Vol. 59, No. 9

INFECTION AND IMMUNITY, Sept. 1991, p. 3284-3286

0019-9567/91/093284-03$02.00/0 Copyright C 1991, American Society for Microbiology

Inhibitory Effects of Human Salivary Histatins and Lysozyme on Coaggregation between Porphyromonas gingivalis and Streptococcus mitis YUKITAKA MURAKAMI,1 HIDEKI NAGATA,1 ATSUO AMANO,1 MASARU TAKAGAKI,1 SATOSHI SHIZUKUISHI,l AKIRA TSUNEMITSU,1* AND SABURO AIMOTO2 Department of Preventive Dentistry, Faculty of Dentistry, Osaka University, 1-8,1 and Institute for Protein Research, Osaka University, 3-2,2 Yamadaoka, Suita, Osaka 565, Japan Received 1 April 1991/Accepted 5 June 1991

The effects of histatins on coaggregation between Porphyromonas gingivalis 381 and Streptococcus mitis ATCC 9811 were investigated by using a turbidimetric assay. The coaggregation activity was significantly inhibited by histatins 5 and 8 and strongly by lysozyme. Tritium-labeled histatin 8 bound to P. gingivalis cells but not to S. mitis cells.

Porphyromonas (Bacteroides) gingivalis, which colonizes the oral cavity, has often been shown to possess adhesive properties that enable it to bind to epithelial cells, salivary pellicle, and gram-positive bacterial species (2, 3, 12, 15). Slots and Gibbons (15) have reported that adherence of P. gingivalis to hydroxyapatite surfaces and buccal epithelial cells is inhibited by human whole saliva. However, little attention has been paid to the identity of the inhibitory substances or to their effect upon bacterial coaggregation activity. We have reported that coaggregation between P. gingivalis and Streptococcus mitis can be identified by a turbidimetric assay (11). Concerning adsorption to hydroxyapatite, the most studied factor, histidine-rich protein (HRP), was described by Hay (4) and Baum et al. (1). MacKay et al. (7) and Pollock et al. (14) have shown that HRPs have antimicrobial effects, including growth inhibition of Candida albicans and Streptococcus mutans. Oppenheim et al. (13) and Troxler et al. (18) have established that there are 12 HRPs in human parotid secretions and have clarified their structural interrelationships. Their primary structures have been determined, and the salivary HRPs were named histatins 1 to 12. Oppenheim et al. (13) have suggested that histatins 1 and 3 are derived from different structural genes, whereas histatin 5 is a proteolytic product of histatin 3. Also, histatin 5 is a more potent inhibitor of C. albicans germination than are histatins 1 and 3 (13). The present report describes the inhibitory effects of synthetic histatins 5 and 8 on coaggregation between P. gingivalis and S. mitis. The inhibitory effects of lysozyme and basic peptides on coaggregation were also studied. P. gingivalis 381 was a gift from the Research Laboratories of Oral Biology, Sunstar Inc., Osaka, Japan. The strain was maintained and the bacterial cells were cultured anaerobically as previously described (5). S. mitis ATCC 9811 was purchased from the American Type Culture Collection, Rockville, Md. The strain was maintained on brain heart infusion agar (BBL, Becton Dickinson Microbiology Systems, Cockeysville, Md.) with four weekly transfers. For use, cells were grown in 10 ml of brain heart infusion broth (BBL). The first series of cultures were transferred into 100 ml of the same broth media used for precultures and incu*

bated at 37°C for 24 h in air. Both species of bacteria were harvested by centrifugation at 8,000 x g for 20 min at 4°C, washed three times with 10 mM phosphate buffer containing 0.15 M NaCl (PBS, pH 6.0), and suspended in the same buffer. The suspension of P. gingivalis was adjusted to an A550 of 1.0, and S. mitis ATCC 9811 was adjusted to an A550 of 0.35. Two peptides, Lys-Phe-His-Glu-Lys-His-His-SerHis-Arg-Gly-Tyr (histatin 8) and Asp-Ser-His-Ala-Lys-Arg-

His-His-Gly-Tyr-Lys-Arg-Lys-Phe-His-Glu-Lys-His-HisSer-His-Arg-Gly-Tyr (histatin 5), were synthesized by a solid-phase method as previously described (10). Synthesized active peptides of adrenocorticotropic hormone (ACTH, residues 1 to 24) and parathyroid hormone (PTH, residues 13 to 34) derived from humans were supplied by Peptide Institute, Inc., Osaka, Japan. Human milk lysozyme and salmine from salmon (grade X), a kind of protamine, were obtained from Sigma Chemical Co., St. Louis, Mo. The reaction mixture for the coaggregation assay contained P. gingivalis 381 and S. mitis ATCC 9811 (0.5 ml each, containing 5 x 108 cells) and 10 mM PBS (pH 6.0) or inhibitors in a final volume of 2 ml. The progress of coaggregation was monitored by measuring the decrease in A550 with time by using a UV-visible recording spectrophotometer and a temperature-controlled cuvette compartment with a magnetic 0.4 -------------------------

0 Ln

0.2

0 0

5 2.5 Time (min)

7.5

FIG. 1. Coaggregation inhibition by synthetic histatin 5. The reaction mixture contained P. gingivalis 381 and S. mitis ATCC 9811 (0.5 ml each, containing 5 x 108 cells) and 10 mM PBS (pH 6.0) in ). Histatin 5 was added to the reaction a final volume of 2 ml ( mixture at 5 x 10-4 M (---).

Corresponding author. 3284

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TABLE 1. Inhibition of bacterial coaggregation by histatins and basic polypeptides Peptide

Primary structure

1 5 10 Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr 1 5 10 15 Asp-Ser-His-Ala-Lys-Arg-His-His-Gly-Tyr-Lys-Arg-Lys-Phe-His20 Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr 1 5 10 15 Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys20 Leu-Gln-Asp-Val-His-Asn-Phe 1 5 10 15 Pro-Arg-Arg-Arg-Arg-Ser-Ser-Ser-Arg-Pro-Val-Arg-Arg-Arg-Arg20 25 30 Arg-Pro-Arg-Val-Ser-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Gly-Arg-Arg-

Histatin 8 Histatin 5

PTH active

fragment Salmine

Arg-Arg 1 5 10 15 Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-Gly-Lys20 Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro

ACTH active fragment Lysozyme

Concn (j.iM) for 50% inhibition' 62

14

11

5.6

3.7

1.3

a The values are the means of duplicate determinations.

stirrer (UV-265FW; Shimadzu Co., Kyoto, Japan) at 37°C. On the basis of the change of A550 at 5 min after the start of the reaction, the precent inhibition was calculated. Tritium labeling of histatin 8 was carried out using N-[ethyl-2-3H]ethylmaleimide (NEM) by the method described by Leslie et al. (6). Cys-histatin 8 (Cys-Lys-Phe-HisGlu-Lys-His-His-Ser-His-Arg-Gly-Tyr) was synthesized by a solid-phase method. This peptide and histatin 8 had almost the same inhibitory activities (data not shown). The Cyspeptide (0.18 ,umol) was dissolved in 500 ,ul of 50 mM phosphate buffer (pH 7.0), and 0.023 ,umol (1 mCi) of labeled NEM (NEN Research Products, Boston, Mass.) was added. An excess of unlabeled NEM was added after the mixture stood for 2 h at room temperature, and the mixture was then

04

3 C

2 4.

10

0

.,{ 1

4.J

4,Jcn 0 0

1

2

Histatin 8 added (106 dpm) FIG. 2. Ability of radiolabeled histatin 8 to bind to P. gingivalis 381 and S. mitis ATCC 9811. Differences in disintegrations per minute between native cells and heated cells (100°C, 10 min) of P. gingivalis (0) and S. mitis (A) are shown for increasing amounts of histatin 8. Each value is the average of duplicated assays.

maintained for another 2 h. After incubation, 1 mg of Cys-HCI was added (9). The assay of binding of labeled histatin 8 to P. gingivalis and/or S. mitis was carried out by the same method (9). The coaggregation curve is shown in Fig. 1. When synthetic histatin 5 was added to the reaction mixture at 5 x l0' M, no change in A550 was found in the coaggregation curve. The results of the assay of inhibition by histatins and basic polypeptides are summarized in Table 1. The data are expressed as the 50% inhibition of coaggregation. Lysozyme was the most potent substance tested as an inhibitor. ACTH, salmine, PTH, and histatin 5 also had significant inhibitory activities; histatin 8 had a lower inhibitory activity. The radiolabeled histatin 8 had almost the same inhibitory activity as the unlabeled peptide. These phenomena were confirmed by microscopic observations (data not shown). Although the histatin 8 showed low inhibition of coaggregation, the tritium-labeled histatin 8 clearly bound to P. gingivalis but not to S. mitis (Fig. 2). The findings obtained here suggest that the basic peptide and protein containing histidine, arginine, and lysine residues may function by binding to the P. gingivalis cell surfaces. It seems unlikely that only histatins bind to the bacterial cells specifically. Previous studies have shown that 6 to 10 mg of the histatin and 0.5 to 5 mg of lysozyme can be isolated from 100 ml of stimulated saliva from human beings (8, 16, 17). The concentration of histatin added to the reaction mixture in vitro might exceed the in vivo concentration in human saliva. The determination of the exact content of histatin in saliva is required to clarify the physiological role of histatin in the oral cavity. The contents of ACTH and PTH in human saliva have not yet been determined. Based on the observed inhibition of coaggregation, it is quite possible that histatins 5 and 8, together with the other histatins and lysozyme, because of their properties as basic peptides and protein, may modulate the attachment of P. gingivalis to grampositive bacteria present in the mouth.

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REFERENCES 1. Baum, B. J., J. L. Bird, D. B. Millar, and R. W. Longton. 1976. Studies on histidine-rich polypeptides from human parotid saliva. Arch. Biochem. Biophys. 177:427-436. 2. Boyd, J., and B. C. McBride. 1984. Fractionation of hemagglutinating and bacterial binding adhesins of Bacteroides gingivalis. Infect. Immun. 45:403-409. 3. Gibbons, R. J., and I. Etherdern. 1983. Comparative hydrophobicities of oral bacteria and their adherence to salivary pellicles. Infect. Immun. 41:1190-1196. 4. Hay, D. I. 1975. Fractionation of human parotid salivary proteins and the isolation of an histidine-rich acidic peptide which shows high affinity for hydroxyapatite surfaces. Arch. Oral Biol. 20:553-558. 5. Inoshita, E., A. Amano, T. Hanioka, H. Tamagawa, S. Shizukuishi, and A. Tsunemitsu. 1986. Isolation and some properties of exohemagglutinin from the culture medium of Bacteroides gingivalis 381. Infect. Immun. 52:421-427. 6. Leslie, J., D. L. Williams, and G. Gorin. 1962. Determination of mercapto groups in proteins with N-ethylmaleimide. Anal. Biochem. 3:257-263. 7. MacKay, B. J., L. Denepitiya, V. J. Iacono, S. B. Krost, and J. J. Pollock. 1984. Growth-inhibitory and bactericidal effects of human parotid salivary histidine-rich peptides on Streptococcus mutans. lnfect. Immun. 44:695-701. 8. MacKay, B. J., J. J. Pollock, V. J. Iacono, and B. J. Baum. 1984. Isolation of milligram quantities of a group of histidine-rich polypeptides from human parotid saliva. Infect. Immun. 44:688694. 9. Murakami, Y., A. Amano, M. Takagaki, S. Shizukuishi, A. Tsunemitsu, and S. Aimoto. 1990. Purification and characterization of a peptide from human parotid secretion which inhibits hemagglutination of Bacteroides gingivalis 381. FEMS Microbiol. Lett. 72:275-280. 10. Murakami, Y., T. Takeshita, S. Shizukuishi, A. Tsunemitsu, and S. Aimoto. 1990. Inhibitory effects of synthetic histidine-rich

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Inhibitory effects of human salivary histatins and lysozyme on coaggregation between Porphyromonas gingivalis and Streptococcus mitis.

The effects of histatins on coaggregation between Porphyromonas gingivalis 381 and Streptococcus mitis ATCC 9811 were investigated by using a turbidim...
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