Inhibition of adhesion of viridans streptococci to fibronectin-coated hydroxyapatite beads by lipoteichoic acid

Fibronectin-coated hydroxyapatite (FnHA) beads were used in a model adhesion assay to isolate the lipoteichoic acid (LTA) mediated adhesion of oral streptococci. Representative strains of the commonly isolated viridans streptococci were incubated with FnHA beads in the presence and absence of exogenous LTA. The LTA inhibited the adhesion of all strains to a greater or lesser extent, but only a very few strains were inhibited by more than 90%. Strains of Streptococcus sanguis Type II and Streptococcus mitis which synthesize an amphiphile other than LTA were also inhibited. The findings provided circumstantial evidence for the involvement of LTA in the adhesion of this group of oral bacteria.     

Inhibition of adhesion of viridans streptococci to fibronectin-coated hydroxyapatite beads by lipoteichoic acid

Fibronectin-coated hydroxyapatite (FnHA) beads were used in a model adhesion assay to isolate the lipoteichoic acid (LTA) mediated adhesion of oral streptococci. Representative strains of the commonly isolated viridans streptococci were incubated with FnHA beads in the presence and absence of exogenous LTA. The LTA inhibited the adhesion of all strains to a greater or lesser extent, but only a very few strains were inhibited by more than 90%. Strains of Streptococcus sanguis Type II and Streptococcus mitis which synthesize an amphiphile other than LTA were also inhibited. The findings provided circumstantial evidence for the involvement of LTA in the adhesion of this group of oral bacteria.     

A 28-kilodalton fibronectin-binding protein of group a streptococci

Lipoteichoic acid (LTA) has been implicated as a major adhesin of group A streptococci that interacts with fibronectin (Fn). It has been suggested that protein adhesins may also be involved in the binding of Fn to streptococci. We searched for such a protein by transblotting membrane preparations from M types 5, 19, and 24 group A streptococci to nitrocellulose and reacting the blot with¹²⁵I-Fn. The Fn reacted with a 28-kDa polypeptide from all three serotypes of streptococci. Using affinity-purified antibodies to the 28-kDa protein in immunoblots of membrane preparations from various streptococci, we demonstrated that the 28-kDa protein is present in all 17 strains tested. Affinity-purified antibodies to the 28-kDa protein also reacted in varying degrees with intact streptococci, demonstrating that the antigen is exposed on the surface of intact organisms. Our results suggest that, in addition to LTA, group A streptococci contain a common Fn-binding moiety that is expressed as a major component of membrane preparations and that is accessible on the surface of streptococci for interactions with Fn.     

Formation of Molecular Complexes Between a Structurally Defined M Protein and Acylated or Deacylated Lipoteichoic Acid of Streptococcus pyogenes

The orientation of lipoteichoic acid (LTA) molecules on the surface of bacterial cells undoubtedly is determined by the ability of the LTA, during its transit through the cell wall, to bind via its polyglycerophosphate backbone or its glycolipid moieties to other constituents of the cytoplasmic membrane and the cell wall. We have investigated the possibility that LTA may become anchored to the cell surface by binding through its polyanionic backbone to positively charged regions of cell wall proteins. LTA was found to prevent the precipitation of partially purified HCl extracts of several strains of streptococci as well as a structurally defined streptococcal M protein molecule (pep M24) in 83% solutions of ethanol. The formation of complexes between LTA and M protein was demonstrated further by immunoelectrophoresis of pep M24 protein with increasing concentrations of radiolabeled LTA and by using antiserum against pep M24 to develop precipitin arcs. Pep M24 electrophoresed alone produced a single precipitin arc close to the origin. In contrast, when electrophoresed as a mixture with LTA or deacylated LTA, the M protein produced a second precipitin arc toward the anode coinciding with the area of migration of the radioactive LTA. Increasing concentrations of LTA or deacylated LTA shifted increasing amounts of the pep M24 antigen to the region of the second arc. Maleylation of M protein to block the positively charged free amino groups before mixing it with LTA prevented the formation of complexes. The complexes formed by the M protein with LTA, but not with deacylated LTA, showed the capacity to bind bovine serum albumin; LTA had been shown previously to bind to the fatty acid binding sites on bovine serum albumin. These results indicate that the LTA molecule is able to bind via its polyanionic backbone to positively charged residues of surface proteins of cells of S. pyogenes. The implications of such interaction as to the orientation of LTA molecules on the surface of cells of S. pyogenes are discussed.     

Relationship between Expression of the Family of M Proteins and Lipoteichoic Acid to Hydrophobicity and Biofilm Formation in Streptococcus pyogenes

Background

Hydrophobicity is an important attribute of bacteria that contributes to adhesion and biofilm formation. Hydrophobicity of Streptococcus pyogenes is primarily due to lipoteichoic acid (LTA) on the streptococcal surface but the mechanism(s) whereby LTA is retained on the surface is poorly understood. In this study, we sought to determine whether members of the M protein family consisting of Emm (M protein), Mrp (M-related protein), Enn (an M-like protein), and the streptococcal protective antigen (Spa) are involved in anchoring LTA in a manner that contributes to hydrophobicity of the streptococci and its ability to form biofilms.

Methodology/Principal Findings

Isogenic mutants defective in expression of emm, mrp, enn, and/or spa genes of eight different serotypes and their parental strains were tested for differences in LTA bound to surface proteins, LTA released into the culture media, and membrane-bound LTA. The effect of these mutations on the ability of streptococci to form a hydrophobic surface and to generate biofilms was also investigated. A recombinant strain overexpressing Emm1 was also engineered and similarly tested. The serotypes tested ranged from those that express only a single M protein gene to those that express two or three members of the M protein family. Overexpression of Emm1 led to enhanced hydrophobicity and biofilm formation. Inactivation of emm in those serotypes expressing only a single emm gene reduced biofilm formation, and protein-bound LTA on the surface, but did not alter the levels of membrane-bound LTA. The results were more varied in those serotypes that express two to three members of the M protein family.

Conclusions/Significance

Our findings suggest that the formation of complexes with members of the M protein family is a common mechanism for anchoring LTA on the surface in a manner that contributes to hydrophobicity and to biofilm formation in S. pyogenes, but these activities in some serotypes are dependent on a trypsin-sensitive protein(s) that remains to be identified. The need for interactions between LTA and M proteins may impose functional constraints that limit variations in the sequence of the M proteins, major virulence factors of S. pyogenes.     

Isolation and study of fosfomycin-resistant mutants of group A and B streptococci

Eighty-nine fosfomycin-resistant mutants of group A and B streptococci were isolated. The mutants differed essentially from the parent strains in cell-wall characteristics, such as morphology of cocci and chains, sensitivity to detergents and hydrophobic properties. At the same time, the recipient activity of the investigated mutants was not changed. It is supposed that resistance of mutants to fosfomycin is connected with a transport block of glycerol 3-phosphate--important lipoteichoic acid (LTA) structural component. Fosr mutants can be useful for experimental examination of Beachey's hypothesis about the importance of LTA or its complex with the M protein for adhesion.     

Physico-chemical and structural properties of the surfaces of Peptostreptococcus micros and Streptococcus mitis as compared to those of mutans streptococci, Streptococcus sanguis and Streptococcus salivarius.

The surface properties of nine Streptococcus mitis and four Peptostreptococcus micros strains from the oral cavity were examined and compared with a large group of oral streptococci. Zeta potential and contact angle measurements were employed to determine physico-chemical cell surface properties. In addition, elemental surface concentration ratios were obtained via X-ray photoelectron spectroscopy, and surface structures were examined with transmission electron microscopy. The S. mitis and P. micros strains were found to have higher isoelectric points, higher hydrophobicities and higher N/C surface concentration ratios than some other oral streptococci. The combined data suggest that both species possess large amounts of surface protein. All the S. mitis strains displayed abundant surface fibrils in negative staining, but the P. micros strains were devoid of surface appendages indicating that surface protein is present in different forms in the two species. The surfaces of S. mitis and P. micros type strains differed significantly from the other strains examined.     

Molecular analysis of lipoteichoic acid from Streptococcus agalactiae.

A method for the analysis of lipoteichoic acid (LTA) by polyacrylamide gel electrophoresis (PAGE) is described. Purified LTA from Streptococcus agalactiae tended to smear in the upper two-thirds of a 30 to 40% linear polyacrylamide gel, while the chemically deacylated form (cdLTA) migrated as a ladder of discrete bands, reminiscent of lipopolysaccharides. The deacylated polymer appeared to separate in this system on the basis of size, as evident from results obtained from PAGE analysis of cdLTA subjected to limited acid hydrolysis and LTA that had been fractionated by gel filtration. A survey of cdLTA from other streptococci revealed similarities in molecular weight ranges. The polymer from Enterococcus hirae was of a higher molecular weight. This procedure was used to examine the effect of penicillin and chloramphenicol on the synthesis, turnover, and heterogeneity of LTA in S. agalactiae. Penicillin appeared to enhance LTA synthesis while causing the release of this polymer into the supernatant fluid. In contrast, chloramphenicol inhibited the synthesis of this molecule and resulted in its depletion from the cell surface. Penicillin did not alter the heterogeneity of this polymer, but chloramphenicol caused an apparent shift to a lower-molecular-weight from of the LTA, as determined by PAGE. This shift in the heterogeneity of LTA did not appear to be due to increased carbohydrate substitution, since chloramphenicol did not alter the electrophoretic migration profile of LTA from E. hirae. From a pulse-chase study, it was determined that LTA was released as a consequence of deacylation.     

Enzymic activity of salivary amylase when bound to the surface of oral streptococci

The enzymatic activity of salivary amylase bound to the surface of several species of oral streptococci was determined by the production of acid from starch and by the degradation of maltotetraose to glucose in a coupled, spectrophotometric assay. Most strains able to bind amylase exhibited functional enzyme on their surface and produced acid from the products of amylolytic degradation. These strains were unable to utilise starch in the absence of salivary amylase. Two strains failed to produce acid from starch, despite the presence of functional salivary amylase, because they could not utilise maltose. Strains that could not bind salivary amylase failed to produce acid from starch. In no case was all the bound salivary amylase active, and two strains of Streptococcus mitis which bound amylase did not exhibit any enzyme activity on their cell surface. The ability to bind amylase may confer a survival advantage on oral bacteria which inhabit hosts that consume diets containing starch.     

Characterization of aid1, a Novel Gene Involved in Fusobacterium nucleatum Interspecies Interactions

The oral opportunistic pathogen Fusobacterium nucleatum is known to interact with a large number of different bacterial species residing in the oral cavity. It adheres to a variety of Gram-positive bacteria, including oral streptococci via the arginine-inhibitable adhesin RadD. In this study, we describe a novel protein encoded by the predicted open reading frame FN1253 that appears to play a role in interspecies interactions of F. nucleatum, particularly with oral streptococci and related Gram-positive species. We designated FN1253 as aid1 (Adherence Inducing Determinant 1). Expression analyses demonstrated that this gene was induced in F. nucleatum single species biofilms, while the presence of representative members of the oral microbiota known to adhere to F. nucleatum triggered its suppression. Inactivation as well as overexpression of aid1 affected the ability of F. nucleatum to coaggregate with oral streptococci and the closely related Enterococcus faecalis, but not other Gram-positive oral species tested. Furthermore, overexpression of aid1 led to a drastic change in the structure of dual species biofilms of F. nucleatum with oral streptococci. Aid1 function was abolished in the presence of arginine and found to be dependent on RadD. Interestingly, differential expression of aid1 did not affect messenger RNA and protein levels of RadD. These findings indicate that RadD-mediated adhesion to oral streptococci involves more complex cellular processes than the simple interaction of adhesins on the surface of partner strains. Aid1 could potentially play an important role in facilitating RadD-mediated interaction with oral streptococci by increasing binding specificity of F. nucleatum to other microbial species.     

Adherence ofStaphylococcus epidermidis to pharyngeal epithelial cells mediated by lipoteichoic acid

Prior treatment of pharyngeal epithelial cells (PEC) with lipoteichoic acid (LTA) derived fromStaphylococcus epidermidis produced a marked inhibition of adherence of the homologous strain and two heterologous strains. The inhibition was dose dependent and saturable with 100 µg/ml of LTA. However, pretreatment of PEC with deacylated LTA did not block the adherence of the three strains tested. A similar but less marked blocking effect on the adherence ofS. epidermidis to PEC was also observed with LTAs derived fromS. aureus andStreptococcus pyogenes. On treatment of bacteria with substances capable of binding to LTA, such as polyclonal mouse anti-LTA antibodies or with human albumin, a marked inhibition of bacterial adherence was observed. Immunofluorescence studies showed that anti-LTA antiserum bound readily to the surface of bacterial cells. These findings provide clear evidence that the lipid component of LTA located on the bacterial surface is centrally involved in the adherence ofS. epidermidis to human mucosal cells.     

Effect of medium composition on the susceptibility of oral streptococci to mercuric chloride

Although there is considerable interest in identifying mercury-resistant bacteria, no standardized assay exists for this purpose. In this study, the effect of the composition of the medium on the susceptibility of oral streptococci to HgCl₂ was investigated. The minimum inhibitory concentration (MIC) of HgCl₂ for 52 streptococcal strains and the reproducibility of MIC values for Hg-sensitive and Hg-resistant strains was determined with 11 different media. Addition of blood increased the MIC values, and some media (tryptone soya agar, with or without blood) could not discriminate between Hg-sensitive and Hg-resistant strains. The proportion of streptococci that appeared to be resistant to Hg was very high (>70%) on some media (mitis-salivarius, tryptone soya, Columbia), but not on others (Mueller-Hinton, Brain Heart Infusion, Isosensitest). The MICs of the control strains varied considerably on different testing occasions for tryptone soya agar (with and without blood), Isosensitest agar, and Columbia agar (with blood). Mueller-Hinton (without blood) appeared to be the most suitable medium for isolating Hg-resistant oral streptococci. Received: 21 December 2001 / Accepted: 17 January 2002     

Selectivity of Mitis Salivarius agar and a new selective medium for oral streptococci in dogs

An evaluation on the applicability of Mitis Salivarius agar (MS) medium, commonly used for the detection of oral streptococci in human and animals, to dog specimens and the development of a new selective medium for isolating streptococci from the canine oral cavity are described. Oral samples from dogs were cultured on MS medium under anaerobic conditions. The predominant facultative anaerobic bacteria on MS plates were gram-negative rods. Selectivity of streptococci on MS medium was 21.2%. A new selective medium, designated MS-CAN-AE, was developed for the isolation of streptococci from the canine oral cavity. The average growth recovery of laboratory and clinically isolated strains of streptococci on MS-CAN-AE medium was 84.1% of that on MS medium. Gram-positive rods and gram-negative rods and cocci rarely grew on the MS-CAN-AE. The selectivity of MS-CAN-AE was 95.0% for clinical samples. MS-CAN-AE medium will be helpful for investigations of streptococci in the canine oral cavity.     

In-vitro bacteriocin-mediated antagonism by oral streptococci against human carrier strains of staphylococci

All strains of oral streptococci tested and specially those of Streptococcus mutans, Strep. sanguis and Strep. mitior produced more than one distinct bacteriocin-like substance with variable inhibitory activity on 20 indicator staphylococci. Inhibitory activity was comparatively higher on nasal strains of Staph. aureus and Staph. epidermidis than on strains of both species isolated from the mouth. Nineteen of 20 staphylococcal indicators were inhibited by 1-12 of the 12 effector streptococci. Sensitivity of nasal staphylococci to bacteriocins (frequency of positive inhibitory tests and total inhibition zone diameters) was significantly higher (P less than 0.001, chi 2 test and P less than 0.05, t test respectively) than that of oral ones. The sensitivity of nasal over oral Staph. aureus (P less than 0.001 and P less than 0.01) and of oral Staph. epidermidis over oral Staph. aureus (P less than 0.01 and P less than 0.05) was also significantly higher. The evaluation of variability of inhibitory patterns of bacteriocins produced by streptococci (p-typing), of sensitivity patterns of staphylococci to bacteriocins (s-typing) and of the significantly higher sensitivity of nasal over oral staphylococci to bacteriocins from the epidemiological and ecological viewpoints are discussed.     

Surface free energies of oral streptococci

Abstract The surface free energies ( γ _b) of a variety of oral streptococci were determined from contact angle measurements on bacterial deposits, using the concept of dispersion and polar components. At least four strains of each species were tested. Strains of Streptococcus mutans, S. sanguis and S. salivarius possessed relatively high surface free energies (103 ± 12 mJ · m⁻²) and at the species level no significant difference was found. In contrast, the strains of S. mitis had remarkably low surface free energies (45 ± 14 mJ · m⁻²). S. milleri appeared to be a heterogeneous species, showing surface free energies over a range of 32–119 mJ · m⁻². No significant differences were observed between laboratory strains and strains freshly isolated from the oral cavity.     

Xylitol and Erythritol Decrease Adherence of Polysaccharide-Producing Oral Streptococci

Xylitol consumption decreases counts of mutans streptococci. However, the mechanism behind this decrease is not well understood. We studied not only type strains and clinical isolates of mutans streptococci, but also other polysaccharide-forming oral streptococci. Growth inhibition and adherence of cells to a smooth glass surface—reflecting synthesis of water-insoluble polysaccharides were studied in the presence of 2% (0.13 mol/l) and 4% (0.26 mol/l) xylitol. The effect of xylitol was compared to a novel polyol sweetener, erythritol. Except for Streptococcus mutans 10449 and S. sobrinus OMZ 176, the glass surface adhesion of most polysaccharide-forming streptococci was reduced by the presence of both 4% xylitol and erythritol. For the S. mutans and S. sobrinus type strains, the growth inhibition with 4% xylitol and erythritol was 36–77% and for the clinical S. mutans isolates 13–73%. Of the other oral streptococci, only S. sanguinis was inhibited with 4% xylitol (45–55%). For both polyols, the magnitude of the growth inhibition observed was not associated with the magnitude of the decrease in adherence (xylitol: r = −0.18; erythritol: r = 0.49). In conclusion, both xylitol and erythritol can decrease polysaccharide-mediated cell adherence contributing to plaque accumulation through a mechanism not dependent on growth inhibition.     

Surface hydrophobicity of "rheumatogenic" and "nephritogenic" strains of group A streptococci and the ultrastructural surface feature of pharyngeal cells exposed to group A streptococci.

The present study was carried out to determine the surface hydrophobicity of group A streptococcal strains responsible for rheumatic fever (RF), "rheumatogenic" strains (RG strains) and strains causing glomerulonephritis, "nephritogenic" strains (NG strains) in relation to their adhesion to human pharyngeal cells. Scanning electronmicroscopic (SEM) studies were carried out to the difference, if any, in the adherence of group A streptococci (M type 5) to pharyngeal and buccal cells (PEC and BEC). By employing two techniques for hydrophobicity determination, salt aggregation titre (SAT) and n-hexadecane binding technique, it was observed that RG strains (M5, M1 and M6) were more hydrophobic than NG strain, M49. However, NG strain M12 was almost equally as hydrophobic as RG strains. The adherence of RG strains, except M1 and M24, to PEC was greater in number than that of NG strains. Although M1 strain was hydrophobic, its adherence to PEC was less. Pepsin and trypsin treatment with streptococci reduced the hydrophobicity and adherence of RG and NG strains to PEC. SEM studies revealed firmly adhered indigenous bacteria on PEC and BEC. Streptococci (M5) adhered more to PEC than to BEC. SEM studies also showed that PEC had a peculiar ultrastructural surface feature to which streptococci adhered. These findings suggest that streptococcal hydrophobicity alone does not determine their adhesion to PEC. The surface nature of PEC might be a characteristic feature of the epithelial cells that allows streptococci to adhere and colonize or it might be a consequence of streptococcal adhesion.     

In-vitro bacteriocin-mediated antagonism by oral streptococci against human carrier strains of staphylococci

All strains of oral streptococci tested and specially those of Streptococcus mutans, Strep. sanguis and Strep. minor produced more than one distinct bacteriocin-like substance with variable inhibitory activity on 20 indicator staphylococci. Inhibitory activity was comparatively higher on nasal strains of Staph. aureus and Staph. epidermidis than on strains of both species isolated from the mouth. Nineteen of 20 staphylococcal indicators were inhibited by 1–12 of the 12 effector streptococci. Sensitivity of nasal staphylococci to bacteriocins (frequency of positive inhibitory tests and total inhibition zone diameters) was significantly higher ( P < 0·001, χ² test and P < 0·05, t test respectively) than that of oral ones. The sensitivity of nasal over oral Staph. aureus ( P < 0·001 and P < 0·01) and of oral Staph. epidermidis over oral Staph. aureus ( P < 0·01 and P < 0·05) was also significantly higher. The evaluation of variability of inhibitory patterns of bacteriocins produced by streptococci (p-typing), of sensitivity patterns of staphylococci to bacteriocins (s-typing) and of the significantly higher sensitivity of nasal over oral staphylococci to bacteriocins from the epidemiological and ecological veiwpoints are discussed.     

Lymphocytes binding and T cell mitogenic properties of group A streptococcal lipoteichoic acid.

We previously reported that lipoteichoic acid (LTA) of group A streptococci binds spontaneously to mammaliam cell membranes via lipid moieties ester-linked to the LTA molecule. We now describe biochemical and immunologic evidence that LTA binds to human and murine lymphocytes as an early event in the induction of mitogenesis in T lymphocytes. The biochemical studies showed that binding of radiolabeled LTA to lymphocytes was lymphocyte-concentration, and temperature dependent, and it reached a maximum in 15 min. Binding was reversible and specific with a dissociation constant of 89 micrometer for adult lymphocytes and 57 micrometer for cord blood lymphocytes. Immunologic studies showed that the LTA was mitogenic only for T lymphocytes. Dose response curves of lymphocyte mitogenesis induced by LTA and the binding of LTA to intact lymphocytes were shown to be related. The results suggest that LTA binds to specific receptor sites on T lymphocytes to trigger the mitogenic response.     

The hydrophobicity of 'viridans' streptococci isolated from the human mouth

The hydrophobicity of human oral streptococci was measured with the hexadecane assay modified by the incorporation of polyethylene glycol 6000. Large variability in the hydrophobicity between cultures of some strains grown on different occasions was observed whereas other strains were less variable. The variation in hydrophobicity was significantly reduced by growing the cells in continuous culture in a chemostat under glucose-limiting conditions. The Streptococcus mutans strains used all had low hydrophobicity and the mean hydrophobicity of this species was significantly lower ( P < 0.05) than the mean hydrophobicity of Strep. salivarius, Strep. sanguis Type I and Strep. sanguis Type II strains. This finding supports the view that hydrophobicity is a contributing factor in the adhesion of viridans streptococci to oral surfaces.