Purification and characterization of a Bacteroides loeschei adhesin that interacts with procaryotic and eucaryotic cells.

The adhesin of Bacteroides loeschei PK1295 that mediates coaggregation with Streptococcus sanguis 34 and hemagglutination of erythrocytes was purified to electrophoretic homogeneity. The lectinlike protein has an estimated native Mr of 450,000 and consists of six subunits of identical molecular weight (Mr 75,000). The purified adhesin appears to be a basic protein with a pI between 7.4 and 8.0. Amino acid and N-terminal sequence analyses were carried out with the purified protein. These indicated that the protein contains a large number of Asx and Glx residues as well as basic amino acid residues. The binding site of the pure adhesin retained its native configuration during purification. When preincubated with streptococcal partner cells at pH 4.6, the adhesin prevented B. loeschei cells from coaggregating with the streptococci. An adhesin preparation adjusted to a pH of 6.8 rapidly agglutinated both streptococci and neuraminidase-treated erythrocytes. Galactosides inhibited the agglutination reactions.     

Coaggregation between Prevotella nigrescens and Prevotella intermedia with Actinomyces naeslundii strains

Abstract Using a visual coaggregation assay, 43% (6 of 14) of Prevotella nigrescens and 50% (4 of 8) of Prevotella intermedia strains coaggregated with Actinomyces naeslundii strains which represented the six Actinomyces coaggregation groups (A to F). For both species, coaggregation occurred most frequently with A. naeslundii strains from coaggregation groups C, D and E. No coaggregation was observed with Actinomyces israelii , Actinomyces odontolyticus or six oral Streptococcus species. Coaggregation was not inhibited by lactose, saliva or serum. Pretreatment of Prevotella strains with heat, SDS and proteinase K abolished coaggregation when the treated cells were added to untreated Actinomyces strains. The same pretreatment of the Actinomyces strains had no effect on their ability to coaggregate with untreated Prevotella strains. Pretreatment of all coaggregating P. nigrescens strains with trypsin abolished coaggregation, whereas the coaggregation ability of the P. intermedia and Actinomyces strains was resistant to trypsin pretreatment. Pretreatment of the strains of both Prevotella species and the Actinomyces with periodate abolished coaggregation in all cases. These results suggest that the Prevotella strains each possess a protein coaggregation adhesin, which for the P. intermedia strains is resistant to trypsin, that interacts with a non-protein receptor on the A. naeslundii strains.     

Shear rate moderates community diversity in freshwater biofilms

The development of freshwater multispecies biofilms at solid-liquid interfaces occurs both in quiescent waters and under conditions of high shear rates. However, the influence of hydrodynamic shear rates on bacterial biofilm diversity is poorly understood. We hypothesized that different shear rates would significantly influence biofilm diversity and alter the relative proportions of coaggregating and autoaggregating community isolates. In order to study this hypothesis, freshwater biofilms were developed at five shear rates (<0.1 to 305 S(-1)) in a rotating concentric cylinder reactor fed with untreated potable water. Eubacterial diversity was assessed by denaturing gradient gel electrophoresis (DGGE) and culturing on R2A agar. Fifty morphologically distinct biofilm strains and 16 planktonic strains were isolated by culturing and identified by partial 16S rRNA gene sequencing, and their relatedness was determined by the construction of a neighbor-joining phylogenetic tree. Phylogenetic and DGGE analyses showed an inverse relationship between shear rate and bacterial diversity. An in vitro aggregation assay was used to assess the relative proportions of coaggregating and autoaggregating species from each biofilm. The highest proportion of autoaggregating bacteria was present at high shear rates (198 to 305 S(-1)). The intermediate shear rate (122 S(-1)) selected for the highest proportion of coaggregating bacteria (47%, or 17 of a possible 36 coaggregation interactions). Under static conditions (<0.1 S(-1)), 41 (33%) of a possible 125 coaggregation interactions were positive. Few coaggregation (3.3%) or autoaggregation (25%) interactions occurred between the 16 planktonic strains. In conclusion, these data show that shear rates affect biofilm diversity as well as the relative proportions of aggregating bacteria.     

Oral streptococcal glyceraldehyde-3-phosphate dehydrogenase mediates interaction with Porphyromonas gingivalis fimbriae

Interaction of Porphyromonas gingivalis with plaque-forming bacteria is necessary for its colonization in periodontal pockets. Participation of Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and P. gingivalis fimbriae in this interaction has been reported. In this investigation, the contribution of various oral streptococcal GAPDHs to interaction with P. gingivalis fimbriae was examined. Streptococcal cell surface GAPDH activity was measured by incubation of a constant number of streptococci with glyceraldehyde-3-phosphate and analysis for the conversion of NAD+ to NADH based on the absorbance at 340 nm. Coaggregation activity was measured by a turbidimetric assay. Cell surface GAPDH activity was correlated with coaggregation activity (r = 0.854, P < 0.01) with Spearman's rank correlation coefficient. S. oralis ATCC 9811 and ATCC 10557, Streptococcus gordonii G9B, Streptococcus sanguinis ATCC 10556, and Streptococcus parasanguinis ATCC 15909 exhibited high cell surface GAPDH activity and coaggregation activity; consequently, their cell surface GAPDHs were extracted with mutanolysin and purified on a Cibacron Blue Sepharose column. Subsequently, their DNA sequences were elucidated. Purified GAPDHs bound P. gingivalis recombinant fimbrillin by Western blot assay, furthermore, their DNA sequences displayed a high degree of homology with one another. Moreover, S. oralis recombinant GAPDH inhibited coaggregation between P. gingivalis and the aforementioned five streptococcal strains in a dose-dependent manner. These results suggest that GAPDHs of various plaque-forming streptococci may be involved in their attachment to P. gingivalis fimbriae and that they may contribute to P. gingivalis colonization.     

Isolation of a coaggregation-inhibiting cell wall polysaccharide from Streptococcus sanguis H1.

Coaggregation between Streptococcus sanguis H1 and Capnocytophaga ochracea ATCC 33596 cells is mediated by a carbohydrate receptor on the former and an adhesin on the latter. Two methods were used to release the carbohydrate receptor from the gram-positive streptococcus, autoclaving and mutanolysin treatment. The polysaccharide released from the streptococcal cell wall by either treatment was purified by ion-exchange chromatography; this polysaccharide inhibited coaggregation when preincubated with the gram-negative capnocytophaga partner. After hydrolysis of the polysaccharide by hydrofluoric acid (HF), the major oligosaccharide of the polysaccharide was purified by high-performance liquid chromatography. By analysis of the HF hydrolysis of the polysaccharide and the purified oligosaccharide, this major oligosaccharide appeared to be the repeating unit of the polysaccharide, with minor components resulting from internal hydrolysis of the major oligosaccharide. Gas chromatography results showed that the oligomer was a hexasaccharide, consisting of rhamnose, galactose, and glucose, in the ratio of 2:3:1, respectively. By weight, the purified hexasaccharide was a fourfold-more-potent inhibitor of coaggregation than the native polysaccharide. Resistance to hydrolysis by sulfuric acid alone and susceptibility to hydrolysis by HF suggested that oligosaccharide chains of the polysaccharide are linked by phosphodiester bonds. Studies with a coaggregation-defective mutant of S. sanguis H1 revealed that the cell walls of the mutant contained neither the polysaccharide nor the hexasaccharide repeating unit. The purification of both a polysaccharide and its constituent hexasaccharide repeating unit, which both inhibited coaggregation, and the absence of this polysaccharide or hexasaccharide on a coaggregation-defective mutant strongly suggest that the hexasaccharide derived from the polysaccharide functions as the receptor for the adhesin from C. ochracea ATCC 33596.     

Streptococcal IgA-binding proteins bind in the Calpha 2-Calpha 3 interdomain region and inhibit binding of IgA to human CD89

Certain pathogenic bacteria express surface proteins that bind to the Fc part of human IgA or IgG. These bacterial proteins are important as immunochemical tools and model systems, but their biological function is still unclear. Here, we describe studies of three streptococcal proteins that bind IgA: the Sir22 and Arp4 proteins of Streptococcus pyogenes and the unrelated beta protein of group B streptococcus. Analysis of IgA domain swap and point mutants indicated that two loops at the Calpha2/Calpha3 domain interface are critical for binding of the streptococcal proteins. This region is also used in binding the human IgA receptor CD89, an important mediator of IgA effector function. In agreement with this finding, the three IgA-binding proteins and a 50-residue IgA-binding peptide derived from Sir22 blocked the ability of IgA to bind CD89. Further, the Arp4 protein inhibited the ability of IgA to trigger a neutrophil respiratory burst via CD89. Thus, we have identified residues on IgA-Fc that play a key role in binding of different streptococcal IgA-binding proteins, and we have identified a mechanism by which a bacterial IgA-binding protein may interfere with IgA effector function.     

Probing of Microbial Biofilm Communities for Coadhesion Partners

Investigations of interbacterial adhesion in dental plaque development are currently limited by the lack of a convenient assay to screen the multitude of species present in oral biofilms. To overcome this limitation, we developed a solid-phase fluorescence-based screening method to detect and identify coadhesive partner organisms in mixed-species biofilms. The applicability of this method was demonstrated using coaggregating strains of type 2 fimbrial adhesin-bearing actinomyces and receptor polysaccharide (RPS)-bearing streptococci. Specific adhesin/receptor-mediated coadhesion was detected by overlaying bacterial strains immobilized to a nitrocellulose membrane with a suspended, fluorescein-labeled bacterial partner strain. Coadhesion was comparable regardless of which cell type was labeled and which was immobilized. Formaldehyde treatment of bacteria, either in suspension or immobilized on nitrocellulose, abolished actinomyces type 2 fimbrial adhesin but not streptococcal RPS function, thereby providing a simple method for assigning complementary adhesins and glycan receptors to members of a coadhering pair. The method''s broader applicability was shown by overlaying colony lifts of dental plaque biofilm cultures with fluorescein-labeled strains of type 2 fimbriated Actinomyces naeslundii or RPS-bearing Streptococcus oralis. Prominent coadhesion partners included not only streptococci and actinomyces, as expected, but also other bacteria not identified in previous coaggregation studies, such as adhesin- or receptor-bearing strains of Neisseria pharyngitis, Rothia dentocariosa, and Kingella oralis. The ability to comprehensively screen complex microbial communities for coadhesion partners of specific microorganisms opens a new approach in studies of dental plaque and other mixed-species biofilms.     

Aggregative behavior of bacteria isolated from canine dental plaque

Interbacterial adhesion of bacteria isolated from canine dental plaque was assessed by performing a visual coaggregation assay. Using conditions mimicking those likely to be encountered in vivo, the entire cultivable plaque microbiota from a single dog was assessed, and eight (6.7%) unique coaggregation interactions were detected for 120 crosses. Transmission electron microscopy was used to visualize several of the bacteria in isolation and as coaggregates, which revealed surface structures that may be involved in adhesion and coaggregation. The results of this study indicate that the prevalence of coaggregating pairs of dental plaque bacteria in dogs is similar to the prevalence of coaggregating pairs of dental plaque bacteria reported in humans. In addition, genera found in both hosts generally exhibited similar coaggregation reactions; however, autoaggregation was found to be more common among oral bacteria isolated from dogs.     

A calorimetric investigation of the interaction of the lac repressor with inducer

A calorimetric study has been made of the interaction between the lac repressor and isopropyl-1-thio-beta-D-galactopyranoside (IPTG). The buffer-corrected enthalpy of reaction at 25 degrees C was found to be -15.6, -24.7, -4.6 kJ/mol of bound IPTG at pH 7.0, pH 8.1, and pH 9.0, respectively. This large range of enthalpy values is in contrast to a maximum difference in the free energy of the reaction of only 1.5 kJ/mol of bound IPTG between these pH values. The reaction was found by calorimetric measurements in different buffers to be accompanied by an uptake of 0.29 mol of protons/mol of bound IPTG at pH 8.1. The pH dependency of the reaction enthalpy suggests differences in the extent of protonation of the binding site and the involvement of H bonding with IPTG. The lack of strong hydrophobic contributions in the IPTG binding process is revealed by the absence of any determinable heat capacity change for the reaction at pH 7.0. The presence of phosphate buffer significantly alters the enthalpy of IPTG binding at higher pH values, but has little effect upon the binding constant. This implies that highly negative phosphate species change the nature of the IPTG binding site without any displacement of phosphate upon IPTG binding.     

Structural relationship between the enzymatic and streptococcal binding sites of human salivary alpha-amylase

Previous studies have demonstrated that human salivary alpha-amylase specifically binds to the oral bacterium Streptococcus gordonii. This interaction is inhibited by substrates such as starch and maltotriose suggesting that bacterial binding may involve the enzymatic site of amylase. Experiments were performed to determine if amylase bound to the bacterial surface possessed enzymatic activity. It was found that over one-half of the bound amylase was enzymatically active. In addition, bacterial-bound amylase hydrolyzed starch to glucose which was then metabolized to lactic acid by the bacteria. In further studies, the role of amylase's histidine residues in streptococcal binding and enzymatic function was assessed after their selective modification with diethyl pyrocarbonate. DEP-modified amylase showed a marked reduction in both enzymatic and streptococcal binding activities. These effects were diminished when DEP modification occurred in the presence of maltotriose. DEP-modified amylase had a significantly altered secondary structure when compared with native enzyme or amylase modified in the presence of maltotriose. Collectively, these results suggest that human salivary alpha-amylase may possess multiple sites for bacterial binding and enzymatic activity which share structural similarities.     

Shear Rate Moderates Community Diversity in Freshwater Biofilms

The development of freshwater multispecies biofilms at solid-liquid interfaces occurs both in quiescent waters and under conditions of high shear rates. However, the influence of hydrodynamic shear rates on bacterial biofilm diversity is poorly understood. We hypothesized that different shear rates would significantly influence biofilm diversity and alter the relative proportions of coaggregating and autoaggregating community isolates. In order to study this hypothesis, freshwater biofilms were developed at five shear rates (<0.1 to 305 S⁻¹) in a rotating concentric cylinder reactor fed with untreated potable water. Eubacterial diversity was assessed by denaturing gradient gel electrophoresis (DGGE) and culturing on R2A agar. Fifty morphologically distinct biofilm strains and 16 planktonic strains were isolated by culturing and identified by partial 16S rRNA gene sequencing, and their relatedness was determined by the construction of a neighbor-joining phylogenetic tree. Phylogenetic and DGGE analyses showed an inverse relationship between shear rate and bacterial diversity. An in vitro aggregation assay was used to assess the relative proportions of coaggregating and autoaggregating species from each biofilm. The highest proportion of autoaggregating bacteria was present at high shear rates (198 to 305 S⁻¹). The intermediate shear rate (122 S⁻¹) selected for the highest proportion of coaggregating bacteria (47%, or 17 of a possible 36 coaggregation interactions). Under static conditions (<0.1 S⁻¹), 41 (33%) of a possible 125 coaggregation interactions were positive. Few coaggregation (3.3%) or autoaggregation (25%) interactions occurred between the 16 planktonic strains. In conclusion, these data show that shear rates affect biofilm diversity as well as the relative proportions of aggregating bacteria.     

Aggregative Behavior of Bacteria Isolated from Canine Dental Plaque

Interbacterial adhesion of bacteria isolated from canine dental plaque was assessed by performing a visual coaggregation assay. Using conditions mimicking those likely to be encountered in vivo, the entire cultivable plaque microbiota from a single dog was assessed, and eight (6.7%) unique coaggregation interactions were detected for 120 crosses. Transmission electron microscopy was used to visualize several of the bacteria in isolation and as coaggregates, which revealed surface structures that may be involved in adhesion and coaggregation. The results of this study indicate that the prevalence of coaggregating pairs of dental plaque bacteria in dogs is similar to the prevalence of coaggregating pairs of dental plaque bacteria reported in humans. In addition, genera found in both hosts generally exhibited similar coaggregation reactions; however, autoaggregation was found to be more common among oral bacteria isolated from dogs.     

Fibronectin binding to a Streptococcus pyogenes strain.

In previous studies, Staphylococcus aureus has been shown to bind fibronectin (P. Kuusela, Nature (London) 276:718-720, 1978), an interaction that may be important in bacterial attachment and opsonization. Recently some strains of streptococci of serological groups A, C, and G were also found to bind fibronectin. The binding to one selected strain of Streptococcus pyogenes has been characterized here. The binding of [125I]fibronectin to streptococcal cells resembles that to staphylococcal cells and was found to be time dependent, functionally irreversible, and specific in the sense that unlabeled proteins other than fibronectin did not block binding. Bacteria incubated with proteases largely lost their ability to bind fibronectin, and material released from the streptococci by a brief trypsin digestion contained active fibronectin receptors. This material inhibited the binding of [125I]fibronectin to the streptococci. The inhibitory activity was adsorbed on a column of fibronectin-Sepharose but not on a column of unsubstituted Sepharose 4B or egg albumin Sepharose. The receptor appeared to be a protein nature since the inhibitory activity of the trypsinate was destroyed by papain and was not absorbed on a column containing monoclonal antibodies directed against lipoteichoic acid bound to protein A-Sepharose. Binding sites in fibronectin for streptococci and staphylococci, respectively, were localized by analyzing the ability of isolated fragments to inhibit [125I]fibronectin binding to bacteria and by adsorbing 125I-labeled tryptic fragments with staphylococcal and streptococcal cells. Both species of bacteria appeared to preferentially bind a fragment (Mr = approximately 25,000) originating from the N-terminal region of the protein. In addition, streptococci also bound a slightly smaller fragment (Mr = approximately 23,000). Fibronectin receptors solubilized from either streptococci or staphylococci inhibited the binding of fibronectin to both species of bacteria.     

Coaggregation of urogenital bacteria in vitro and in vivo

The working hypotheses of the present study were that (1) bacterial coaggregates exist in the urogenital tract of healthy and infected women, and (2) coaggregation reactions can occur in vitro between members of the urogenital flora. Examination of urogenital specimens from 25 healthy women showed that lactobacilli were the dominant organisms colonizing the epithelia and coaggregating with other Gram-positive and Gram-negative bacteria. In vitro light and electron microscopic studies confirmed that members of the urogenital flora could coaggregate. An examination of specimens from 9 women with urinary tract infection showed the presence of autoaggregated uropathogens free-floating in the urine and attached to epithelial cells. The phenomenon of autoaggregation was also noted in vitro for various uropathogens, suggestive that this may represent a virulence factor. It is evident that bacterial cell-to-cell binding within a strain and among different genera occurs in the urogenital tract. Further studies of the mechanisms that maintain and disrupt these microbial interactions will help to improve our understanding of disease initiation.     

Characterization of a Streptococcus sp.-Veillonella sp. community micromanipulated from dental plaque

Streptococci and veillonellae occur in mixed-species colonies during formation of early dental plaque. One factor hypothesized to be important in assembly of these initial communities is coaggregation (cell-cell recognition by genetically distinct bacteria). Intrageneric coaggregation of streptococci occurs when a lectin-like adhesin on one streptococcal species recognizes a receptor polysaccharide (RPS) on the partner species. Veillonellae also coaggregate with streptococci. These genera interact metabolically; lactic acid produced by streptococci is a carbon source for veillonellae. To transpose these interactions from undisturbed dental plaque to an experimentally tractable in vitro biofilm model, a community consisting of RPS-bearing streptococci juxtaposed with veillonellae was targeted by quantum dot-based immunofluorescence and then micromanipulated off the enamel surface and cultured. Besides the expected antibody-reactive cell types, a non-antibody-reactive streptococcus invisible during micromanipulation was obtained. The streptococci were identified as Streptococcus oralis (RPS bearing) and Streptococcus gordonii (adhesin bearing). The veillonellae could not be cultivated; however, a veillonella 16S rRNA gene sequence was amplified from the original isolation mixture, and this sequence was identical to the sequence of the previously studied organism Veillonella sp. strain PK1910, an oral isolate in our culture collection. S. oralis coaggregated with S. gordonii by an RPS-dependent mechanism, and both streptococci coaggregated with PK1910, which was used as a surrogate during in vitro community reconstruction. The streptococci and strain PK1910 formed interdigitated three-species clusters when grown as a biofilm using saliva as the nutritional source. PK1910 grew only when streptococci were present. This study confirms that RPS-mediated intrageneric coaggregation occurs in the earliest stages of plaque formation by bringing bacteria together to create a functional community.     

Effects of trifluoperazine on calcium binding by calmodulin. A microcalorimetric study

Microcalorimetric titrations of calmodulin with Ca2+ and trifluoperazine (TFP) at various molar ratios have been carried out at 25 degrees C and at pH 7.0. Ca2+ binding to calmodulin produces heat (-delta H) in the presence of TFP, while heat is absorbed in the absence of TFP. The total heat produced by Ca2+ binding to all four sites is increased at increasing TFP-to-calmodulin ratios, attaining a plateau at about 7. These results indicate that at the higher ratios, the enthalpy changes (delta H) associated with Ca2+ binding are affected by TFP molecules bound at both high- and low-affinity sites. In addition, the Ca2+ binding reaction of the calmodulin-TFP complex is driven solely by a favorable enthalpy change of -27 kJ/mol of site; the entropy change (delta S) is -35 J/mol/K. These thermodynamic changes are opposite to those for TFP-free calmodulin and distinctly different from other Ca2+ binding proteins such as skeletal and cardiac troponin C and parvalbumin, where the reaction is driven by favorable changes of entropy as well as enthalpy.     

Coaggregation between aquatic bacteria is mediated by specific-growth-phase-dependent lectin-saccharide interactions

Coaggregating strains of aquatic bacteria were identified by partial 16S rRNA gene sequencing. The coaggregation abilities of four strains of Blastomonas natatoria and one strain of Micrococcus luteus varied with culture age but were always maximum in the stationary phase of growth. Each member of a coaggregating pair carried either a heat- and protease-sensitive protein (lectin) adhesin or a saccharide receptor, as coaggregation was reversed by sugars.     

Is plasminogen deployed as a Streptococcus pyogenes virulence factor?

Streptococcus pyogenes (group A streptococcus) causes human skin and throat infections as well as highly invasive diseases including necrotizing fasciitis. Group A streptococcal infections and invasive disease have made a resurgence in developed countries during the past two decades. S. pyogenes use multiple pathways for the acquisition and activation of human plasminogen, securing potent proteolytic activity on the bacterial cell surface. Recent experimental evidence using a humanized transgenic mouse model suggests a crucial role for human plasminogen in the dissemination of S. pyogenes in vivo.     

The SpeB virulence factor of Streptococcus pyogenes, a multifunctional secreted and cell surface molecule with strepadhesin, laminin-binding and cysteine protease activity

The interactions between pathogenic bacteria and the host need to be resolved at the molecular level in order to develop novel vaccines and drugs. We have previously identified strepadhesin, a novel glycoprotein-binding activity in Streptococcus pyogenes, which is regulated by Mga, a regulator of streptococcal virulence factors. We have now identified the protein responsible for the strepadhesin activity and find that (i) strepadhesin activity is carried by SpeB, streptococcal pyrogenic exotoxin with cysteine protease activity; (ii) SpeB carries laminin-binding activity of the bacteria; and (iii) SpeB is not only a secreted molecule but also occurs unexpectedly tightly bound to the bacterial cell surface. Thus, in contrast to the previous view of SpeB as mainly an extracellular protease, it is also present as a streptococcal surface molecule with binding activity to laminin and other glycoproteins.     

Acquisition of complement factor H is important for pathogenesis of Streptococcus pyogenes infections: evidence from bacterial in vitro survival and human genetic association

Streptococcus pyogenes (or group A streptococcus [GAS]) is a major human pathogen causing infections, such as tonsillitis, erysipelas, and sepsis. Several GAS strains bind host complement regulator factor H (CFH) via its domain 7 and, thereby, evade complement attack and C3b-mediated opsonophagocytosis. Importance of CFH binding for survival of GAS has been poorly studied because removal of CFH from plasma or blood causes vigorous complement activation, and specific inhibitors of the interaction have not been available. In this study, we found that activation of human complement by different GAS strains (n = 38) correlated negatively with binding of CFH via its domains 5-7. The importance of acquisition of host CFH for survival of GAS in vitro was studied next by blocking the binding with recombinant CFH5-7 lacking the regulatory domains 1-4. Using this fragment in full human blood resulted in death or radically reduced multiplication of all of the studied CFH-binding GAS strains. To study the importance of CFH binding in vivo (i.e., for pathogenesis of streptococcal infections), we used our recent finding that GAS binding to CFH is diminished in vitro by polymorphism 402H, which is also associated with age-related macular degeneration. We showed that allele 402H is suggested to be associated with protection from erysipelas (n = 278) and streptococcal tonsillitis (n = 209) compared with controls (n = 455) (p < 0.05). Taken together, the bacterial in vitro survival data and human genetic association revealed that binding of CFH is important for pathogenesis of GAS infections and suggested that inhibition of CFH binding can be a novel therapeutic approach in GAS infections.