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Use of a High-throughput <em>In Vitro</em> Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Newcastle University

The Sca permease in the oral bacterium Streptococcus gordonii is a member of a family of ATP-binding cassette (ABC)-type transporters for manganese (Mn(2+)) and related cations that are associated with streptococcal virulence in a number of infection models. Since Mn(2+) has a protective function against oxidative damage in a variety of bacteria, we have investigated the role of Sca permease in oxidative stress tolerance in Streptococcus gordonii. A single Mn(2+)-dependent superoxide dismutase (SOD), encoded by sodA, is expressed by S. gordonii and was >10-fold up-regulated under oxidative stress conditions. Inactivation of sodA resulted in increased susceptibility of S. gordonii cells to growth inhibition by dioxygen (O(2)), and to killing by paraquat (a superoxide anion generator) and by hydrogen peroxide (H(2)O(2)). Expression of thiol peroxidase, encoded by the tpx gene located immediately downstream of the scaCBA operon, was also up-regulated under oxidative conditions. Inactivation of tpx led to increased susceptibility of cells to H(2)O(2), but not to O(2) or paraquat. In low-Mn(2+) medium (0.01 micro M Mn(2+)) sodA and tpx genes were transcriptionally down-regulated, SOD activity was reduced and cells were more sensitive to growth inhibition by O(2). A Sca permease-deficient (scaC) mutant showed further reduced SOD activity and hypersensitivity to O(2) in medium containing <0.1 micro M Mn(2+). These results demonstrate that the Sca (Mn(2+)) permease in S. gordonii is essential for protection against oxidative stress.

Oxidative stress tolerance is manganese (Mn(2+)) regulated in Streptococcus gordonii.

There is increasing recognition of the emerging role of manganese regulation and acquisition in some pathogenic bacteria. Expression of the Enterococcus faecalis endocarditis-associated virulence factor EfaA is induced by growth in serum. It is demonstrated here that expression of the efaCBA operon encoding a putative ABC-type transporter is regulated by Mn(2+). Transcription of efaCBA and EfaA production were repressed in Mn(2+)-supplemented medium. A Mn(2+)-responsive transcriptional regulator, EfaR, sharing 27 % identity with the Corynebacterium diphtheriae diphtheria toxin repressor (DtxR), was identified. In the presence of Mn(2+), EfaR protein bound in vitro to the efaC promoter region. Analysis of the E. faecalis V583 genome revealed ten additional putative EfaR-binding sites, suggesting that manganese availability could have a broader regulatory role in infection. The results identify a new Mn(2+)-sensing regulator in enterococci that regulates the expression of a virulence factor implicated in enterococcal endocarditis.

Manganese-dependent regulation of the endocarditis-associated virulence factor EfaA of Enterococcus faecalis.

Microbial interactions with host molecules, and programmed responses to host environmental stimuli, are critical for colonization and initiation of pathogenesis. Bacteria of the genus Streptococcus are primary colonizers of the human mouth. They express multiple cell-surface adhesins that bind salivary components and other oral bacteria and enable the development of polymicrobial biofilms associated with tooth decay and periodontal disease. However, the mechanisms by which streptococci invade dentine to infect the tooth pulp and periapical tissues are poorly understood. Here we show that production of the antigen I/II (AgI/II) family polypeptide adhesin and invasin SspA in Streptococcus gordonii is specifically upregulated in response to a collagen type I signal, minimally the tri-peptide Gly-Pro-Xaa (where Xaa is hydroxyproline or alanine). Increased AgI/II polypeptide expression promotes bacterial adhesion and extended growth of streptococcal cell chains along collagen type I fibrils that are characteristically found within dentinal tubules. These observations define a new model of host matrix signal-induced tissue penetration by bacteria and open the way for novel therapy opportunities for oral invasive diseases.

Host collagen signal induces antigen I/II adhesin and invasin gene expression in oral Streptococcus gordonii.

The antigen I/II (AgI/II) family polypeptides, ranging from 1310 to 1653 amino acid (aa) residues, are cell wall anchored adhesins expressed by most indigenous species of oral streptococci. The polypeptides interact with a wide range of host molecules, in particular salivary agglutinin glycoprotein (SAG or gp340), and with ligands on other oral bacteria. To determine the receptor recognition properties of six different AgI/II family polypeptides from strains of Streptococcus gordonii, Streptococcus intermedius and Streptococcus mutans, the genes were cloned and expressed on the surface of the surrogate host Lactococcus lactis. The S. gordonii SspA and SspB polypeptides mediated higher binding levels of L. lactis cells to surface immobilized gp340 than did S. intermedius Pas protein, or S. mutans SpaP or PAc proteins. However, the AgI/II proteins were all similar in their abilities to mediate aggregation of lactococci by fluid phase gp340. The SpaP(I) polypeptide from S. mutans Ingbritt, which was C-terminally truncated by approximately 400 aa residues, did not bind gp340. Lactococci expressing AgI/II proteins, including SpaP(I), were aggregated by a synthetic 16 aa residue peptide SRCRP2 derived from the aa repeat block sequences within gp340. In coaggregation assays, SspB from S. gordonii was unique in mediating coaggregation with only group A and group E strains of Actinomyces naeslundii. All the other AgI/II polypeptides mediated coaggregation with group C and group D strains of A. naeslundii. Analysis of chimeric protein constructs revealed that coaggregation specificity was determined by sequences within the N-terminal half of AgI/II protein. A synthetic peptide (20 aa residues), which defines a putative adhesion epitope within the C-terminal region of polypeptide, inhibited AgI/II-mediated aggregation by gp340 but did not affect coaggregation with A. naeslundii. These results suggest that different mechanisms operate in interactions of AgI/II family polypeptides with native gp340, gp340 SRCR domain peptide, and A. naeslundii. Specificity of these interactions appears to be determined by discontinuous but interacting regions of the polypeptides, thus providing flexibility in receptor recognition for streptococcal colonization of the human host.

Differential binding specificities of oral streptococcal antigen I/II family adhesins for human or bacterial ligands.

Streptococcus gordonii colonizes multiple sites within the human oral cavity. This colonization depends upon the initial interactions of streptococcal adhesins with host receptors. The adhesins that bind salivary agglutinin glycoprotein (gp340) and human cell surface receptors include the antigen I/II (AgI/II) family polypeptides SspA and SspB and a sialic acid-binding surface protein designated Hsa or GspB. In this study we determined the relative functions of the AgI/II polypeptides and Hsa in interactions of S. gordonii DL1 (Challis) with host receptors. For an isogenic mutant with the sspA and sspB genes deleted the levels of adhesion to surface-immobilized gp340 were reduced 40%, while deletion of the hsa gene alone resulted in >80% inhibition of bacterial cell adhesion to gp340. Adhesion of S. gordonii DL1 cells to gp340 was sialidase sensitive, verifying that Hsa has a major role in mediating sialic acid-specific adhesion to gp340. Conversely, aggregation of S. gordonii cells by fluid-phase gp340 was not affected by deletion of hsa but was eliminated by deletion of the sspA and sspB genes. Deletion of the AgI/II polypeptide genes had no measurable effect on hsa mRNA levels or Hsa surface protein expression, and deletion of hsa did not affect AgI/II polypeptide expression. Further analysis of mutant phenotypes showed that the Hsa and AgI/II proteins mediated adhesion of S. gordonii DL1 to human HEp-2 epithelial cells. Hsa was also a principal streptococcal cell surface component promoting adhesion of human platelets to immobilized streptococci, but Hsa and AgI/II polypeptides acted in concert in mediating streptococcal cell-platelet aggregation. The results suggest that Hsa directs primary adhesion events for S. gordonii DL1 (Challis) with immobilized gp340, epithelial cells, and platelets. AgI/II polypeptides direct gp340-mediated aggregation, facilitate multimodal interactions necessary for platelet aggregation, and modulate S. gordonii-host engagements into biologically productive phenomena.

Functions of cell surface-anchored antigen I/II family and Hsa polypeptides in interactions of Streptococcus gordonii with host receptors.

Bacterial interactions and successions during plaque development.

Streptococcus gordonii colonization of damaged heart surfaces in infective endocarditis is dependent upon the recognition of host receptors by specific bacterial surface proteins. However, despite several attempts to identify the mechanisms involved in this interaction, the nature of the bacterial proteins required remains poorly understood. This study provides clear evidence that several S. gordonii surface proteins participate in the interaction with platelets to support platelet adhesion and induce platelet aggregation. S. gordonii strains were found to support strong (DL1-Challis, SK12, SK184, and Blackburn) or moderate (UB1545 delta hsa and CH1-Challis) adhesion or failed to support platelet adhesion (M5, M99, and Channon). In addition, under flow conditions, platelets rolled and subsequently adhered to immobilized S. gordonii at low shear (50 s(-1)) in an Hsa-dependent manner but did not interact with S. gordonii DL1 at any shear rate of >50 s(-1). S. gordonii strains either induced (DL1-Challis, SK12, SK184, UB1545 delta hsa, and M99) or failed to induce (M5, CH1-Challis, Channon, and Blackburn) platelet aggregation. Using a proteomic approach to identify differential cell wall protein expression between aggregating (DL1) and nonaggregating (Blackburn) strains, we identified antigen I/antigen II family proteins SspA and SspB. The overexpression of SspA or SspB in platelet-nonreactive Lactococcus lactis induced GPIIb/GPIIIa-dependent platelet aggregation similar to that seen with S. gordonii DL1. However, they failed to support platelet adhesion. Thus, S. gordonii has distinct mechanisms for supporting platelet adhesion and inducing platelet aggregation. Differential protein expression between strains may be important for the pathogenesis of invasive diseases such as infective endocarditis.

Role of Streptococcus gordonii surface proteins SspA/SspB and Hsa in platelet function.

Interactions involving genetically distinct bacteria, for example, between oral streptococci and actinomyces, are central to dental plaque development. A DNA microarray identified Streptococcus gordonii genes regulated in response to coaggregation with Actinomyces naeslundii. The expression of 23 genes changed >3-fold in coaggregates, including that of 9 genes involved in arginine biosynthesis and transport. The capacity of S. gordonii to synthesize arginine was assessed using a chemically defined growth medium. In monoculture, streptococcal arginine biosynthesis was inefficient and streptococci could not grow aerobically at low arginine concentrations. In dual-species cultures containing coaggregates, however, S. gordonii grew to high cell density at low arginine concentrations. Equivalent cocultures without coaggregates showed no growth until coaggregation was evident (9 h). An argH mutant was unable to grow at low arginine concentrations with or without A. naeslundii, indicating that arginine biosynthesis was essential for coaggregation-induced streptococcal growth. Using quantitative reverse transcriptase PCR, the expression of argC, argG, and pyrA(b) was strongly (10- to 100-fold) up-regulated in S. gordonii monocultures after 3 h of growth when exogenous arginine was depleted. Cocultures without induced coaggregation showed similar regulation. However, within 1 h after coaggregation with A. naeslundii, the expression of argC, argG, and pyrA(b) in S. gordonii was partially up-regulated although arginine was plentiful, and mRNA levels did not increase further when arginine was diminished. Thus, A. naeslundii stabilizes S. gordonii expression of arginine biosynthesis genes in coaggregates but not cocultures and enables aerobic growth when exogenous arginine is limited.

Regulation of gene expression in a mixed-genus community: stabilized arginine biosynthesis in Streptococcus gordonii by coaggregation with Actinomyces naeslundii.

In dental plaque alpha-haemolytic streptococci, including Streptococcus gordonii, are considered beneficial for oral health. These organisms produce hydrogen peroxide (H(2)O(2)) at concentrations sufficient to kill many oral bacteria. Streptococci do not produce catalase yet tolerate H(2)O(2). We recently demonstrated that coaggregation with Actinomyces naeslundii stabilizes arginine biosynthesis in S. gordonii. Protein arginine residues are sensitive to oxidation by H(2)O(2). Here, the ability of A. naeslundii to protect S. gordonii against self-produced H(2)O(2) was investigated. Coaggregation with A. naeslundii enabled S. gordonii to grow in the absence of arginine, and promoted survival of S. gordonii following growth with or without added arginine. Arginine-replete S. gordonii monocultures contained 20-30 microM H(2)O(2) throughout exponential growth. Actinomyces naeslundii did not produce H(2)O(2) but synthesized catalase, removed H(2)O(2) from coaggregate cultures and decreased protein oxidation in S. gordonii. On solid medium, S. gordonii inhibited growth of A. naeslundii; exogenous catalase overcame this inhibition. In coaggregate cultures, A. naeslundii cell numbers were >90% lower than in monocultures after 24 h. These results indicate that coaggregation with A. naeslundii protects S. gordonii from oxidative damage. However, high cell densities of S. gordonii inhibit A. naeslundii. Therefore, H(2)O(2) may drive these organisms towards an ecologically balanced community in natural dental plaque.

Role of hydrogen peroxide in competition and cooperation between Streptococcus gordonii and Actinomyces naeslundii.

The ability to control intracellular levels of transition metals such as Mn(2+), Fe(2+) and Zn(2+) is critical for the virulence of many pathogenic bacteria. In this issue of Molecular Microbiology, Rosch et al. describe the first identification of a Mn(2+) efflux system in bacteria, MntE of Streptococcus pneumoniae, and demonstrate that it is required for virulence in an animal model. Disruption of the mntE gene leads to widespread transcriptional changes that are distinct from responses to extracellular Mn(2+). These findings reveal, for the first time, that a bacterial trace metal efflux system plays a role in disease. Thus, MntE represents a new lead for the development of antimicrobials specifically aimed at disrupting microbial metal ion homeostasis.

A new direction for manganese homeostasis in bacteria: identification of a novel efflux system in Streptococcus pneumoniae.

Adhesion of bacterial cells to fibronectin (FN) is thought to be a pivotal step in the pathogenesis of invasive infectious diseases. Viridans group streptococci such as Streptococcus gordonii are considered commensal members of the oral microflora, but are important pathogens in infective endocarditis. S. gordonii expresses a battery of cell-surface adhesins that act alone or in concert to bind host receptors. Here, we employed molecular genetic approaches to determine the relative contributions of five known S. gordonii surface proteins to adherence to human FN. Binding levels to FN by isogenic mutants lacking Hsa glycoprotein were reduced by 70 %, while mutants lacking CshA and CshB fibrillar proteins showed approximately 30 % reduced binding. By contrast, disruption of antigen I/II adhesin genes sspA and sspB in a wild-type background did not result in reduced FN binding. Enzymic removal of sialic acids from FN led to reduced S. gordonii DL1 adhesion (>50 %), but did not affect binding by the hsa mutant, indicating that Hsa interacts with sialic acid moieties on FN. Conversely, desialylation of FN did not affect adherence levels of Lactococcus lactis cells expressing SspA or SspB polypeptides. Complementation of the hsa mutant partially restored adhesion to FN. A model is proposed for FN binding by S. gordonii in which Hsa and CshA/CshB are primary adhesins, and SspA or SspB play secondary roles. Understanding the basis of oral streptococcal interactions with FN will provide a foundation for development of new strategies to control infective endocarditis.

Multiple adhesin proteins on the cell surface of Streptococcus gordonii are involved in adhesion to human fibronectin.

The streptococcal coaggregation regulator (ScaR) of Streptococcus gordonii is a manganese-dependent transcriptional regulator. When intracellular manganese concentrations become elevated, ScaR represses transcription of the scaCBA operon, which encodes a manganese uptake transporter. A member of the DtxR/MntR family of metalloregulators, ScaR shares sequence similarity with other family members, and many metal-binding residues are conserved. Here, we show that ScaR is an active dimer, with two dimers binding the 46 base pair scaC operator. Each ScaR subunit binds two manganese ions, and the protein is activated by a variety of other metal ions, including Cd(2+), Co(2+), and Ni(2+) but not Zn(2+). The crystal structure of apo-ScaR reveals a tertiary and quaternary structure similar to its homologue, the iron-responsive regulator DtxR. While each DtxR subunit binds a metal ion in two sites, labeled primary and ancillary, crystal structures of ScaR determined in the presence of Cd(2+) and Zn(2+) show only a single occupied metal-binding site that is novel to ScaR. The site analogous to the primary site in DtxR is unoccupied, and the ancillary site is absent from ScaR. Instead, metal ions bind to ScaR at a site labeled "secondary", which is composed of Glu80, Cys123, His125, and Asp160 and lies roughly 5 A away from where the ancillary site would be predicted to exist. This difference suggests that ScaR and its closely related homologues are activated by a mechanism distinct from that of either DtxR or MntR.

Characterization and structure of the manganese-responsive transcriptional regulator ScaR.

Growth of oral bacteria in situ requires adhesion to a surface because the constant flow of host secretions thwarts the ability of planktonic cells to grow before they are swallowed. Therefore, oral bacteria evolved to form biofilms on hard tooth surfaces and on soft epithelial tissues, which often contain multiple bacterial species. Because these biofilms are easy to study, they have become the paradigm of multispecies biofilms. In this Review we describe the factors involved in the formation of these biofilms, including the initial adherence to the oral tissues and teeth, cooperation between bacterial species in the biofilm, signalling between the bacteria and its role in pathogenesis, and the transfer of DNA between bacteria. In all these aspects distance between cells of different species is integral for oral biofilm growth.

Oral multispecies biofilm development and the key role of cell-cell distance.

The persistent colonization of paranasal sinus mucosa by microbial biofilms is a major factor in the pathogenesis of chronic rhinosinusitis (CRS). Control of microorganisms within biofilms is hampered by the presence of viscous extracellular polymers of host or microbial origin, including nucleic acids. The aim of this study was to investigate the role of extracellular DNA in biofilm formation by bacteria associated with CRS.

Efficacy of a marine bacterial nuclease against biofilm forming microorganisms isolated from chronic rhinosinusitis.

It is now clear that the most common oral diseases, dental caries and periodontitis, are caused by mixed-species communities rather than by individual pathogens working in isolation. Oral streptococci are central to these disease processes since they are frequently the first microorganisms to colonize oral surfaces and they are numerically the dominant microorganisms in the human mouth. Numerous interactions between oral streptococci and other bacteria have been documented. These are thought to be critical for the development of mixed-species oral microbial communities and for the transition from oral health to disease. Recent metagenomic studies are beginning to shed light on the co-occurrence patterns of streptococci with other oral bacteria. Refinements in microscopy techniques and biofilm models are providing detailed insights into the spatial distribution of streptococci in oral biofilms. Targeted genetic manipulation is increasingly being applied for the analysis of specific genes and networks that modulate interspecies interactions. From this work, it is clear that streptococci produce a range of extracellular factors that promote their integration into mixed-species communities and enable them to form social networks with neighboring taxa. These "community integration factors" include coaggregation-mediating adhesins and receptors, small signaling molecules such as peptides or autoinducer-2, bacteriocins, by-products of metabolism including hydrogen peroxide and lactic acid, and a range of extracellular enzymes. Here, we provide an overview of various types of community interactions between oral streptococci and other microorganisms, and we consider the possibilities for the development of new technologies to interfere with these interactions to help control oral biofilms.

Nicholas S. Jakubovics

Centre for Oral Health Research,
School of Dental Sciences,
Centre for Oral Health Research, School of Dental Sciences

Newcastle University

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

Nicholas S. Jakubovics

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Newcastle University

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

Centre for Oral Health Research,
School of Dental Sciences,
Centre for Oral Health Research, School of Dental Sciences