Journal of Dental Research

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Effect of Transferrin on the Growth of Porphyromonas gingivalis E. Inoshita, K. Iwakura, A. Amano, H. Tamagawa and S. Shizukuishi J DENT RES 1991 70: 1258 DOI: 10.1177/00220345910700090501 The online version of this article can be found at: http://jdr.sagepub.com/content/70/9/1258

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Effect of Transferrin on the Growth of Porphyromonas gingivalis E. INOSHITA, K. IWAKURA, A. AMANO, H. TAMAGAWA, and S. SHIZUKUISHI1 Department of Preventive Dentistry, Osaka University Faculty of Dentistry, 1-8, Yamadaoka, Suita, Osaka 565, Japan

This study describes the effect of transferrin as an iron source on the growth of Porphyromonas (formally Bacteroides) gingivalis. Bacterial growth was monitored spectrophotometrically. All strains of P. gingivalis tested grew well in medium containing transferrin. The growth of P. gingivalis depended not only on the concentration of transferrin, but also on the iron saturation level of the protein. However, growth was not stimulated with either the ferrous or ferric iron salts tested. The addition of dipyridyl to the medium containing transferrin suppressed the growth of P. gingivalis, which also did not show species-specificity for human transferrin. Transferrinbinding activity was found in P. gingivalis by solid-phase assay with peroxidase-conjugated human transferrin. These results suggest that P. gingivalis may be capable of utilizing transferrin as an iron source for growth in vivo. J Dent Res 70(9):1258-1261, September, 1991

Introduction. Several studies have demonstrated that an important factor in the establishment of a bacterial infection is the ability of the bacteria to acquire iron from an infected host (Griffiths, 1987). It is well-known that black-pigmented anaerobes, which have a role in the pathogenesis of periodontal diseases (Slots and Genco, 1984), can utilize heme and heme-compounds as iron sources for growth in vitro (Gibbons and MacDonald, 1960; Mayrand and McBride, 1980; McKee et al., 1986; Bramanti and Holt, 1990). In the periodontal pocket, the growth of several of these organisms may be iron-limited or may be severely restricted by the low availability of iron. When gingival bleeding occurs in the periodontal pocket, hemoglobin may act as an iron source for the growth of these organisms. However, since the majority of hemoglobin liberated by hemolysis is rapidly bound by haptoglobin in plasma, haptoglobin may sequester hemoglobin in a form unavailable for growth (Eaton et al., 1982). Some strains of black-pigmented anaerobes, including Porphyrononas (formally Bacteroides) gingivalis, have been shown to be effective in degrading plasma proteins such as haptoglobin, hemopexin, and albumin (Carlsson et al., 1984). However, plasma and crevicular fluid contain potent host-derived protease inhibitors (Sandholm, 1986) that might block the breakdown of plasma-derived proteins by these bacteria. More than 90% of iron in plasma is bound by transferrin (Aisen, 1980). Transferrin functions in the absorption, transport, and exchange of iron in tissues (Awai and Brown, 1963). The presence of transferrin in plasma accounts, in part, for the bacteriostatic activity in the fluid for some bacteria (Bullen et al., 1978). However, it has been found that some bacteria can use transferrin as an iron source for growth in vitro (Kochan et al., 1977). One of the major iron sources available to blackReceived for publication January 15, 1991 Accepted for publication April 3, 1991 This investigation was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan. 'To whom correspondence and reprint requests should be addressed

pigmented anaerobes in the periodontal pocket is probably transferrin, and transferrin may support the pathogens during periodontal infection. Thus, our purpose in this study was to determine whether P. gingivalis utilizes transferrin as an iron source for growth.

Materials and methods. Bacterial strains and growth conditions.-Eight strains of black-pignented anaerobes were obtained from stock strains at the Research Laboratory of Oral Biology, Sunstar Inc., Osaka, Japan. The strains were maintained by weekly transfer on plates containing trypticase soy agar (BBL Microbiology Systems, Cockeysville, MD) supplemented with 5% sheep blood, 1 mg of yeast extract (BBL) per mL, 5 jig of hemin per mL, and 1 ,ug of menadione per mL. Freshly grown cells from the blood agar plates were grown to the late-logarithmic phase in 5 mL of trypticase soy broth (BBL) supplemented with 1 mg of yeast extractper mL, 1 jig of menadioneper mL, and 5 pg of hemin per mL. Unless otherwise stated, this culture was further transferred as a 1% (v/v) inoculum to the same medium without hemin. This culture was then used as a 1% (v/v) inoculum in a medium supplemented with various iron sources, as described below. The cultures were incubated in an Anaerobic System 1024 (Forma, Marietta, OH) in a N2-H2-CO2 (80:10:10) atmosphere at 35TC. Bacterial growth was monitored as the absorbance at 600 nm in a Bausch & Lomb spectrophotometer (Shimadzu Scientific Instruments, Kyoto, Japan). Transferrin binding assay. -The dot enzyme assay for transferrin was performed according to the method of Schryvers and Morris (1988). Cell suspensions (from 0.1 to 5 pug of protein) were applied directly to nitrocellulose/cellulose acetate paper (0.45 prm HA paper, Millipore Corporation, Bedford, MA) and allowed to dry. The paper was blocked by incubation with Block Ace (Yukijirushi Co., Sapporo, Japan) as a blocking solution for one h. After the blocking solution was removed, the paper was washed with 50 mmol/L tris-HCI buffer (pH 7.5) containing 0.15 mol/L NaCl and was exposed to the same buffer containing 0.5 pig/mL of horseradish-peroxidase-conjugated transferrin (Jackson Immunoresearch Labs., Avondale, PA). For competition experiments, the indicated amount of unconjugated transferrin (100% iron-saturated) was added to the mixture. The binding mixture was incubated at 370C for one h, and the paper was washed three times. The paper was then developed with a chloronaphthol/hydrogen-peroxide substrate mixture (HRP reagent, Bio-Rad Labs., Richmond, CA) at room temperature for five to 20 min and washed with water. Protein concentration was estimated by Bradford's method (1976), with reagent from Bio-Rad Labs. Chemicals.-Human transferring, apotransfeffmn, and transferrins from other animals were obtained from Sigma Chemical Co., St. Louis, MO. The human transferrmns were > 98% pure, as estimated by the supplier and confirmed by us by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Partiallyiron-saturated transferring were prepared by the addition of iron-free transferrin to 100% saturated protein. The iron saturation of transferrin was checked by measurement of the iron content in transferrin (The International Committee for Stan-

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TRANSFERRIN UTILIZATION BY P. gingivalis

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Fig. 1-Ability of black-pigmented anaerobes to obtain iron from hemin and transferrin. [E). control growth without addition; ~,5 pLg/mL hemin; 0, 5 p~mol/L transferrin. The final absorbance at 600 mu was measured after 72 h of growth in medium with the indicated addition.

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dardization in Hematology, 1971). Iron salts were from Wako Pure Chemical Industries, Ltd., Osaka, Japan.

Results. The strains of Prevotella intermedia and Prevotella denticola tested did not grow in the first passage in the hemin-free medium. Thus, these strains could not be further passaged in hemin-free media. All strains tested, except these two strains, grew well in the first passage in hemin-free media, but did not grow on subsequent transfer to iron-free medium. Fig. 1 shows the ability of eight black-pigmented anaerobes to use hemin or transferrin as an iron source. All strains tested grew well in the hemin medium. The strains of P. gingivalis and P. denticola exhibited growth in the medium containing transferrin,

whereas growth of strains such as P. intennedia, P. melaninogenica, and P. loescheii was not observed in the medium containing transferrin. The growth of P. gingivalis and P. denticola was maintained for at least four passages with transferrin. Fig. 2 shows the growth of P. gingivalis 381 in medium containing a variety of 100% iron-saturated transferrin additions. The growth depended on the concentration of transferrin which, up to a concentration of approximately 5 ,umol/L, was capable of limiting the growth. Fig. 3 shows the growth of P. gingivalis 381 in the presence of 10 pmol/L transferrin at various degrees of iron saturation. Growth also depended on the

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Time (hours) Fig. 2-Effect of transferrin (TF) concentration on the growth of P. gingivals 381. .e---., control growth without addition; -0--, 0.5 pmol/ L TF; 2.5 pmol/L TF; -A- -, 5 mol/L TF; --A--, 10 rmol/ L TF; *--, 20 pmoV/L TF; and -4, 5 mg/L hemin. -j-,

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Time (hours) Fig. 3-Effect of percent iron saturation of 10 pLmol/L transferrin (TF) on the growth of P. gingivalis 381. a-*, 100% saturated TF; - , 50% saturated TF; -A-, 20% saturated TF; -A-, 10% saturated TF; -0-, 0% saturated TF; and --0--, control growth without addition.


J Dent Res September 1991

INOSHITA et al.

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70 30 50 Time (hours) Fig. 5-Species-specificity of transferrin (TF)(5 pumolJL) for the growth of P. gingivalis 381. *-e-, human TF; -U , rabbit TF; -A-, horse TF; and -f-, bovine TF. 10

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Time (hours) Fig. 4-Inhibitory effect of dipyridyl (DP) dn the growth of P. gingivalis 381 in medium containing 10 pmol/L transferrin (TF). -* , no 100 pmoVfL DP; --, 200 pmol/L DP; addition of DP; *An -a-, 300 pmol/L DP; -i-, 400 ,umol/L DP; and ....*.-., no addition of both DP and TF.

saturation level of the transferrin. When 50% iron-saturated transferrin was added to a concentration of 10 jxmol/L, growth was almost equal to that obtained with 100% iron-saturated transferrin added to a concentration of 5 ,umol/L. As shown in Fig. 4, dipyridyl effectively suppressed the growth of P. gingivalis. Growth was reduced to approximately 35% with 200 pLmol/L dipyridyl after 48 h of growth. Further increases in dipyridyl concentration, up to 300 pumol/L, caused more than 90% suppression of bacterial growth. Addition of iron salts such as ferrous chloride, ferric chloride, ferrous sulfate, ferric sulfate, ferrous ammonium sulfate, and ferric ammonium

sulfate did not stimulate the growth of P. gingivalis 381. Fig. 5 illustrates the results from a growth experiment in which transferring derived fom various animals were provided as iron sources. Although P. gingivalis grew slowly in the medium containing bovine transferrin, stimulation of growth by transferrins of other animals was comparable with that by human transferrin. For examination of transferrin binding activity in P. gingivalis, dot enzyme assays with horseradish-peroxidase-conjugated transferrin were performed, as shown in Fig. 6-A. An increase in cell amount resulted in an increase in transferrin binding activity. When competition dot assays-in which unconjugated transferrin was mixed with conjugated transferrin prior to exposure to filter-bound intact bacterial cells-were also performed, unconjugated transferrin inhibited conjugated transferrin binding activity (Fig. 6-B).

Discussion.

Fig. 6-(A) Dot enzyme assay for binding of transferrin to P. gingivalis 381 cells. Control, 5 pg protein of heated cells. (B) Competition binding assay.

Although the ability of micro-organisms to obtain iron from various iron compounds in vitro has been extensively studied in aerobic and facultatively anaerobic bacteria, there have been few studies on iron uptake by anaerobic organisms. Some Bacteroides species have been shown to utilize hemin, transferrin, and iron salts as sources of essential iron (Verweij-van Vught et al., 1988). Our results indicated that hemin was a requirement of the oral black-pigmented anaerobes used in this study. This indication is similar to observations made by other investigators on P. gingivalis (Mayrand and McBride, 1980; McKee et al., 1986; Bramanti and Holt, 1990) and on blackpigmented anaerobes (Gibbons and MacDonald, 1960) in general. However, differences in ability to grow in medium containing transferrin were observed among the black-pigmented anaerobes used in this study. It is interesting that all strains of P. gingivalis were able to grow well in the medium containing transferrin, although it is unclear whether the ability to utilize transferrin as a source of iron contributes to the pathogenicity of P. gingivalis. P. gingivalis produces cytochromes, one of the major components of the electron transport system, from hemin (Shah and Williams, 1987). However, growth with transferrin was not so different from that with hemin, indica-


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ting that the bacterium grown with transferrin might utilize other metabolic pathways. The growth of P. gingivalis with transferrin was dependent on both the amount of iron and the iron-saturation level of the transferrin. The concentration of transferrin in vivo is usually from 20 to 40 pmol/L, and transferrin is usually saturated to approximately 35% (Aisen, 1980). The transferrin concentration in gingival crevicular fluid has been reported to be about 70% of that in serum (Schenkein and Genco, 1977). Thus, our results suggest that P. gingivalis might be capable of using transferrin as an iron source for growth in the periodontal pocket. The growth of P. gingivalis in the presence of transferrin was limited by addition of the ferrous chelator, dipyridyl. This result was consistent with those obtained for P. gingivalis (Barua et al., 1990) and some Bacteroides species (Verweij-van Vught et al., 1988; Otto et al., 1988) grown with hemin. Although one transferrin molecule can bind two molecules of ferric iron (Aisen, 1980), dipyridyl may chelate a ferrous iron from transferrin under anaerobic and reducing conditions. P. gingivalis was not able to use for growth any of the ferric or ferrous iron salts tested. However, it has been reported that the addition of ferrous ammonium sulfate to medium under bacterial-growth restriction by dipyridyl resulted in immediate restoration of growth by P. gingivalis (Barua et al., 1990). Although it is difficult to explain this discrepancy, P. gingivalis might be capable of utilizing iron that is released from dipyridyl by the addition of ferrous ammonium sulfate. The mechanism by which P. gingivalis sequesters iron from transferrin is not known. Under normal physiological conditions, bacteria might be expected to assimilate the iron bound by transferrin in four ways. One way is by the production of iron-chelating compounds, known as siderophores, that are able to remove iron from transferrin (Griffiths, 1987). However, so far there is no evidence for the production of such chelators by P. gingivalis. The second way is through proteolytic cleavage of transferrin, which disrupts the iron-binding site and releases iron. Carlsson et al. (1984) reported that some strains of black-pigmented anaerobes were able to degrade transferring. The third way involves the removal of iron from transferrin by a reductive pathway, not dependent on a direct transferrin/ cell-wall interaction (Cowart and Foster, 1985). It is possible that these last two mechanisms for iron acquisition are operative in P. gingivalis in vivo. The fourth mechanism is a direct interaction between transferrin and a transferrin receptor on the bacterial cell wall. This mechanism is thought to operate in Neisseria gonorrhoeae and in Neisseria meningitidis (Mickelsen and Sparling, 1981). Recent studies have indicated an iron-regulated outer membrane protein in N. meningitidis as a transferrin receptor that is specific for human transferrin (Schryvers and Morris, 1988). By means of an enzyme dot assay, we also showed that P. gingivalis cells were able to bind transferrin. However, P. gingivalis did not show speciesspecificity for human transferrin. The binding of P. gingivalis to transferrin may not be as specific as that reported for Neisseria. However, our results suggest that a direct interaction of P. gingivalis with transferrin might be involved in its ironacquisition from transferring. REFERENCES AISEN, P. (1980): The Transferrins. In: Iron in Biochemistry and Medicine, Vol. 2, A. Jacobs and M. Worwood, Eds., New York: Academic Press, Inc., pp. 87-129. AWAI, M. and BROWN, E.B. (1963): Studies of the Metabolism of I131-labeled Human Transferrin, J Lab Clin Med 61:363-396.

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