The binding of human salivary α-amylase by oral strains of streptococcal bacteria

doi:10.1016/0003-9969(83)90003-1

Archives of Oral Biology

Volume 28, Issue 7, 1983, Pages 567-573

https://doi.org/10.1016/0003-9969(83)90003-1 Get rights and content

Abstract

The ability of various oral streptococci to bind salivary α-amylase to their cell surfaces was investigated. Samples of cells were mixed with whole cleared saliva and the α-amylase remaining after removal of the cells was assayed by radial diffusion in starch-containing agarose. Seventy-five per cent of Streptococcus sanguis strains bound the enzyme but strains of Streptococcus mutans and Streptococcus mitior did not. SDS-polyacrylamide gel electrophoretic analysis of Strep. sanguis cells which had been mixed with saliva showed that α-amylase is bound to the surface of the cells and can be recovered from them intact. The ability of Strep. sanguis strains to bind the enzyme did not correlate with biotype, IPS production or ability to adhere to saliva-coated hydroxyapatite. The cell-surface receptor responsible for the binding of α-amylase to Strep. sanguis (NCTC 7865) is a heat-stable component, possibly protein in nature, which is closely associated with the cell wall.

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Cited by (61)

Implications of salivary protein binding to commensal and pathogenic bacteria
2013, Journal of Oral Biosciences
Citation Excerpt :
It catalyzes the hydrolysis of α-1,4-glucosidic bonds of starch, glycogen and other polysaccharides [79]. The enzyme not only binds to S. gordonii, but also to Streptococcus mitis, Streptococcus cristatus, Streptococcus parasanguinis, Streptococcus salivarius, and several other species [80–83], collectively referred to as the α-amylase-binding streptococci (ABS). To date, ABS have only been found to colonize animals that are able to secrete α-amylase in their saliva [84].
An important function of salivary proteins is to interact with microorganisms that enter the oral cavity. For some microbes, these interactions promote microbial colonization. For others, these interactions are deleterious and result in the elimination of the microbe from the mouth, This paper reviews recent studies of the interaction of salivary proteins with two model bacteria; the commensal species Streptococcus gordonii, and the facultative pathogen Staphylococcus aureus. These organisms selectively interact with a variety of salivary proteins to influence important functions such as bacterial adhesion to surfaces, evasion of host defense, bacterial nutrition and metabolism and gene expression.
Enhancement of carbachol-induced amylase secretion in parotid glands from rats with experimental periodontitis
2011, Archives of Oral Biology
Citation Excerpt :
It would thus be appropriate to seek other functions of amylase in line with the concept of multifunctional saliva proteins.2 In this respect, it has been reported that amylase displays inhibitory activity against microorganisms.3,4 Amylase is increased in parotid saliva from patients with periodontitis5,6 and in whole saliva from patients with generalised aggressive periodontitis.7
In a previous study we observed that parotid glands from rats with experimental periodontitis showed an increase in basal amylase release as a result of an increase in cAMP accumulation induced by PGE₂ production. The aim of this work was to study whether this change in amylase release influences the secretory effect of carbachol.
Experimental periodontitis was induced through placing a black thread around the cervix of the two lower first molars. Experiments were done 22 days after ligature induced periodontitis. Amylase release was evaluated in vitro and determined using a colorimetric method which uses starch as substrate.
The effect of carbachol was increased in parotid glands from periodontitis rats. The effect of 10⁻⁶ M carbachol was inhibited by 4-DAMP (10⁻⁶ M), U-73122 (5 × 10⁻⁶ M) and trifluoperazine (5 × 10⁻⁶ M) in both groups. No changes were observed in the binding sites and affinity in parotid membranes from rats with experimental periodontitis. The inhibition of the adenylyl cyclase and the cyclooxygenase induced a right shift of the carbachol concentration–response curve in periodontitis group whilst the opposite effect was observed in control group in the presence of db-cAMP and PGE₂.
Parotid glands from rats with experimental periodontitis release more amylase in response to carbachol suggesting an interaction between Ca²⁺ and cAMP in the fusion/exocytosis step of secretory vesicles.
Probing the Role of Aromatic Residues at the Secondary Saccharide-Binding Sites of Human Salivary α-Amylase in Substrate Hydrolysis and Bacterial Binding
2008, Journal of Molecular Biology
Human salivary α-amylase (HSAmy) has three distinct functions relevant to oral health: (1) hydrolysis of starch, (2) binding to hydroxyapatite (HA), and (3) binding to bacteria (e.g., viridans streptococci). Although the active site of HSAmy for starch hydrolysis is well-characterized, the regions responsible for bacterial binding are yet to be defined. Since HSAmy possesses several secondary saccharide-binding sites in which aromatic residues are prominently located, we hypothesized that one or more of the secondary saccharide-binding sites harboring the aromatic residues may play an important role in bacterial binding. To test this hypothesis, the aromatic residues at five secondary binding sites were mutated to alanine to generate six mutants representing either single (W203A, Y276A, and W284A), double (Y276A/W284A and W316A/W388A), or multiple [W134A/W203A/Y276A/W284A/W316A/W388A; human salivary α-amylase aromatic residue multiple mutant (HSAmy-ar)] mutations. The crystal structure of HSAmy-ar as an acarbose complex was determined at a resolution of 1.5 Å and compared with the existing wild-type acarbose complex. The wild-type and the mutant enzymes were characterized for their abilities to exhibit enzyme activity, starch-binding activity, HA-binding activity, and bacterial binding activity. Our results clearly showed that (1) mutation of aromatic residues does not alter the overall conformation of the molecule; (2) single or double mutants showed either moderate or minimal changes in both starch-binding activity and bacterial binding activity, whereas HSAmy-ar showed significant reduction in these activities; (3) starch-hydrolytic activity was reduced by 10-fold in HSAmy-ar; (4) oligosaccharide-hydrolytic activity was reduced in all mutants, but the action pattern was similar to that of the wild-type enzyme; and (5) HA binding was unaffected in HSAmy-ar. These results clearly show that the aromatic residues at the secondary saccharide-binding sites in HSAmy play a critical role in bacterial binding and in starch-hydrolytic functions of HSAmy.
Identification and analysis of the amylase-binding protein B (AbpB) and gene (abpB) from Streptococcus gordonii
2002, FEMS Microbiology Letters
The binding of salivary amylase to Streptococcus gordonii has previously been shown to involve a 20-kDa amylase-binding protein (AbpA). S. gordonii also releases an 82-kDa protein into the supernatant that binds amylase. To study this 82-kDa component, proteins were precipitated from bacterial culture supernatants by the addition of acetone or purified amylase. Precipitated proteins were separated by SDS–PAGE and transferred to a sequencing membrane. The P2 kDa band was then sequenced, yielding a 25 N-terminal amino acid sequence, CGFIFGRQLTADGSTMFGPTEDYP. Primers derived from this sequence were used in an inverse PCR strategy to clone the full-length gene from S. gordonii chromosomal DNA. An open reading frame of 1959 bp was noted that encoded a 652 amino acid protein having a predicted molecular mass of 80 kDa. The first 24 amino acid residues were consistent with a hydrophobic signal peptide, followed by a 25 amino acid N-terminal sequence that shared identity (24 of 25 residues) with the amino acid sequence of purified AbpB. The abpB gene from strains of S. gordonii was interrupted by allelic exchange with a 420-bp fragment of the abpB gene linked to an erythromycin cassette. The 82-kDa protein was not detected in supernatants from these mutants. These abpB mutants retained the ability to bind soluble amylase. Thus, AbpA, but not AbpB, appears sufficient to be the major receptor for amylase binding to the streptococcal surface. The role of AbpB in bacterial colonization remains to be elucidated.
Expression, characterization, and biochemical properties of recombinant human salivary amylase
2002, Protein Expression and Purification
Human salivary amylase, a major component of human salivary secretions, possesses multiple functions in the oral cavity. It is the only enzyme in saliva capable of degrading oligosaccharides, which are used by the oral microflora for nutritional purposes. In order to understand its role in disease processes such as caries, we have undertaken the structure-function analyses of amylase. In this regard, the nonglycosylated human salivary amylase was expressed in a baculovirus expression system. The native and the recombinant amylases exhibit similar biochemical as well as biophysical properties. Unlike recombinant human pancreatic amylase, recombinant human salivary amylase is not glycosylated when expressed in a baculovirus system as determined from the crystal structure determination of the recombinant enzyme. Therefore, this system is suitable for further structure-function work without resorting to enzymatic removal of the carbohydrate chain. Details of the expression, purification, and biophysical properties will be presented.
Oral colonization and cariogenicity of Streptococcus gordonii in specific pathogen-free TAN:SPFOM(OM)BR rats consuming starch or sucrose diets
2001, Archives of Oral Biology
Citation Excerpt :
α-amylase, the most abundant enzyme in saliva (Aguirre et al., 1987, 1993), degrades starch to glucose and maltose (Merritt and Karn, 1977; Karn and Malacinski, 1978; Zakowski and Bruns, 1985), making them, at least theoretically, available for fermentation by S. gordonii and other glycolytic oral bacteria. Other oral streptococci also bind α-amylase, including S. mitis, S. crista, some Streptococcus salivarius, Streptococcus vestibularis and S. parasanguis, but not S. sanguis, S. oralis, S. mutans or, as far as is known, other species of oral bacteria (Douglas, 1983; Scannapieco et al., 1989; Douglas et al., 1990; Kilian and Nyvad, 1990; Scannapieco et al., 1990, 1992; Gwynn and Douglas, 1994; Scannapieco 1994; Rudney et al., 1995). Interestingly, α-amylase-binding bacteria are found only in the mouths of 14 mammalian species which have ability to produce salivary α-amylase, suggesting its significance ecologically and, perhaps, cariologically (Scannapieco et al., 1989).
The significance of Streptococcus gordonii in dental caries is undefined, as is that of other α-amylase-binding bacteria (ABB) commonly found in the mouth. To clarify the ecological and cariological roles of S. gordonii our specific pathogen-free Osborne–Mendel rats, TAN:SPFOM(OM)BR, were fed either diet 2000 (containing 56% confectioner's sugar, most of which is sucrose) or diet 2000CS (containing 56% cornstarch, in lieu of confectioner's sugar) and inoculated with S. gordonii strains. Uninoculated rats were free of both indigenous mutans streptococci (MS) and ABB, including S. gordonii, as shown by culture on mitis salivarius and blood agars of swabs and sonicates of dentitions after weanlings had consumed these diets for 26 days. ABB were detected by radiochemical assay using [¹²⁵I]-amylase reactive to α-amylase-binding protein characteristic of the surface of S. gordonii and other ABB. No ABB were detected (detection limit <1 colony-forming units in 10⁶ colony-forming units). Thus the TAN:SPFOM(OM)BR colony presents a ‘clean animal model’ for subsequent study. Consequently, S. gordonii strains Challis or G9B were used to inoculate weanling rat groups consuming either the high-sucrose diet 2000 or the cornstarch diet 2000CS. Two additional groups fed each of these diets remained unioculated. Recoveries of inoculants were tested 12 and 26 days later by oral swabs and sonication of the molars of one hemimandible of each animal, respectively. Uninoculated animals were reconfirmed to be free of ABB and mutans streptococci, but inoculated ones eating diet 2000CS had S. gordonii recoveries of 1–10% or, if eating diet 2000, 10–30% of total colony-farming units in sonicates. There were no statistically significant differences among the inoculated and uninoculated animal groups’ caries scores when they ate the cornstarch diet. Lesion scores for sucrose-eating rats were, however, from 2.4–5.1-fold higher than for cornstarch-eating rats, P<0.001, and were still higher if animals had been inoculated with either Challis (1.41-fold) or G9B (1.64-fold), than if uninoculated, both P<0.001, so long as the rats ate the sucrose diet. Therefore, TAN:SPFOM(OM)BR rats do not harbour ABB or S. gordonii but can be colonized by S. gordonii. Colonization levels of S. gordonii on the teeth are higher in the presence of high sucrose than with high starch-containing diets. Caries scores are augmented by sucrose compared with starch, and are further augmented by S gordonii colonization. S. gordonii is thus cariologically significant in the presence of sucrose, at least in this rat.

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Abstract

References (29)

Parotid-amylase activity: a possible role for proline-rich proteins

FEBS Letts.

The isolation and partial characterisation of a sulphated glycoprotein from human whole saliva which aggregates strains of Streptococcus sanguis but not Streptococcus mutans

Archs oral Biol.

Genetic polymorphism of proline-rich human salivary proteins

Science

Epithelial cell binding of group A streptococci by lipoteichoic acid on fimbrial denuded of M protein

J. exp. Med.

Manganese antagonizes the inhibitory effect of fluoride on the glucose metabolism of S. mutans

Microbios

Zooglea-forming streptococci, resembling Streptococcus sanguis isolated from dental plaque in man

Odont. Revy

Comparative estimates of bacterial affinities and adsorption sites on hydroxyapatite surfaces

Infect. Immun.

Taxonomy of some human viridans streptococci

Measurement of intracellular iodophilic polysaccharide in the cariogenic strains of Streptococcus mutans by cytochemical and chemical methods

Infect. Immun.

Effect of nutrients upon Streptococcus mutans BHT and Streptococcus mitior LPA-1 growing in pure or mixed culture on human teeth in an artificial mouth

J. appl. Bact.

The interaction of salivary components with oral streptococci

The nature of secretory agglutinins and aggregating factors I. Secretory conglutinin-like factor, secretory bacterial aggregating factors and secretory IgA antibody in human saliva and amniotic fluid

Int. Archs Allergy appl. Immun.

The role of anionic glyco-conjugates, particularly sulphated glycoproteins in relation to the oral cavity

Cited by (61)

Implications of salivary protein binding to commensal and pathogenic bacteria

Enhancement of carbachol-induced amylase secretion in parotid glands from rats with experimental periodontitis

Probing the Role of Aromatic Residues at the Secondary Saccharide-Binding Sites of Human Salivary α-Amylase in Substrate Hydrolysis and Bacterial Binding

Identification and analysis of the amylase-binding protein B (AbpB) and gene (abpB) from Streptococcus gordonii

Expression, characterization, and biochemical properties of recombinant human salivary amylase

Oral colonization and cariogenicity of Streptococcus gordonii in specific pathogen-free TAN:SPFOM(OM)BR rats consuming starch or sucrose diets