced "petite" characteristics. In addition, respiratory measurements using a biological oxygen monitor (Yellow Springs Instruments, Yellow Springs, Ohio) (7) of cells grown in the presence of 56, 112, or 225 μg/ml of histatin 5 indicated that the oxygen consumption of these cells in their respective supernatants was certainly not lower than that from cells that had been cultured in the absence of histatin 5 (10.6, 10.6, 10.2, and 9.0 nmol O2/min/OD620, respectively).
In previous studies it was demonstrated that cellular respiration is important in C. albicans sensitivity to histatin 5 (8). It was hypothesized that C. glabrata might escape histatin 5 activity by utilizing fermentative pathways, since theoretically dextrose (glucose) can either be fermented or assimilated. Furthermore, C. glabrata, but not C. albicans, is a Crabtree-positive fungus (22), and its respiration may be negatively affected by certain levels of glucose (15). To exclude the possibility of substrate fermentation by C. glabrata, growth inhibition assays were also conducted in broth containing only glycerol as the carbon source. C. glabrata was insensitive in either glycerol- or glucose-limited broth, exhibiting 50% inhibitory concentrations (IC50s) higher than 225 μg/ml (Fig. 3). This result shows that the resistance of this fungus to histatin 5 is not explained by mitochondrial dysfunction or lack of respiration. In contrast, C. albicans was sensitive to histatin 5 in glycerol- and glucose-containing broths, exhibiting IC50s of 13.8 ± 4.8 and 21.2 ± 2.5 μg/ml, respectively. The sensitivity of C. albicans in glucose broth is consistent with the fact that respiration of C. albicans is not suppressed by glucose (10).
Previous studies have shown that the site-directed mutation in C. glabrata of CgCDR1, CgCDR2, or both affects sensitivity to fluconazole, in particular when both genes were disrupted simultaneously (20). Using the standardized antifungal susceptibility protocol M27-A2 of the National Committee for Clinical Laboratory Standards (NCCLS) (14), we confirmed published results (20) that strains in which either CgCDR1 or both CgCDR genes were abolished showed a marked increased sensitivity to fluconazole (Table 1). Since the intracellular accumulation of histatins is required for their activity toward C. albicans (8, 13, 25), it was hypothesized that C. glabrata resistance could be related to the fact that histatin 5 fails to accumulate intracellularly as a result of the activity of efflux pumps. While it is unknown whether histatin 5 could be transported by the CgCDR1 and CgCDR2 pumps that are so effective in exporting fluconazole, it was assessed whether the mutants lacking one or both of these genes would show an increased sensitivity to histatin 5. Growth inhibition assays with histatin 5 were conducted in diluted SDB (9), since salts in RPMI medium interfere with histatin activity (9). As indicated in Table 1, the efficacy of fluconazole in RPMI medium and in diluted SDB was in most cases comparable, allowing the comparison of C. glabrata sensitivities to fluconazole and histatin 5 in diluted SDB. The results indicated that while the various C. glabrata mutants showed differential sensitivities to fluconazole, all C. glabrata strains were resistant to histatin 5 growth inhibition (IC50 > 225 μg/ml). Notably, the observed histatin 5 resistance was relative to the histatin 5 sensitivity of C. albicans, which displayed IC50 values of 9.5 ± 0.5 μg/ml. In killing assays conducted in 5 mM potassium phosphate buffer, pH 7.0 (9), similar results of C. glabrata resistance were obtained, except that the ura3 C. glabrata strain (DSY 1029) was somewhat sensitive (Table 1). These results combined indicated that C. glabrata resistance to histatin 5 is independent of mutations in CgCDR1 or CgCDR2.
The data obtained here and in our previous study (9) reveal a seemingly fundamental and widespread resistance of C. glabrata to histatin 5. While the resistance of C. glabrata to fluconazole is known to occur in cells exhibiting petite mutations and/or overexpressing specific efflux pumps, the resistance to histatin 5 is not related to either of these two mechanisms. The absence of a correlation between the responses to fluconazole and histatin 5 of the various C. glabrata mutants evaluated suggests that the cellular characteristics providing resistance against azoles and cationic antifungal proteins in this fungus are not identical.
ACKNOWLEDGMENTS
We thank Nerline Grand-Pierre and Ana S. Fraga for technical assistance.
This study is supported by NIH/NIDCR grants DE05672, DE07652, and DE14950.
EFERENCES
Bennett, J. E., K. Izumikawa, and K. A. Marr. 2004. Mechanism of increased fluconazole resistance in Candida glabrata during prophylaxis. Antimicrob. Agents Chemother. 48:1773-1777.
Borst, A., M. T. Raimer, D. W. Warnock, C. J. Morisson, and B. A. Arthington-Skaggs. 2005. Rapid acquisition of stable azole resistance by Candida glabrata isolates obtained before the clinical introduction of fluconazole. Antimicrob. Agents Chemother. 49:783-787.
Brun, S., C. Aubry, O. Lima, R. Filmon, T. Bergès, D. Chabasse, and J. P. Bouchara. 2003. Relationship between respiration and susceptibility to azole antifungals in Candida glabrata. Antimicrob. Agents Chemother. 47:847-853.
Defontaine, A., J. P. Bouchara, P. Declerk, C. Planchenault, D. Chabasse, and J. N. Hallet. 1999. In vitro resistance to azoles associated with mitochondrial DNA deficiency in Candida glabrata. J. Med. Microbiol. 48:663-670.
Edgerton, M., and S. E. Koshlukova. 2000. Salivary histatin 5 and its similarities to the other antimicrobial proteins in human saliva. Adv. Dent. Res. 14:16-21.
Feng, Z., B. Jiang, J. Chandra, M. Ghannoum, S. Nelson, and A. Weinberg. 2005. Human beta-defensins: differential activity against candidal species and regulation by Candida albicans. J. Dent. Res. 84:445-450.
Gyurko, C., U. Lendenmann, R. F. Troxler, and F. G. Oppenheim. 2000. Candida albicans mutants deficient in respiration are resistant to the small cationic salivary antimicrobial peptide histatin 5. Antimicrob. Agents Chemother. 44:348-354.
Helmerhorst, E. J., and F. G. Oppenheim. 2004. The antifungal mechanisms of antimicrobial proteins, p. 245-277. In R. E. W. Hancock and D. Devine (ed.), Mammalian antimicrobial proteins, 1st ed. Cambridge University Press, Cambridge, United Kingdom.
Helmerhorst, E. J., C. Venuleo, A. Beri, and F. G. Oppenheim. 2005. Candida glabrata is unusual with respect to its resistance to cationic antifungal proteins. Yeast 22:705-714.
Helmerhorst, E. J., M. Stan, M. P. Murphy, F. Sherman, and F. G. Oppenheim. 2005. The concomitant expression and availability of conventional and alternative, cyanide-insensitive, respiratory pathways in Candida albicans. Mitochondrion 5:200-211.
Joly, S., C. Maze, P. B. McCray Juniorperiod, and J. M. Guthmiller. 2004. Human -defensins 2 and 3 demonstrate strain-selective activity against oral microorganisms. J. Clin. Microbiol. 42:1024-1029.
Kaur, R., I. Castano, and B. P. Cormack. 2004. Functional genomic analysis of fluconazole susceptibility in the pathogenic yeast Candida glabrata: roles of calcium signaling and mitochondria. Antimicrob. Agents Chemother. 48:1600-1613.
Koshlukova, S. E., M. W. Araujo, D. Baev, and M. Edgerton. 2000. Released ATP is an extracellular cytotoxic mediator in salivary histatin 5-induced killing of Candida albicans. Infect. Immun. 68:6848-6856.
National Committee for Clinical Laboratory Standards. 2002. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard, 2nd ed. M27-A2. National Committee for Clinical Laboratory Standards, Wayne, Pa.
Niimi, M., A. Kamiyama, and M. Tokunaga. 1988. Respiration of medically important Candida species and Saccharomyces cerevisiae in relation to glucose effect. J. Med. Vet. Mycol. 26:195-198.
Oppenheim, F. G., T. Xu, F. M. McMillian, S. M. Levitz, R. D. Diamond, G. D. Offner, and R. F. Troxler. 1988. Histatins, a novel family of histidine-rich proteins in human parotid secretion. Isolation, characterization, primary structure, and fungistatic effects on Candida albicans. J. Biol. Chem. 263:7472-7477.
Oppenheim, F. G. 1989. Salivary histidine-rich proteins, p. 151-160. In J. O. Tenovuo (ed.), Human saliva: clinical chemistry and microbiology. CRC Press, Boca Raton, Fla.
Pfaller, M. A., S. A. Messer, R. J. Hollis, R. N. Jones, and D. J. Diekema. 2002. In vitro activities of ravuconazole and voriconazole compared with those of four approved systemic antifungal agents against 6,970 clinical isolates of Candida spp. Antimicrob. Agents Chemother. 46:1723-1727.
Sanglard, D., F. Ischer, D. Calabrese, P. A. Majcherczyk, and J. Bille. 1999. The ATP binding cassette transporter gene CgCDR1 from Candida glabrata is involved in the resistance of clinical isolates to azole antifungal agents. Antimicrob. Agents Chemother. 43:2753-2765.
Sanglard, D., F. Ischer, and J. Bille. 2001. Role of ATP-binding cassette transporter genes in high frequency acquisition of resistance to azole antifungals in Candida glabrata. Antimicrob. Agents Chemother. 45:1174-1183.
Tsai, H., and L. A. Bobek. 1998. Human salivary histatins: promising anti-fungal therapeutic agents. Crit. Rev. Oral Biol. Med. 9:480-497.
Van Urk, H., W. S. Leopold Voll, W. A. Scheffers, and J. P. van Dijken. 1990. Transient-state analysis of metabolic fluxes in Crabtree-positive and Crabtree-negative yeasts. Appl. Environ. Microbiol. 56:281-287.
Vermitsky, J.-P., and T. D. Edlind. 2004. Azole resistance in Candida glabrata: coordinate upregulation of multidrug transporters and evidence for a Pdr1-like transcription factor. Antimicrob. Agents Chemother. 48:3773-3781.
Wei, G. X., and L. A. Bobek. 2004. In vitro synergistic antifungal effect of MUC7 12-mer with histatin 5 12-mer or miconazole. J. Antimicrob. Chemother. 53:750-758.
Xu, Y., I. Ambudkar, H. Yamagishi, W. Swaim, T. J. Walsh, and B. C. O'Connell. 1999. Histatin 3-mediated killing of Candida albicans: effect of extracellular salt concentration on binding and internalization. Antimicrob. Agents Chemother. 43:2256-2262.
Department of Periodontology and Oral Biology, Goldman School of Dental Medicine, Boston University, 700 Albany Street, Boston, Massachusetts 02118,1 Institute of Microbiology, University Hospital Lausanne (CHUV), Rue de Bugnon 48, CH-1011 Lausanne, Switzerland2(Eva J. Helmerhorst, Cater)