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Saliva affects the antifungal activity of exogenously added histatin 3 towards Candida albicans
FEMS Microbiology Letters 244 (2005) 207–212 www.fems-microbiology.org
Saliva affects the antifungal activity of exogenously added histatin 3 towards Candida albicans Hisako Yamagishi a,b, Deirdre H. Fitzgerald c,*, Tin Sein d, Thomas J. Walsh d, Brian C. OÕConnell a,c a
Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA b Department of Pharmacology, Tokyo Dental College, Chiba, Japan c Department of Restorative Dentistry & Periodontology, School of Dental Science, Trinity College, Dublin, Ireland d Pharmacokinetics Section, Pediatric Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Received 8 November 2004; received in revised form 24 January 2005; accepted 25 January 2005 First published online 2 February 2005 Edited by D.J. Jamieson
Abstract Antifungal activity of histatin 3 against two Candida albicans clinical isolates was determined in assays containing rabbit submandibular gland saliva. Histatin 3 inhibited the cell growth and germination of both isolates dose-dependently (10–100 lg ml 1) with maximum inhibition occurring after 60 min incubation. Adding fresh histatin 3 after 60 min caused further reduction in the viable cell count. Higher histatin 3 concentrations (50–100 lg ml 1) and prolonged exposure to peptide were required to inhibit germination. Histatin 3 was rapidly degraded in rabbit submandibular gland saliva and this may explain why fresh addition of histatin 3 increases candidacidal activity. 2005 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies. Keywords: Antifungal; Candida; Histatin; Saliva; Histatin degradation; Rabbit submandibular gland saliva
1. Introduction Oral candidiasis is a common infection caused by the opportunistic pathogen Candida albicans and other candida species. It has been reported that oral candidiasis becomes clinically apparent in the prodromal stages of HIV-infection/AIDS, with 62–75% of infected patients presenting with candidiasis during the course of the disease [1,2]. Oral candidiasis is a potential reservoir of organisms for spreading the disease locally in * Corresponding author. Present address: Department II, Dublin Dental School and Hospital, Trinity College, Dublin 2, Ireland. Tel.: +353 0 612 7260; fax: +353 0 612 7295. E-mail address: [email protected] (D.H. Fitzgerald).
the compromised host, which may result in oropharyngeal and esophageal candidiasis in AIDS [3,4]. Hence the early treatment of oral candidiasis is important to limit later, more serious infection. Commonly used therapies for treatment of oral candidiasis include fluconazole or itraconazole. However, the increase in the occurrence of azole-resistant strains of Candida in HIV-infected patients [5–8] highlights the need for alternative therapies. Histatins are a family of 3–4 kDa histidine-rich, basic proteins found in the saliva of humans and old world monkeys but not any other species [9]. In vitro the histatins have potent antifungal activity against C. albicans though the role of the histatins in vivo has not been established [10,11]. There are 12 known
0378-1097/$22.00 2005 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies. doi:10.1016/j.femsle.2005.01.045
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histatins of which histatin-5 is the most candidacidal followed by histatin 3 [12,13]. However, histatin 3 is most effective at inhibiting the germination of C. albicans blastoconidia [14]. It has been reported that the concentration of histatin in HIV-infected patients is significantly decreased compared to that of healthy adults [15] and that this may contribute to the increased occurrence of oral candidiasis in HIV-infected patients. The antifungal properties of histatin make it a potential alternative to the use of antifungal azole drugs in the treatment of candidiasis. However, much remains to be understood about its mechanism of action and the conditions that affect its candidacidal activity, before it can be developed as an alternative antifungal therapeutic agent. In the present study, we determined the candidacidal activity of exogenously added histatin 3 towards two strains of C. albicans, in a reaction mixture containing rabbit submandibular gland saliva. To date, most in vitro studies of histatin activity have been carried out using non-organic buffers in the candidacidal assay, despite the fact that histatin activity is known to be very sensitive to the conditions used including the overall salt concentration and the concentration of ions such as calcium and magnesium [16,17]. Since the saliva of the rabbit does not contain histatins, the use of rabbit submandibular gland saliva in the assay buffer allowed us to study the candidacidal activity of histatin 3 in an environment similar to that of the human oral cavity in terms of pH, ion concentration, glycoprotein and proteolytic environment. Although the dynamic range and characteristics of proteins in rabbit and human submandibular gland saliva may differ, rabbit saliva was appropriate for use here since it is difficult to obtain consistently histatin–poor saliva from humans and furthermore, removing histatin from human saliva by dialysis or autodegradation would also remove other small molecules such as defensins and salts, which would affect the candidacidal assay.
2. Materials and methods 2.1. Animals Female New Zealand White rabbits (Hazelton, Rockville, MD), weighing 2.5–3.5 kg, were used. Silastic central venous catheters were surgically placed under sterile operative conditions of non-traumatic venous access, as previously described [18]. All animals were individually housed and provided with food and water ad libitum in accordance with National Institutes of Health Guidelines on the Care and Use of Laboratory Animals. General anesthesia was maintained by intravenous injection of ketamine and xylazine during saliva collection.
2.2. C. albicans isolates and growth conditions Two clinical isolates of C. albicans recovered from separate patients were used in this study. The isolate CA8621 was susceptible to fluconazole at the minimum inhibitory concentration (MIC) of 0.25 lg ml 1, determined by macrodilution methods (National Committee for Clinical Laboratory Standards, NCCLS, 1992). The isolate CA119 was resistant to fluconazole with MIC > 64 lg ml 1. Both isolates were grown overnight at 37 C on sabouraud dextrose agar (SDA). Three colonies of C. albicans were suspended in 50 ml of EmmomÕs modified sabouraud dextrose broth and incubated at 37 C in a shaking incubator at 80 rpm for 16–18 h. Cells were washed 3 times with sterile saline and centrifuged at 3500 rpm for 10 min. The pellets were resuspended in saline and cell numbers adjusted to 1 · 105 cells ml 1 for candidacidal assays and 1 · 106 cells ml 1 for germination inhibition assays. 2.3. Collection of saliva from rabbit submandibular gland Submandibular gland saliva was collected from animals in 1.5-ml microtubes, by cannulating to the ducts with polyethylene tubes, and stimulating salivary flow by intraductual injection of pilocarpine (0.5 mg per gland). To terminate salivary secretion, animals were administered atropine (100 lg per animal) by intravenous injection [18]. All samples were immediately centrifuged at 1000g for 10 min at 4 C and then stored at 70 C until required. 2.4. Sequential timed candidacidal assay Histatin 3 (Asp-Ser-His-Ala-Lys-Arg-His-His-GlyTyr-Lys-Arg-Lys-Phe-His-Glu-Lys-His-His-Ser-HisArg-Gly-Tyr-Arg-Ser-Asn-Tyr-Leu-Tyr-Asp-Asn) was custom-synthesised and purified by Genemed Synthesis Inc. (San Francisco, CA). C. albicans cells (2 · 104) were added to 24-well flat-bottom microtitre plates (Costar, Cambridge, MA) in a 2 ml reaction mixture (35 mM NaCl in 3-[N-morpholino]propanesulfonic acid (MOPS)-buffered RPMI-1640 supplemented with 2 mM glutamine. Saliva was centrifuged at 1000g for 5 min before adding to the assay. Saliva was diluted 1/10 into the reaction mixture. Histatin 3 was added to wells to give a final concentration of 0, 10, 20, 50 and 100 lg ml 1. The range of pH in the reaction mixture was 6.0–6.5. Plates were incubated at 37 C at 5% (v/v) CO2 and duplicate 100 ll samples were taken from each well at 0, 30, 60, 90 and 120 min time points. Each sample was serially diluted 10- and 100-fold for quantitative cultures and the diluted suspension spread onto SDA plates, which were incubated at 37 C for 24 h. Colonies were counted and cfu ml 1 was plotted as a function of time. To determine the concentration of
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2.7. Test of statistical significance
histatin 3 in the reaction mixture, a sample of the media at each time point was collected in a 1.5-ml microtube and stored on dry ice immediately for later processing.
Data are expressed as means ± SEM. BonferroniÕs multiple comparison test following one-way ANOVA was used to compare sets of data. P values of 5% or less were considered significant.
2.5. Germination inhibition assay Cells were diluted to a final concentration of 1 · 105 cfu ml 1 in 24-well flat-bottom microtitre plates in duplicate, using the same buffer as for sequential timed candidacidal assays saliva (1/10 dilution) was also added. Histatin 3 was added to plates to give a final concentration of 0, 50 and 100 lg ml 1. Plates were incubated at 37 C at 5% (v/v) CO2 for 120 and 240 min, and then examined microscopically. One hundred C. albicans cells were examined for the presence or absence of germination and percentage germination calculated as [germinated cells/(germinated cells + ungerminated cells)] · 100%.
3. Results 3.1. Candidacidal activity of histatin 3 peptide in rabbit submandibular saliva towards C. albicans isolates Incubation of C. albicans isolates CA8621 and CA119 with rabbit submandibular gland saliva caused a time-dependent reduction in the viable cell count which reached 68.9% (CA119) and 59.3% (CA8621) killing activity after 120 min incubation with saliva (Fig. 1). Killing activity increased dramatically in both C. albicans isolates when exogenous histatin 3 was added to the incubation mixture and was found to be concentration dependent over the histatin 3 range 10–100 lg ml 1. For both isolates, maximum killing activity was observed after incubation with histatin 3 (100 lg ml 1) and saliva for 60 min. Under these conditions, the viable cell count was reduced by 96.3% (CA119 and CA8621).
2.6. Measurement of histatin 3 concentration by capillary electrophoresis The histatin 3 concentration in the saliva-containing reaction mixture from candidacidal assays was measured by capillary electrophoresis (CE) with UV detection [19]. Rabbit submandibular gland saliva alone was used as a negative control. Briefly, the reaction mixture was centrifuged at 3500g for 10 min to remove cells. The supernatant or thawed saliva was adjusted to pH 4.5 by 2 N acetic acid and boiled for 4 min to inhibit proteolytic degradation [11]. After cooling on ice for 20 min, samples were centrifuged at 14,000g at 4 C for 5 min and 0.1% (v/v) trifluoroacetic acid was added. CE was performed in a Beckman P/ACE system using a Beckman 87/80, 50 lm uncoated capillary tube. CE conditions were 30 C, 20 kV for 60 min. The running buffer was 50 mM phosphate buffer, pH 2.5 (Beckman) and the absorption wavelength was 200 nm. Saliva samples were spiked with histatin 3 to identify the electrophoretic position of this peptide.
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3.2. The effect of repeat addition of histatin 3 at 60 min time-point in rabbit submandibular saliva against C. albicans isolates The effect of adding exogenous histatin 3 (10 lg ml 1) to saliva in the assay mixture at 0 min was compared to addition of 2 doses of histatin 3 (10 lg ml 1) at 0 and 60 min for both C. albicans isolates (Fig. 2). Addition of the second histatin 3 dose after 60 min resulted in further significant reduction in the viable cell count at later time-points (90 and 120 min) compared to the addition of a single dose of histatin 3 at 0 min.
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Fig. 1. Time-course of candidacidal activity of histatin 3 in saliva against azole-susceptible C. albicans CA119 (A) and azole-resistant C. albicans CA8621 (B) isolates. Histatin 3 was added to the reaction mixture (containing rabbit submandibular gland saliva) at 0 min time point. Data are expressed as means ± SEM of three independent experiments. d, control – no saliva; n, saliva alone; j, saliva + 10 lg ml 1 histatin 3; e, saliva + 20 lg ml 1 histatin 3; s, saliva + 50 lg ml 1 histatin 3; m, saliva + 100 lg ml 1 histatin 3). *P < 0.05, **P < 0.01 vs. saliva without histatin at each time-point.
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Time (min) Fig. 2. Candidacidal effect of histatin 3 added to saliva-containing medium at time 0 min and 60 min, in C. albicans isolates CA119 (A) and CA8621 (B). Histatin 3 (H3) was added to the reaction mixture at 0 and/or 60 min time points and incubation was continued for a further 60 min. Data are expressed as means ± SEM of three independent experiments. Black bar, saliva without histatin 3; grey bar, saliva + histatin 3 (10 lg ml 1) added at time 0; clear bar, saliva + histatin 3 (10 lg ml 1) added at time 0 and 60 min. *P < 0.05, **P < 0.01 vs. saliva without histatin group at each time point. #P < 0.05, ##P < 0.01 vs. saliva + histatin 3 (10 lg ml 1) added at time 0 at each time point.
3.3. Inhibition of germination of C. albicans isolates CA8621 and CA119 by histatin 3
3.4. The time course of degradation of exogenously added histatin 3 in rabbit submandibular gland saliva
The inhibition of germination of both C. albicans isolates by histatin 3 was determined over 3 h. Saliva alone had no effect on germ tube production in either isolate (Fig. 3). Germination was significantly inhibited when either 50 or 100 lg ml 1 histatin 3 was added to the assay in 2 or 3 separate doses (0, 2 h or 0, 1, 2 h), but not with a single dose of peptide at 0 h. Addition of lower concentration of histatin 3 (10 and 20 lg ml 1) had no effect on the inhibition of the germ tube production of either C. albicans CA8621 or CA119 (data not shown).
Histatin 3 peptide was not detectable in rabbit submandibular gland saliva (Fig. 4A). When saliva was spiked with histatin 3 (20 lg ml 1) a sharp peak appeared on the electropherogram at t = 32 min (Fig. 4B). The time-course of degradation of histatin 3, when incubated with rabbit submandibular gland saliva, was measured. The histatin 3 concentration decreased by 50% after 90 min of incubation and had decreased to 0% after 180 min (Fig. 5). When saliva was spiked with a second dose of histatin 3 (50 lg ml 1) after 60 min the added histatin 3 was also degraded gradually over the course of the incubation to a minimum concentration of 19.02 ± 0.53 lg ml 1 (Fig. 5).
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Fig. 3. Effect of histatin 3 on germination of C. albicans isolates CA8621 (black bar) and CA119 (grey bar). Histatin (H3) (50 or 100 lg ml 1) was added to the reaction mixture at time 0, 1, and/or 2 h. Data are expressed as means ± SEM of three independent experiments. *P < 0.05, **P < 0.01 vs. saliva without histatin 3 group. #P < 0.05 vs. double addition of 50 lg ml 1 histatin 3 group. bP < 0.05 vs. double addition of 100 lg ml 1 histatin 3 group.
Fig. 4. Electropherogram showing detection of histatin 3 in saliva. (A) Normal rabbit submandibular gland saliva. (B) 20 lg ml 1 of histatin 3 in saliva. The arrow shows the histatin 3 peak at a migration time of 32 min. A representative electropherogram is shown.
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Fig. 5. Degradation of histatin 3 in rabbit submandibular gland saliva. Data are expressed as means ± SEM of three independent experiments. d, 50 lg ml 1 of histatin 3 added at t = 0 min. n, 50 lg ml 1 of histatin 3 added at t = 0 and 60 min (arrow shown).
4. Discussion Histatin 3 is known to have potent candidacidal activity in vitro but it has been shown that this activity is highly sensitive to the assay environment such as pH, ion concentration and temperature [16,17,20,21]. To mimic the environment of the human oral cavity, we investigated the candidacidal activity of histatin 3 against C. albicans in medium containing rabbit submandibular gland saliva. Rabbit saliva does not contain histatin peptides [22] but contains broad-spectrum antimicrobial agents such as defensins, lysozyme, chitinase and lactoferrin. The results presented here suggest that rabbit saliva has considerable anticandidacidal activity but the candidacidal activity and inhibition of germination found in the presence of rabbit submandibular gland saliva is due mainly to histatin 3 added to the assay, as a concentration-dependent decrease in CFU ml 1 was found. The candidacidal activity of histatin in medium containing rabbit submandibular gland saliva is closer to the in vivo activity of histatin 3 in human saliva than an assay carried out in buffer alone because it includes not only effects due to pH and salt concentration but also effects due to protein and glycoprotein environment. It should be noted however that the characteristics and dynamic concentrations of proteins such as mucins, defensins and proteolytic enzymes may differ between rabbit and human saliva. Although the mechanism of candidacidal activity of the histatins remains unclear [23], it is apparent that this peptide uses pathways distinct from those of amphotericin-b or the azole drugs in therapeutic use currently [16,24]. Unlike such antifungals, histatins do not disrupt ergosterol biosynthesis but use a multistep pathway involving binding to a specific receptor on the yeast cell membrane, internalization and interaction with intracellular targets including the mitochondria [16,17,25,26]. It has been shown that histatin-5 is active against both
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amphotericin- and azole-resistant C. albicans isolates [14,24,27] and we have shown here that after a 60-min incubation, histatin 3 is also effective in killing and inhibiting the germination of the azole-resistant isolate CA119 even in the presence of rabbit submandibular gland saliva (Figs. 1 and 2). It has been reported that the concentration of total histatin peptides in healthy human parotid saliva ranges from 8.3 to 190.7 and 6.3 to 37.2 lg ml 1 in submandibular saliva [15]. In the present study, a histatin 3 concentration as low as 10 lg ml 1 was sufficient to cause inhibition of candidal cell growth (Fig. 1) after 60 min incubation with rabbit submandibular gland saliva. Germ tube production was inhibited by much higher concentrations of histatin 3 (50 and 100 lg ml 1) and only when at least two fresh additions of histatin 3 were made to the incubation mixture (Fig. 3). The results described in this study highlight the increased candidacidal activity and inhibition of germination achieved on exposure of both C. albicans isolates CA8621 and CA119 to fresh doses of histatin (Figs. 2 and 3). This situation might arise in vivo where in healthy human saliva the histatin peptides are continually secreted, proteolytically processed and degraded. Incubation of synthetic histatins with human parotid saliva has revealed a series of peptide degradation products, and sequence analysis has suggested that the activities involved include trypsin-like and chymotrypsin-like enzymatic activities, representing the most active salivary proteases and also alanine–lysine endopeptidase and histidine peptidase activities [28]. Furthermore, serine–protease and acidic-protease activities in the oral cavity may originate from the microflora or the blood leucocytes. We have shown here that exogenously added histatin 3 is degraded almost completely over a 2-h period in the presence of rabbit submandibular saliva. Therefore rabbit submandibular gland saliva may also contain protease activities arising from salivary secretions. The candidacidal activity of histatin 3 reached a maximum after 60 min incubation in the presence of saliva (Fig. 1). The slight increase in viable cell count after this time might be explained by the degradation of this peptide in saliva. This continual degradation of histatins in saliva may also explain why adding fresh histatin to rabbit submandibular gland saliva result in increased candidacidal activity and inhibition of germination in both C. albicans isolates tested. We have reported that histatin 3 has effective candidacidal activity in vitro in a medium with a similar salt and pH concentration to that of the oral cavity (35 mM NaCl and pH 6.0–6.5 [15]). Our laboratory has also reported that gene therapy using recombinant adenovirus encoding human histatin 3 effectively reduces C. albicans infection [29]. The results presented here suggest that for most effective candidacidal activity of histatin 3 in the oral cavity, peptide concentration must be maintained
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over time and furthermore, inhibition of germ-tube formation requires higher histatin concentrations and continual exposure. These are important findings to consider if the histatin peptide were to be further developed as a therapeutic antifungal agent.
Acknowledgements The authors thank the National Institutes of Health (NIH) for financial support.
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Saliva affects the antifungal activity of exogenously added histatin 3 towards Candida albicans Hisako Yamagishi a,b, Deirdre H. Fitzgerald c,*, Tin Sein d, Thomas J. Walsh d, Brian C. OÕConnell a,c a
Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA b Department of Pharmacology, Tokyo Dental College, Chiba, Japan c Department of Restorative Dentistry & Periodontology, School of Dental Science, Trinity College, Dublin, Ireland d Pharmacokinetics Section, Pediatric Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Received 8 November 2004; received in revised form 24 January 2005; accepted 25 January 2005 First published online 2 February 2005 Edited by D.J. Jamieson
Abstract Antifungal activity of histatin 3 against two Candida albicans clinical isolates was determined in assays containing rabbit submandibular gland saliva. Histatin 3 inhibited the cell growth and germination of both isolates dose-dependently (10–100 lg ml 1) with maximum inhibition occurring after 60 min incubation. Adding fresh histatin 3 after 60 min caused further reduction in the viable cell count. Higher histatin 3 concentrations (50–100 lg ml 1) and prolonged exposure to peptide were required to inhibit germination. Histatin 3 was rapidly degraded in rabbit submandibular gland saliva and this may explain why fresh addition of histatin 3 increases candidacidal activity. 2005 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies. Keywords: Antifungal; Candida; Histatin; Saliva; Histatin degradation; Rabbit submandibular gland saliva
1. Introduction Oral candidiasis is a common infection caused by the opportunistic pathogen Candida albicans and other candida species. It has been reported that oral candidiasis becomes clinically apparent in the prodromal stages of HIV-infection/AIDS, with 62–75% of infected patients presenting with candidiasis during the course of the disease [1,2]. Oral candidiasis is a potential reservoir of organisms for spreading the disease locally in * Corresponding author. Present address: Department II, Dublin Dental School and Hospital, Trinity College, Dublin 2, Ireland. Tel.: +353 0 612 7260; fax: +353 0 612 7295. E-mail address: [email protected] (D.H. Fitzgerald).
the compromised host, which may result in oropharyngeal and esophageal candidiasis in AIDS [3,4]. Hence the early treatment of oral candidiasis is important to limit later, more serious infection. Commonly used therapies for treatment of oral candidiasis include fluconazole or itraconazole. However, the increase in the occurrence of azole-resistant strains of Candida in HIV-infected patients [5–8] highlights the need for alternative therapies. Histatins are a family of 3–4 kDa histidine-rich, basic proteins found in the saliva of humans and old world monkeys but not any other species [9]. In vitro the histatins have potent antifungal activity against C. albicans though the role of the histatins in vivo has not been established [10,11]. There are 12 known
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histatins of which histatin-5 is the most candidacidal followed by histatin 3 [12,13]. However, histatin 3 is most effective at inhibiting the germination of C. albicans blastoconidia [14]. It has been reported that the concentration of histatin in HIV-infected patients is significantly decreased compared to that of healthy adults [15] and that this may contribute to the increased occurrence of oral candidiasis in HIV-infected patients. The antifungal properties of histatin make it a potential alternative to the use of antifungal azole drugs in the treatment of candidiasis. However, much remains to be understood about its mechanism of action and the conditions that affect its candidacidal activity, before it can be developed as an alternative antifungal therapeutic agent. In the present study, we determined the candidacidal activity of exogenously added histatin 3 towards two strains of C. albicans, in a reaction mixture containing rabbit submandibular gland saliva. To date, most in vitro studies of histatin activity have been carried out using non-organic buffers in the candidacidal assay, despite the fact that histatin activity is known to be very sensitive to the conditions used including the overall salt concentration and the concentration of ions such as calcium and magnesium [16,17]. Since the saliva of the rabbit does not contain histatins, the use of rabbit submandibular gland saliva in the assay buffer allowed us to study the candidacidal activity of histatin 3 in an environment similar to that of the human oral cavity in terms of pH, ion concentration, glycoprotein and proteolytic environment. Although the dynamic range and characteristics of proteins in rabbit and human submandibular gland saliva may differ, rabbit saliva was appropriate for use here since it is difficult to obtain consistently histatin–poor saliva from humans and furthermore, removing histatin from human saliva by dialysis or autodegradation would also remove other small molecules such as defensins and salts, which would affect the candidacidal assay.
2. Materials and methods 2.1. Animals Female New Zealand White rabbits (Hazelton, Rockville, MD), weighing 2.5–3.5 kg, were used. Silastic central venous catheters were surgically placed under sterile operative conditions of non-traumatic venous access, as previously described [18]. All animals were individually housed and provided with food and water ad libitum in accordance with National Institutes of Health Guidelines on the Care and Use of Laboratory Animals. General anesthesia was maintained by intravenous injection of ketamine and xylazine during saliva collection.
2.2. C. albicans isolates and growth conditions Two clinical isolates of C. albicans recovered from separate patients were used in this study. The isolate CA8621 was susceptible to fluconazole at the minimum inhibitory concentration (MIC) of 0.25 lg ml 1, determined by macrodilution methods (National Committee for Clinical Laboratory Standards, NCCLS, 1992). The isolate CA119 was resistant to fluconazole with MIC > 64 lg ml 1. Both isolates were grown overnight at 37 C on sabouraud dextrose agar (SDA). Three colonies of C. albicans were suspended in 50 ml of EmmomÕs modified sabouraud dextrose broth and incubated at 37 C in a shaking incubator at 80 rpm for 16–18 h. Cells were washed 3 times with sterile saline and centrifuged at 3500 rpm for 10 min. The pellets were resuspended in saline and cell numbers adjusted to 1 · 105 cells ml 1 for candidacidal assays and 1 · 106 cells ml 1 for germination inhibition assays. 2.3. Collection of saliva from rabbit submandibular gland Submandibular gland saliva was collected from animals in 1.5-ml microtubes, by cannulating to the ducts with polyethylene tubes, and stimulating salivary flow by intraductual injection of pilocarpine (0.5 mg per gland). To terminate salivary secretion, animals were administered atropine (100 lg per animal) by intravenous injection [18]. All samples were immediately centrifuged at 1000g for 10 min at 4 C and then stored at 70 C until required. 2.4. Sequential timed candidacidal assay Histatin 3 (Asp-Ser-His-Ala-Lys-Arg-His-His-GlyTyr-Lys-Arg-Lys-Phe-His-Glu-Lys-His-His-Ser-HisArg-Gly-Tyr-Arg-Ser-Asn-Tyr-Leu-Tyr-Asp-Asn) was custom-synthesised and purified by Genemed Synthesis Inc. (San Francisco, CA). C. albicans cells (2 · 104) were added to 24-well flat-bottom microtitre plates (Costar, Cambridge, MA) in a 2 ml reaction mixture (35 mM NaCl in 3-[N-morpholino]propanesulfonic acid (MOPS)-buffered RPMI-1640 supplemented with 2 mM glutamine. Saliva was centrifuged at 1000g for 5 min before adding to the assay. Saliva was diluted 1/10 into the reaction mixture. Histatin 3 was added to wells to give a final concentration of 0, 10, 20, 50 and 100 lg ml 1. The range of pH in the reaction mixture was 6.0–6.5. Plates were incubated at 37 C at 5% (v/v) CO2 and duplicate 100 ll samples were taken from each well at 0, 30, 60, 90 and 120 min time points. Each sample was serially diluted 10- and 100-fold for quantitative cultures and the diluted suspension spread onto SDA plates, which were incubated at 37 C for 24 h. Colonies were counted and cfu ml 1 was plotted as a function of time. To determine the concentration of
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2.7. Test of statistical significance
histatin 3 in the reaction mixture, a sample of the media at each time point was collected in a 1.5-ml microtube and stored on dry ice immediately for later processing.
Data are expressed as means ± SEM. BonferroniÕs multiple comparison test following one-way ANOVA was used to compare sets of data. P values of 5% or less were considered significant.
2.5. Germination inhibition assay Cells were diluted to a final concentration of 1 · 105 cfu ml 1 in 24-well flat-bottom microtitre plates in duplicate, using the same buffer as for sequential timed candidacidal assays saliva (1/10 dilution) was also added. Histatin 3 was added to plates to give a final concentration of 0, 50 and 100 lg ml 1. Plates were incubated at 37 C at 5% (v/v) CO2 for 120 and 240 min, and then examined microscopically. One hundred C. albicans cells were examined for the presence or absence of germination and percentage germination calculated as [germinated cells/(germinated cells + ungerminated cells)] · 100%.
3. Results 3.1. Candidacidal activity of histatin 3 peptide in rabbit submandibular saliva towards C. albicans isolates Incubation of C. albicans isolates CA8621 and CA119 with rabbit submandibular gland saliva caused a time-dependent reduction in the viable cell count which reached 68.9% (CA119) and 59.3% (CA8621) killing activity after 120 min incubation with saliva (Fig. 1). Killing activity increased dramatically in both C. albicans isolates when exogenous histatin 3 was added to the incubation mixture and was found to be concentration dependent over the histatin 3 range 10–100 lg ml 1. For both isolates, maximum killing activity was observed after incubation with histatin 3 (100 lg ml 1) and saliva for 60 min. Under these conditions, the viable cell count was reduced by 96.3% (CA119 and CA8621).
2.6. Measurement of histatin 3 concentration by capillary electrophoresis The histatin 3 concentration in the saliva-containing reaction mixture from candidacidal assays was measured by capillary electrophoresis (CE) with UV detection [19]. Rabbit submandibular gland saliva alone was used as a negative control. Briefly, the reaction mixture was centrifuged at 3500g for 10 min to remove cells. The supernatant or thawed saliva was adjusted to pH 4.5 by 2 N acetic acid and boiled for 4 min to inhibit proteolytic degradation [11]. After cooling on ice for 20 min, samples were centrifuged at 14,000g at 4 C for 5 min and 0.1% (v/v) trifluoroacetic acid was added. CE was performed in a Beckman P/ACE system using a Beckman 87/80, 50 lm uncoated capillary tube. CE conditions were 30 C, 20 kV for 60 min. The running buffer was 50 mM phosphate buffer, pH 2.5 (Beckman) and the absorption wavelength was 200 nm. Saliva samples were spiked with histatin 3 to identify the electrophoretic position of this peptide.
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3.2. The effect of repeat addition of histatin 3 at 60 min time-point in rabbit submandibular saliva against C. albicans isolates The effect of adding exogenous histatin 3 (10 lg ml 1) to saliva in the assay mixture at 0 min was compared to addition of 2 doses of histatin 3 (10 lg ml 1) at 0 and 60 min for both C. albicans isolates (Fig. 2). Addition of the second histatin 3 dose after 60 min resulted in further significant reduction in the viable cell count at later time-points (90 and 120 min) compared to the addition of a single dose of histatin 3 at 0 min.
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Fig. 1. Time-course of candidacidal activity of histatin 3 in saliva against azole-susceptible C. albicans CA119 (A) and azole-resistant C. albicans CA8621 (B) isolates. Histatin 3 was added to the reaction mixture (containing rabbit submandibular gland saliva) at 0 min time point. Data are expressed as means ± SEM of three independent experiments. d, control – no saliva; n, saliva alone; j, saliva + 10 lg ml 1 histatin 3; e, saliva + 20 lg ml 1 histatin 3; s, saliva + 50 lg ml 1 histatin 3; m, saliva + 100 lg ml 1 histatin 3). *P < 0.05, **P < 0.01 vs. saliva without histatin at each time-point.
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Time (min) Fig. 2. Candidacidal effect of histatin 3 added to saliva-containing medium at time 0 min and 60 min, in C. albicans isolates CA119 (A) and CA8621 (B). Histatin 3 (H3) was added to the reaction mixture at 0 and/or 60 min time points and incubation was continued for a further 60 min. Data are expressed as means ± SEM of three independent experiments. Black bar, saliva without histatin 3; grey bar, saliva + histatin 3 (10 lg ml 1) added at time 0; clear bar, saliva + histatin 3 (10 lg ml 1) added at time 0 and 60 min. *P < 0.05, **P < 0.01 vs. saliva without histatin group at each time point. #P < 0.05, ##P < 0.01 vs. saliva + histatin 3 (10 lg ml 1) added at time 0 at each time point.
3.3. Inhibition of germination of C. albicans isolates CA8621 and CA119 by histatin 3
3.4. The time course of degradation of exogenously added histatin 3 in rabbit submandibular gland saliva
The inhibition of germination of both C. albicans isolates by histatin 3 was determined over 3 h. Saliva alone had no effect on germ tube production in either isolate (Fig. 3). Germination was significantly inhibited when either 50 or 100 lg ml 1 histatin 3 was added to the assay in 2 or 3 separate doses (0, 2 h or 0, 1, 2 h), but not with a single dose of peptide at 0 h. Addition of lower concentration of histatin 3 (10 and 20 lg ml 1) had no effect on the inhibition of the germ tube production of either C. albicans CA8621 or CA119 (data not shown).
Histatin 3 peptide was not detectable in rabbit submandibular gland saliva (Fig. 4A). When saliva was spiked with histatin 3 (20 lg ml 1) a sharp peak appeared on the electropherogram at t = 32 min (Fig. 4B). The time-course of degradation of histatin 3, when incubated with rabbit submandibular gland saliva, was measured. The histatin 3 concentration decreased by 50% after 90 min of incubation and had decreased to 0% after 180 min (Fig. 5). When saliva was spiked with a second dose of histatin 3 (50 lg ml 1) after 60 min the added histatin 3 was also degraded gradually over the course of the incubation to a minimum concentration of 19.02 ± 0.53 lg ml 1 (Fig. 5).
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Fig. 3. Effect of histatin 3 on germination of C. albicans isolates CA8621 (black bar) and CA119 (grey bar). Histatin (H3) (50 or 100 lg ml 1) was added to the reaction mixture at time 0, 1, and/or 2 h. Data are expressed as means ± SEM of three independent experiments. *P < 0.05, **P < 0.01 vs. saliva without histatin 3 group. #P < 0.05 vs. double addition of 50 lg ml 1 histatin 3 group. bP < 0.05 vs. double addition of 100 lg ml 1 histatin 3 group.
Fig. 4. Electropherogram showing detection of histatin 3 in saliva. (A) Normal rabbit submandibular gland saliva. (B) 20 lg ml 1 of histatin 3 in saliva. The arrow shows the histatin 3 peak at a migration time of 32 min. A representative electropherogram is shown.
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Fig. 5. Degradation of histatin 3 in rabbit submandibular gland saliva. Data are expressed as means ± SEM of three independent experiments. d, 50 lg ml 1 of histatin 3 added at t = 0 min. n, 50 lg ml 1 of histatin 3 added at t = 0 and 60 min (arrow shown).
4. Discussion Histatin 3 is known to have potent candidacidal activity in vitro but it has been shown that this activity is highly sensitive to the assay environment such as pH, ion concentration and temperature [16,17,20,21]. To mimic the environment of the human oral cavity, we investigated the candidacidal activity of histatin 3 against C. albicans in medium containing rabbit submandibular gland saliva. Rabbit saliva does not contain histatin peptides [22] but contains broad-spectrum antimicrobial agents such as defensins, lysozyme, chitinase and lactoferrin. The results presented here suggest that rabbit saliva has considerable anticandidacidal activity but the candidacidal activity and inhibition of germination found in the presence of rabbit submandibular gland saliva is due mainly to histatin 3 added to the assay, as a concentration-dependent decrease in CFU ml 1 was found. The candidacidal activity of histatin in medium containing rabbit submandibular gland saliva is closer to the in vivo activity of histatin 3 in human saliva than an assay carried out in buffer alone because it includes not only effects due to pH and salt concentration but also effects due to protein and glycoprotein environment. It should be noted however that the characteristics and dynamic concentrations of proteins such as mucins, defensins and proteolytic enzymes may differ between rabbit and human saliva. Although the mechanism of candidacidal activity of the histatins remains unclear [23], it is apparent that this peptide uses pathways distinct from those of amphotericin-b or the azole drugs in therapeutic use currently [16,24]. Unlike such antifungals, histatins do not disrupt ergosterol biosynthesis but use a multistep pathway involving binding to a specific receptor on the yeast cell membrane, internalization and interaction with intracellular targets including the mitochondria [16,17,25,26]. It has been shown that histatin-5 is active against both
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amphotericin- and azole-resistant C. albicans isolates [14,24,27] and we have shown here that after a 60-min incubation, histatin 3 is also effective in killing and inhibiting the germination of the azole-resistant isolate CA119 even in the presence of rabbit submandibular gland saliva (Figs. 1 and 2). It has been reported that the concentration of total histatin peptides in healthy human parotid saliva ranges from 8.3 to 190.7 and 6.3 to 37.2 lg ml 1 in submandibular saliva [15]. In the present study, a histatin 3 concentration as low as 10 lg ml 1 was sufficient to cause inhibition of candidal cell growth (Fig. 1) after 60 min incubation with rabbit submandibular gland saliva. Germ tube production was inhibited by much higher concentrations of histatin 3 (50 and 100 lg ml 1) and only when at least two fresh additions of histatin 3 were made to the incubation mixture (Fig. 3). The results described in this study highlight the increased candidacidal activity and inhibition of germination achieved on exposure of both C. albicans isolates CA8621 and CA119 to fresh doses of histatin (Figs. 2 and 3). This situation might arise in vivo where in healthy human saliva the histatin peptides are continually secreted, proteolytically processed and degraded. Incubation of synthetic histatins with human parotid saliva has revealed a series of peptide degradation products, and sequence analysis has suggested that the activities involved include trypsin-like and chymotrypsin-like enzymatic activities, representing the most active salivary proteases and also alanine–lysine endopeptidase and histidine peptidase activities [28]. Furthermore, serine–protease and acidic-protease activities in the oral cavity may originate from the microflora or the blood leucocytes. We have shown here that exogenously added histatin 3 is degraded almost completely over a 2-h period in the presence of rabbit submandibular saliva. Therefore rabbit submandibular gland saliva may also contain protease activities arising from salivary secretions. The candidacidal activity of histatin 3 reached a maximum after 60 min incubation in the presence of saliva (Fig. 1). The slight increase in viable cell count after this time might be explained by the degradation of this peptide in saliva. This continual degradation of histatins in saliva may also explain why adding fresh histatin to rabbit submandibular gland saliva result in increased candidacidal activity and inhibition of germination in both C. albicans isolates tested. We have reported that histatin 3 has effective candidacidal activity in vitro in a medium with a similar salt and pH concentration to that of the oral cavity (35 mM NaCl and pH 6.0–6.5 [15]). Our laboratory has also reported that gene therapy using recombinant adenovirus encoding human histatin 3 effectively reduces C. albicans infection [29]. The results presented here suggest that for most effective candidacidal activity of histatin 3 in the oral cavity, peptide concentration must be maintained
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over time and furthermore, inhibition of germ-tube formation requires higher histatin concentrations and continual exposure. These are important findings to consider if the histatin peptide were to be further developed as a therapeutic antifungal agent.
Acknowledgements The authors thank the National Institutes of Health (NIH) for financial support.
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