Short communication
Oral Mierobiol Itimnmol 1992: 7: 51-52
Antifungal activities of salivary histidine-rich polypeptides against Candida albicans and other oral yeast isolates
R. Rayhan\ L. X u \ R. P. Santarpia lll\ C. A. Tylenda', J. J. Pollock^ 'Department of Oral Biology and Pathology, School of Dental Medicine, State University of New York at Stony Brook, ^Clinical Investigations and Patient Care Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland, USA
Rayhan R, Xu L, Santarpia RP III, Tylenda CA, Polloek JJ. Antifwigal activities of .salivctry histidine-rieh polypeptides against Candida albicans and other oral yeast isolates. Oral Mierobiol htttttitttol 1992: 7: 51-52. Twenty-six oral yeast isolates from 26 donors were tested for their susceptibility to salivary histidine-rich polypeptide-4 (HRP-4) in blastospore viability assays. HRP-4 was observed to inhibit blastospore division in all of the yeast isolates, although inhibition was variable depending upon both species and strain tested. Nine species of Cattdida and 2 strains of Trichosporon pullulatts were included in the study. No significant differences in susceptibility to HRP-4 could be seen, irrespective of where in the oral cavity the yeast isolate was obtained.
Oral candidiasis is seen most commonly in people wearing dentures (7). Recent reports concerning the incidence of fungal infeetions among denture stomatitis patients indicate that 60-100%. of the patients are infected with Candida and other yeast species (11). During the last decade, oral candidiasis has also become prevalent in individuals with AIDS (3). Moreover, Klein et al. (5) observed that HlV-1 positive individuals who also became positive for oral candidiasis progressed more rapidly to opportunistic diseases and AIDS than the individuals who did not acquire this oral infection. Although Candida albieans has been found to be the main causative yeast species both in denture stomatitis (9) and AIDS (4) patients, other yeast species have also been implicated in the development of oral candidiasis (11, 12). Recent immunoadsorption affinity chromatography studies in our laboratory have identified the histidine-rich polypeptides (HRPs) as being tesponsible for the anti-candidal activity of parotid saliva (6). These peptides, termed histatins by Oppenhcim et al. (8), inhibit both blastospore viability (10) and germ tube formation (1). Among the HRPs, HRP-4 is by far the most effective antifungal agent against C albicatis when used under variable
Key words: Candida aibicans: saliva: antifungal: histidine-rich polypeptide Dr. Jerry J. Pollock, Department of Oral Biology and Pathology, School of Dental Medicine, State University of New York at Stony Brook, Stony Brook, NY, 11794-8702, USA Accepted for publication May 21, 1991
conditions of pH and yeast cell concen- path, 1 cm) in either potato dextrose or tration (1, 10). It was the purpose, there- Sabouraud dextrose broth (Difco Labfore, of this investigation to test HRP- oratories, Detroit, MI) and were then 4 in blastospore viability experiments stored lyophilized in 2%) skim milk until ready for use in blastospore viability against a variety of oral yeast isolates. Twenty-six oral yeast isolates from 26 assays. Lyophilized cultures were grown donors were included in the study. Of at room temperature to late log phase these, 7 were isolated frorn normal (optical density at 600 nm, 1.2) and then healthy adult carriers, 10 were fresh iso- freshly grown cells were inoculated a lates of individuals with AIDS and 9 second time to allow growth to again were fresh isolates from the acrylic sur- proceed to late log phase. At this point, face of the maxillary denture of denture yeast cell suspensions were centrifuged, stomatitis patients. Included in the 9 washed and resuspended in 0.025 M denture isolates was our reference HBPBS buffer, pH 7 to a cell concenstrain, C. albicatis GDH 2023, which tration of 10^ colony-forming units per was kindly provided by Dr. L. Julia ml. HRP-4 was then added to a final Douglas. The Candida species tested concentration of 4 /miol per ml and rewere C albicans, C. stellatoidea. C. trop- action mixtures were incubated at 25 °C. icalis. C. glabrata, C. parapsdosis, C. After 15 min and I h, aliquots were guillerttiondii, C. lattibica, C. pseudotro-removed, serially diluted and then icalis and C. kru.sei. Two strains of plated onto potato dextrose agar for Tricho.spot-ott fmllulans were also testedviability determination. Reaction mixfor their susceptibility to HRP-4. Yeast tures without added HRP-4 served as species were identified by both the 20 C controls. Experiments for each yeast API system (Analytab Products, Plain- stt ain were repeated 3 times. HRP-4 was view, NJ) and by the Uni-Yeast-Tek sys- custom synthesized and purified by a tem (Flow Laboratories, McLean, "VA). combination of ion exchange and reThe yeast isolates used in the study were verse-phase high-performance liquid obtained from denture, whole saliva, chromatography (6). buccal, tongue and palatal sites in the Table 1 presents the HRP-4 blastooral cavity. spore inhibitory activity against a varStock cultures of fresh yeast isolates iety of oral yeast strains. Because only were grown to late log phase (optical a limited number of strains were tested density range at 600 nm, 1.2-1.5; light in this study, we have not ranked the
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Ravhan et al.
Taiiic 1. Su.sceptit>tiity0/ veast tiiastosporc idtiltig to HRP-4 Pcrcctitagc itiliitjitiott of viability Yeast speciesC. alhicatis N8W" C. albicatts NIOB
C. aihicans GDH 2023 C. albicans A3W C. u / W r a / u A l l W
C albicans A3T C albicans A7T C albicans A16T C. stellatoidea DSID C tropicatis DS2D C tropicalis DS3D C. tropicalis DS4D C giabrata DS5D C giabrata DS6D C giabrata DS7D C parapsilosis DS8D C. parapsilosis N11W C. parapsilosis A33W C. guillertitondii A3W C /a/»i/7/(a N20W
C latnbica AI4W C pseuclotropicalis A28W C. pseudotropicalis N28T C A:;-w.ve/N15W T. pullulans N I I W r pullulans A 2 4 W
15 tnin
60 tnin
82.9+1.7'' 79.9 ±1.7 89.5 + 0.9 75.0 + 4.7 97.5 + 0.6 91.4 + 2.2 70.6 + 3.2 23.2 + 7.4 46.2 + 9.3
96.4±2.2 75.9±1.3 100±0 100±0 I00±0 97.3±0.2
84.8 ±1.8
54.9 ±2.1 60.0 ±0.9 IO.4±O.l 51.5±6.9 39.0±5.l 38.3 ±4.6 75.0±2.7 64.1 ±1.8 83.3±0.9 100±0 90.0 ±1.0 96.2 ±1.8 59.7 ±2.7 30.4±4.4 11.8±2.6 53.2±3.6
64.3 ±3.4
78.2±9.7 96.9 ±2.4 93.5±0.7 50.6 ±2.0 56.7±1.9 34.3±5.1 82.1 ±0.4 61.2±2.2 45.1±3.5 85.7±1.1 57.8±3.7 89.7 ±1.2 100±0 100±0 100±0 65.2±0.5 32.4±3.7 43.9±4.8 60.0 ±6.1
" Letter designations: N, normal patient; W, whole saliva isolate; B, buccal isolate; T, tongue isolate; P, palatal isolate; D, denture isolate; A, AIDS patient; and DS, denture stomatitis patient. *• Values for each experiment for each yeast strain represent the mean of triplicate samples± the standard error based on comparison with untreated buffer controls. Each experiment was repeated 3 times so that percentage inhibitions are averages of 9 determinations.
yeast species in order of susceptibility to HRP-4. However, under the experimental conditions used in these studies, no yeast strain tested exhibited complete resistance to salivary HRP-4. In addition, no differences in susceptibility to HRP-4 could be seen, irrespective of where in the oral cavity the yeast isolate was obtained. Although all of the yeast isolates showed susceptibility to HRP-4, it is not possible to predict the effects of the HRPs as they exist in native saliva. In the case of denture stomatitis, BudtzJorgensen (2) has hypothesized that major gland salivary access to the potential site of infection, the oral palatal mu-
cosa, is blocked by wearing of the patient's maxillary denture. However, this would not be the case in individuals with AIDS who are infected early on with Candida albieans and other yeast species. Acknowledgement This study was supported by Public Health Service Grant DE07441 from the National Institute of Dental Research. References 1. Brant EC, Santarpia RP III, Pollock JJ.
The role of pH in salivary histidine-rich polypeptide antifungal germ tube inhibitory activity. Oral Microbiol Immunol 1990; 5; 336-339. 2. Budtz-Jorgensen E. The significance of Cattdtda albicatts in the denture sore mouth. Scand J Dent Res 1974; 82; 151-190. 3. Gottlieb MS, Schroff R, Schanker HM et al. Pncumocystis caritni pneumonia and mucosal candidiasis in previously healthy homosexual men. N Engl J Med 1981; 305; 1425-1430. 4. Greenspan D, Greenspan JS. Oral mucosal manifestations of AIDS. Dermatol Clin 1987; 5: 733-737. 5. Klein RS, Harris CA, Small CB, Moll B, Lesser M, Friedland GH. Oral candidiasis in high-risk patients as the initial manifestation of immunodeficiency syndrome. N Engl J Med 1984: 311: 354-358. 6. Lai K, Santarpia RP III. Xu L, Manssuri F, Pollock JJ. One-step purification of histidine-rich polypeptides from human parotid saliva and determination of anticandidal activity. Oral Microbiol Immunol 1992; 7; 44-50. 7. Olsen I. Denture stomatitis occurrence and distribution of fungi. Acta Odontol Scand 1974: 32: 329-333. 8. Oppenheim FG, Xu T, McMillan FM et al. Histatins, a novel family of histidinerich proteins in human parotid secretion; isolation, characterization, primary structure and fungistatic effects on Cattdida albtcatts. J Biol Chem 1988; 263: 7472-7477. 9. Renner RP, Lee M, Andors L, McNamara TF. The role of C. albicatts in denture stomatitis. Oral Surg Oral Med Oral Pathol 1979:47: 323-328. 10. Santarpia RP III, Cho M-I, Pollock JJ. Parameters affecting the inhibition of Cattdida albicatts GDH 2023 and GRI 2773 blastospore viability by purified synthetic salivary histidine-rich polypeptides. Oral Microbiol Immunol 1990: 5; 226-232. 11. Tamamoto M, Miyake Y, Fugita Y, Suginaka M, Hamada T. Frequency and distribution of Candida species from denture wearers. Hiroshima J Med Sci 1986: 35: 39-43. 12. Tylenda CA, Larsen J, Yeh C-K, Lane HC, Fox PC. High levels of oral yeasts in early HIV-1 infection. J Oral Pathol Med 1989; 18: 520-524.
Oral Mierobiol Itimnmol 1992: 7: 51-52
Antifungal activities of salivary histidine-rich polypeptides against Candida albicans and other oral yeast isolates
R. Rayhan\ L. X u \ R. P. Santarpia lll\ C. A. Tylenda', J. J. Pollock^ 'Department of Oral Biology and Pathology, School of Dental Medicine, State University of New York at Stony Brook, ^Clinical Investigations and Patient Care Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland, USA
Rayhan R, Xu L, Santarpia RP III, Tylenda CA, Polloek JJ. Antifwigal activities of .salivctry histidine-rieh polypeptides against Candida albicans and other oral yeast isolates. Oral Mierobiol htttttitttol 1992: 7: 51-52. Twenty-six oral yeast isolates from 26 donors were tested for their susceptibility to salivary histidine-rich polypeptide-4 (HRP-4) in blastospore viability assays. HRP-4 was observed to inhibit blastospore division in all of the yeast isolates, although inhibition was variable depending upon both species and strain tested. Nine species of Cattdida and 2 strains of Trichosporon pullulatts were included in the study. No significant differences in susceptibility to HRP-4 could be seen, irrespective of where in the oral cavity the yeast isolate was obtained.
Oral candidiasis is seen most commonly in people wearing dentures (7). Recent reports concerning the incidence of fungal infeetions among denture stomatitis patients indicate that 60-100%. of the patients are infected with Candida and other yeast species (11). During the last decade, oral candidiasis has also become prevalent in individuals with AIDS (3). Moreover, Klein et al. (5) observed that HlV-1 positive individuals who also became positive for oral candidiasis progressed more rapidly to opportunistic diseases and AIDS than the individuals who did not acquire this oral infection. Although Candida albieans has been found to be the main causative yeast species both in denture stomatitis (9) and AIDS (4) patients, other yeast species have also been implicated in the development of oral candidiasis (11, 12). Recent immunoadsorption affinity chromatography studies in our laboratory have identified the histidine-rich polypeptides (HRPs) as being tesponsible for the anti-candidal activity of parotid saliva (6). These peptides, termed histatins by Oppenhcim et al. (8), inhibit both blastospore viability (10) and germ tube formation (1). Among the HRPs, HRP-4 is by far the most effective antifungal agent against C albicatis when used under variable
Key words: Candida aibicans: saliva: antifungal: histidine-rich polypeptide Dr. Jerry J. Pollock, Department of Oral Biology and Pathology, School of Dental Medicine, State University of New York at Stony Brook, Stony Brook, NY, 11794-8702, USA Accepted for publication May 21, 1991
conditions of pH and yeast cell concen- path, 1 cm) in either potato dextrose or tration (1, 10). It was the purpose, there- Sabouraud dextrose broth (Difco Labfore, of this investigation to test HRP- oratories, Detroit, MI) and were then 4 in blastospore viability experiments stored lyophilized in 2%) skim milk until ready for use in blastospore viability against a variety of oral yeast isolates. Twenty-six oral yeast isolates from 26 assays. Lyophilized cultures were grown donors were included in the study. Of at room temperature to late log phase these, 7 were isolated frorn normal (optical density at 600 nm, 1.2) and then healthy adult carriers, 10 were fresh iso- freshly grown cells were inoculated a lates of individuals with AIDS and 9 second time to allow growth to again were fresh isolates from the acrylic sur- proceed to late log phase. At this point, face of the maxillary denture of denture yeast cell suspensions were centrifuged, stomatitis patients. Included in the 9 washed and resuspended in 0.025 M denture isolates was our reference HBPBS buffer, pH 7 to a cell concenstrain, C. albicatis GDH 2023, which tration of 10^ colony-forming units per was kindly provided by Dr. L. Julia ml. HRP-4 was then added to a final Douglas. The Candida species tested concentration of 4 /miol per ml and rewere C albicans, C. stellatoidea. C. trop- action mixtures were incubated at 25 °C. icalis. C. glabrata, C. parapsdosis, C. After 15 min and I h, aliquots were guillerttiondii, C. lattibica, C. pseudotro-removed, serially diluted and then icalis and C. kru.sei. Two strains of plated onto potato dextrose agar for Tricho.spot-ott fmllulans were also testedviability determination. Reaction mixfor their susceptibility to HRP-4. Yeast tures without added HRP-4 served as species were identified by both the 20 C controls. Experiments for each yeast API system (Analytab Products, Plain- stt ain were repeated 3 times. HRP-4 was view, NJ) and by the Uni-Yeast-Tek sys- custom synthesized and purified by a tem (Flow Laboratories, McLean, "VA). combination of ion exchange and reThe yeast isolates used in the study were verse-phase high-performance liquid obtained from denture, whole saliva, chromatography (6). buccal, tongue and palatal sites in the Table 1 presents the HRP-4 blastooral cavity. spore inhibitory activity against a varStock cultures of fresh yeast isolates iety of oral yeast strains. Because only were grown to late log phase (optical a limited number of strains were tested density range at 600 nm, 1.2-1.5; light in this study, we have not ranked the
52
Ravhan et al.
Taiiic 1. Su.sceptit>tiity0/ veast tiiastosporc idtiltig to HRP-4 Pcrcctitagc itiliitjitiott of viability Yeast speciesC. alhicatis N8W" C. albicatts NIOB
C. aihicans GDH 2023 C. albicans A3W C. u / W r a / u A l l W
C albicans A3T C albicans A7T C albicans A16T C. stellatoidea DSID C tropicatis DS2D C tropicalis DS3D C. tropicalis DS4D C giabrata DS5D C giabrata DS6D C giabrata DS7D C parapsilosis DS8D C. parapsilosis N11W C. parapsilosis A33W C. guillertitondii A3W C /a/»i/7/(a N20W
C latnbica AI4W C pseuclotropicalis A28W C. pseudotropicalis N28T C A:;-w.ve/N15W T. pullulans N I I W r pullulans A 2 4 W
15 tnin
60 tnin
82.9+1.7'' 79.9 ±1.7 89.5 + 0.9 75.0 + 4.7 97.5 + 0.6 91.4 + 2.2 70.6 + 3.2 23.2 + 7.4 46.2 + 9.3
96.4±2.2 75.9±1.3 100±0 100±0 I00±0 97.3±0.2
84.8 ±1.8
54.9 ±2.1 60.0 ±0.9 IO.4±O.l 51.5±6.9 39.0±5.l 38.3 ±4.6 75.0±2.7 64.1 ±1.8 83.3±0.9 100±0 90.0 ±1.0 96.2 ±1.8 59.7 ±2.7 30.4±4.4 11.8±2.6 53.2±3.6
64.3 ±3.4
78.2±9.7 96.9 ±2.4 93.5±0.7 50.6 ±2.0 56.7±1.9 34.3±5.1 82.1 ±0.4 61.2±2.2 45.1±3.5 85.7±1.1 57.8±3.7 89.7 ±1.2 100±0 100±0 100±0 65.2±0.5 32.4±3.7 43.9±4.8 60.0 ±6.1
" Letter designations: N, normal patient; W, whole saliva isolate; B, buccal isolate; T, tongue isolate; P, palatal isolate; D, denture isolate; A, AIDS patient; and DS, denture stomatitis patient. *• Values for each experiment for each yeast strain represent the mean of triplicate samples± the standard error based on comparison with untreated buffer controls. Each experiment was repeated 3 times so that percentage inhibitions are averages of 9 determinations.
yeast species in order of susceptibility to HRP-4. However, under the experimental conditions used in these studies, no yeast strain tested exhibited complete resistance to salivary HRP-4. In addition, no differences in susceptibility to HRP-4 could be seen, irrespective of where in the oral cavity the yeast isolate was obtained. Although all of the yeast isolates showed susceptibility to HRP-4, it is not possible to predict the effects of the HRPs as they exist in native saliva. In the case of denture stomatitis, BudtzJorgensen (2) has hypothesized that major gland salivary access to the potential site of infection, the oral palatal mu-
cosa, is blocked by wearing of the patient's maxillary denture. However, this would not be the case in individuals with AIDS who are infected early on with Candida albieans and other yeast species. Acknowledgement This study was supported by Public Health Service Grant DE07441 from the National Institute of Dental Research. References 1. Brant EC, Santarpia RP III, Pollock JJ.
The role of pH in salivary histidine-rich polypeptide antifungal germ tube inhibitory activity. Oral Microbiol Immunol 1990; 5; 336-339. 2. Budtz-Jorgensen E. The significance of Cattdtda albicatts in the denture sore mouth. Scand J Dent Res 1974; 82; 151-190. 3. Gottlieb MS, Schroff R, Schanker HM et al. Pncumocystis caritni pneumonia and mucosal candidiasis in previously healthy homosexual men. N Engl J Med 1981; 305; 1425-1430. 4. Greenspan D, Greenspan JS. Oral mucosal manifestations of AIDS. Dermatol Clin 1987; 5: 733-737. 5. Klein RS, Harris CA, Small CB, Moll B, Lesser M, Friedland GH. Oral candidiasis in high-risk patients as the initial manifestation of immunodeficiency syndrome. N Engl J Med 1984: 311: 354-358. 6. Lai K, Santarpia RP III. Xu L, Manssuri F, Pollock JJ. One-step purification of histidine-rich polypeptides from human parotid saliva and determination of anticandidal activity. Oral Microbiol Immunol 1992; 7; 44-50. 7. Olsen I. Denture stomatitis occurrence and distribution of fungi. Acta Odontol Scand 1974: 32: 329-333. 8. Oppenheim FG, Xu T, McMillan FM et al. Histatins, a novel family of histidinerich proteins in human parotid secretion; isolation, characterization, primary structure and fungistatic effects on Cattdida albtcatts. J Biol Chem 1988; 263: 7472-7477. 9. Renner RP, Lee M, Andors L, McNamara TF. The role of C. albicatts in denture stomatitis. Oral Surg Oral Med Oral Pathol 1979:47: 323-328. 10. Santarpia RP III, Cho M-I, Pollock JJ. Parameters affecting the inhibition of Cattdida albicatts GDH 2023 and GRI 2773 blastospore viability by purified synthetic salivary histidine-rich polypeptides. Oral Microbiol Immunol 1990: 5; 226-232. 11. Tamamoto M, Miyake Y, Fugita Y, Suginaka M, Hamada T. Frequency and distribution of Candida species from denture wearers. Hiroshima J Med Sci 1986: 35: 39-43. 12. Tylenda CA, Larsen J, Yeh C-K, Lane HC, Fox PC. High levels of oral yeasts in early HIV-1 infection. J Oral Pathol Med 1989; 18: 520-524.
