Oral streptococci are gram-positive bacteria residing in the human oral cavity; they are the earliest attachment bacteria in plaque formation and are abundant in plaques [1]. Oral streptococci are one of the causative agents of dental caries and infective endocarditis [1].

Iron is an essential nutrient for bacterial growth [2]. Approximately 95% of the iron in the body is located in the red blood cells, liver, spleen, and muscles, and iron outside the cell is attached to the transferrin of the serum, lymph, or lactoferrin in mucosal secretion [3]. Iron has a low solubility under physiological conditions. The concentration of iron freely present in the human body is approximately 10^–18 M, much lower than the required concentration for bacterial growth [4]; therefore, bacteria must have a specific mechanism for obtaining iron.

There are three known mechanisms by which bacteria acquire iron. First, bacteria use iron-chelation siderophores to absorb iron from the environment. In this mechanism, iron is obtained when iron-bound siderophores are transferred to bacteria through receptors in the bacterial body [5]. Siderophores have been reported in Mycobacterium smegmatis [6] and Corynebacterium diphtheria [7]; however, they were not detected in Listeria monocytogenes [8] and Streptococcus mutans [9].

The second mechanism is the use of iron-binding proteins in the host. Some bacterial species have low iron-inducible outer membrane proteins, which can be combined with host iron-binding proteins such as transferrin or lactoferrin [10,11]. For instance, Neisseria gonorrhoeae and Neisseria meningitides can obtain iron using iron-binding proteins (transferrin, lactoferrin, among others) of the host [12].

In the third mechanism, iron is obtained through heme compounds. Some bacteria contain heme-binding proteins; iron binds to hemin, and the iron-bound hemin is transported into the bacteria by attaching to the hemin-binding proteins of the bacteria [13]. Heme compounds can provide a sufficient supply of iron for bacterial growth even at low concentrations (<10 μM) [14]. Heme can supply bacteria with iron from heme proteins such as hemoglobin, cytochrome C, haptoglobin-hemoglobin, and hemopexin [14]. The ability to obtain iron from heme or heme compounds for bacterial growth has been reported in oral bacteria such as Porphyromonas gingivalis [15], Treponema denticola [16], and Aggregatibacter actinomycetemcomitans [17] and other bacterial species, such as Helicobacter pylori [18], Neisseria gonorrhoeae [19], and Vibrio cholera [20].

No streptococcal species has been reported to produce siderophores [21]. S. mutans is an oral streptococcus and one of the causative agents of dental caries [22] without siderophores or lactoferrin [9]. In addition, the iron transport systems of streptococci are not precisely understood.

Therefore, this study investigated the hemin-binding properties of streptococci to confirm the ability of oral streptococci to obtain iron through heme compounds.

Bacterial growth conditions

Streptococci were cultured in a carbon dioxide (CO₂) incubator containing 5% CO₂ on Todd-Hewitt agar plates (Becton Dickinson Biosciences, Franklin Lake, NJ, USA) for one day at 37°C, transferred to Todd-Hewitt liquid medium, and incubated for 18 hr. S. criceti E49, S. gordonii DL1, KN105, KN115, G9B, M5, S. mutans KN88, BHT, KPSK2, S. oralis KN116, S. ratti Fa-1f, S. sanguinis 15, 17, 20, 31, 32, 38, 43, 49, 66X49, 804, KN107, HPC1, MPC1, and S. sobrinus KN100 strains were used for the experiments.

Hemin-binding assay

Streptococci were cultured for 18 hr and centrifuged (12,000 ×g, 4°C, 10 min) to obtain the bacterial pellets. The bacterial pellet was washed twice with phosphate buffered saline (PBS) and resuspended in PBS (OD₆₆₀=1.5 [1×10¹⁰ cells/mL]). The bacterial suspension (1 mL) and hemin (0.5 mL) (Sigma-Aldrich, Saint Louis, MO, USA) (final concentration, 30 μg/mL) were incubated in a 37°C water bath for 30 min. The culture was centrifuged in an Eppendorf centrifuge (12,000 ×g, 4°C, 10 min), and the optical density (OD) of the supernatant was analyzed using a spectrophotometer at 400 nm [23,24]. The hemin concentration remaining in the supernatant was determined using the hemin-concentration standard curve. The amount of hemin bound to the bacteria was calculated as the amount initially added and the difference in the amount remaining in the supernatant after the reaction [24].

The hemin-binding of the bacteria strains was analyzed to determine the difference in hemin-binding depending on the species. Staphylococcus aureus Cowan1 was used as a positive control [25]. Hemin-binding of S. gordonii DL1, a bacterium that binds to a large amount of hemin, was analyzed under various conditions. First, to determine whether the hemin-binding of S. gordonii depended on the incubation temperature, the incubation temperature was set at 4°C, 25°C, and 37°C. In addition, experiments were carried out at several concentrations of S. gordonii (0–20×10⁹ cells/mL) to determine how the binding of S. gordonii to hemin differed according to the amount of bacteria added. Experiments were also carried out at various concentrations of hemin (final concentration 0–40 μg/mL) to determine how the binding of S. gordonii to hemin differed according to the amount of hemin added.

Hemin-binding inhibition of trypsin and protease

S. gordonii DL1, which was cultured for 18 hr, was suspended in PBS in the same manner described for the hemin-binding assay. The bacterial suspension (1 mL) was incubated with trypsin from porcine pancreas (0.5 mL) (Sigma-Aldrich) (final concentration 0–111 μg/mL) in a water bath at 37°C for 1 hr and centrifuged in an Eppendorf centrifuge (12,000 ×g, 4°C, 10 min). The resulting pellet was washed three times with PBS, resuspended in PBS (1 mL), and incubated with 0.5 mL of hemin (final concentration 30 μg/mL) in a 37°C water bath for 30 min. The culture was centrifuged (12,000 ×g, 4°C, 10 min), and the OD value of the supernatant was measured using a spectrophotometer at 400 nm [24]. Hemin-binding inhibition of protease from Streptomyces caespitosus (Sigma-Aldrich) was carried out in the same manner as that of trypsin.

Growth analysis

Growth analysis was conducted to determine the ability of streptococci to obtain iron from hemin for growth. Bacterial growth was investigated using an iron chelator and hemin, and the experiment was performed using a modified procedure described by Eichenbaum et al. [26]. Iron was depleted by the addition of 30 mM nitrilotriacetic acid (NTA) trisodium salt (Sigma-Aldrich) to Todd-Hewitt broth (Becton Dickinson Biosciences) medium (pH 7.3) containing 0.1 M Tris-hydrochloride (Tris-HCl). Magnesium chloride (MgCl₂) (Sigma-Aldrich), Calcium chloride (CaCl₂) (Sigma-Aldrich), manganese (II) chloride (MnCl₂) (Sigma-Aldrich), and zinc chloride (ZnCl₂) (Sigma-Aldrich) were each added to the medium at 1 mM. In addition, hemin (10 or 25 μg/mL) was added to the NTA-treated medium. Todd-Hewitt medium without NTA or hemin was used as a control. Each medium (10 mL) was inoculated with 2×10⁸ cells/mL of S. gordonii and cultured for 18 hr. The OD value of the culture medium was measured at 660 nm every 1.5 hr [26]. The experiment was performed with triplicate tubes.

Statistics analysis

Statistical significance was determined using the two-sample Student’s t-test. The student’s t-test was run on the software package for social sciences (SPSS version 23; IBM Corp., Armonk, NY, USA). The criterion for statistical significance was set at p<0.05.

Hemin-binding assay

Hemin-binding was analyzed in S. sanguinis, S. gordonii, S. ratti, S. criceti, S. mutans, S. oralis, and S. sobrinus to determine the hemin-binding ability of streptococci and how they differed according to the species. Hemin-binding was observed in all strains, although it differed according to the species. S. gordonii M5, G9B, DL1, KN115, KN105, S. sanguinis 66X49, S. oralis KN116, S. mutans KN88, and KPSK2 were combined with higher amounts of hemin than S. aureus (the positive control), and S. ratti Fa-1f was combined with a lesser amount of hemin than S. aureus (Fig. 1).

Fig. 1. Hemin-binding of streptococci. Bacteria were assayed to analyze their hemin-binding ability from the reaction mixture, which contained 1 mL of cell suspension (1×10¹⁰ cells/mL) and 0.5 mL of hemin (final concentration, 30 μg/mL). Hemin absorbance was measured at 400 nm after incubation, and hemin concentration was determined by the standard curve of hemin using absorbance. Values indicate the means of duplicate experiment, and the error bars indicate standard deviations of the mean.

To determine the effect of bacterial concentration on the hemin-binding capacity, hemin-binding was analyzed in S. gordonii DL1 at various bacterial concentrations. The hemin-binding ability of bacteria increased in proportion to the concentration of bacteria (Fig. 2). To confirm the difference in the hemin-binding ability of bacteria according to hemin concentration, S. gordonii DL1 was combined with various concentrations of hemin. Hemin-binding to S. gordonii increased with elevated concentrations of hemin. However, when hemin was added at a higher concentration (60 μg/mL), there was no significant difference in the hemin-binding ability of the bacteria compared to the amount of hemin added (Fig. 3). In addition, the hemin-binding assay of S. gordonii DL1 was performed at various incubation temperatures to determine the changes in the hemin-binding ability of bacteria with varying incubation temperatures. The incubation temperature had little effect on the hemin-binding ability of bacteria. (Fig. 4).

Fig. 2. Effects of bacterial concentration on hemin-binding of S. gordonii DL1. S. gordonii cells (0–20×10⁹ cells/mL) were incubated with hemin at 37°C. After incubating the mixture for 30 min, the binding reactions were terminated by centrifugation at 12,000 ×g for 10 min. The quantity of unbound hemin remaining in the supernatant was measured at 400 nm in a spectrophotometer. The bound hemin on DL1 cells was calculated by subtraction. Each datum point represents the mean of results from duplicate experiments, and the vertical bars denote the standard deviation.

Fig. 3. Effects of hemin concentration on the hemin-binding activity of S. gordonii DL1. The reaction mixture contained 1 mL of cell suspension (optical density [OD]₆₆₀=1.5) and 0.5 mL hemin (final concentration 0 to 40 μg/mL). After incubating the mixture at 37°C for 30 min, the binding reactions were terminated by centrifugation at 12,000 ×g for 10 min. The quantity of unbound hemin remaining in the supernatant was measured at 400 nm in a spectrophotometer. The bound hemin on DL1 cells was calculated by subtraction. Each datum point represents the mean of results from duplicate experiments, and the vertical bars denote the standard deviation.

Fig. 4. Effect of incubation temperature on S. gordonii DL1 hemin-binding. S. gordonii DL1 (1×10¹⁰ cells/mL) was bound to hemin for 30 min at 4°C, 25°C, and 37°C, respectively. Values indicate means of duplicate, and the vertical bars denote the standard deviation.

Inhibition of hemin-binding of streptococci by trypsin and protease

To confirm the effect of proteins in the hemin-binding of streptococci, trypsin and protease (which possess proteolytic properties) were pretreated with bacteria and analyzed. Trypsin inhibited the hemin-binding of bacteria (Fig. 5). The hemin-binding of S. gordonii was also inhibited when treated with small amounts of trypsin. However, no significant difference was observed regarding trypsin concentration. Proteases also inhibited the hemin-binding ability of bacteria (Fig. 5). The inhibitory effect on the hemin-binding of S. gordonii increased with elevated protease concentration.

Fig. 5. Effects of trypsin and protease on hemin-binding of S. gordonii DL1. The role of proteins in hemin-binding activity was evaluated by the effect of trypsin or protease treatment on the hemin-binding inhibitory activity of DL1 cells. The reaction mixture contained 1 mL of the cell suspension (OD₆₆₀=1.5) and was pretreated with trypsin or protease (final concentration, 0 to 111 μg/mL). DL1 cells were treated for 1 hr, washed with PBS, and bound to the hemin for 30 min. Values indicate means of duplicate, and the vertical bars denote the standard deviation. PBS, phosphate buffered saline. The statistically significant p-values (<0.05) are indicated by *.

Growth analysis

Growth analysis indicated that S. gordonii growth was not observed in the iron-depleted NTA-treated Todd-Hewitt medium (Fig. 6). However, bacterial growth was observed when hemin was added to the NTA-treated Todd-Hewitt medium (Fig. 6). In addition, bacterial growth increased with elevated hemin concentration.

Fig. 6. Growth curve of S. gordonii DL1. S. gordonii grew in Todd-Hewitt (TH) medium (^◇) and in iron-depleted (with nitrilotriacetic acid [NTA]) TH medium without hemin (x); with hemin (10 μg/mL) (^△); or with hemin (25 μg/mL) (^□). The bacterial growth was not observed after 15 hr in the iron-depleted TH medium. Values indicate means of duplicate, and the vertical bars denote the standard deviation.

Oral streptococci require mechanisms to acquire iron in the environment for growth. Heme compounds can supply sufficient iron for bacterial growth [14], and hemin-binding streptococci tend to use hemin as a source of iron. The ability to use heme compounds has been implicated in the hemin-binding of bacteria [27], as demonstrated by studies on bacteria that require hemin for growth. Therefore, this study analyzed the hemin-binding ability of streptococci. The results showed that streptococci differed in the amount of hemin-binding according to the species; however, all streptococcal strains bound to hemin, suggesting that oral streptococci can utilize hemin-binding as an iron acquisition mechanism. The growth of S. gordonii was also confirmed in an NTA-treated iron-depleted medium after hemin was added. This indicated that S. gordonii can utilize hemin for growth.

This study analyzed the hemin-binding properties of streptococci. The hemin-binding of bacteria increased proportionally with the concentration of hemin added. However, when a higher amount (60 μg/mL) of hemin was added, the amount of hemin-binding to S. gordonii remained almost unchanged. This might be due to a decrease in available binding sites for hemin rather than a reduction in the bacteria-hemin-binding affinity at the binding site [28], suggesting that one bacterium can bind to only a certain amount of hemin, indicating that there is a certain number of hemin-binding sites in a single bacterium. The incubation temperature did not have a significant effect on the hemin-binding of S. gordonii. These results are similar to those of Tai et al. [28], where the hemin-binding activity of Streptococcus pneumoniae was not influenced by incubation temperatures.

Tai et al. [28] identified cell surface proteins as the major component responsible for hemin-binding in S. pneumoniae. To determine the effect of proteins on hemin-binding of oral streptococci, S. gordonii was pretreated with trypsin or protease and analyzed. The hemin-binding ability of S. gordonii was inhibited by pre-treatment with trypsin and protease. This result is similar to that of Tai et al. [28], suggesting that proteins are involved in the hemin-binding of S. gordonii DL1 and may be the main component responsible for hemin-binding.

Overall, differences were observed in the amount of hemin-binding according to the bacterial species, and the bacteria are likely to have a certain number of hemin-binding sites. Proteins are considered to be involved in the hemin-binding mechanism of streptococci. Therefore, this study suggests that streptococci could use hemin to obtain iron. Studies on the mechanism of iron accumulation through hemin-binding in various bacteria have been conducted; however, the mechanism of iron accumulation by oral streptococci using hemin remains unclear.

Similar to other pathogens, streptococci should be able to obtain iron from the environment for growth. In the oral environment, heme compounds (such as hemoglobin) can be obtained from the blood components in the mouth. In the gingival sulcus fluid and periodontal pocket, hemin is not freely available because it exists as compounds, such as haptoglobin, hemopexin, and albumin [29]. Nonetheless, acids degrade hemoglobin to produce four hemin molecules [30-32]. As acids can be produced by bacteria, hemin is potentially present in the oral cavity. Thus, the hemin-binding ability of oral streptococci plays an important role in bacterial growth. Further analysis on the hemin-binding protein of oral streptococci and the mechanism of iron transfer in bacteria is necessary to understand the precise mechanism of iron acquisition in oral streptococci.

None.

The authors declare that they have no competing interests.