Branched amphotericin functional poly(N-isopropyl acrylamide): an antifungal polymer.

Branched poly(N-isopropylacrylamide) was functionalized with Amphotericin B (AmB) at the chain ends to produce an antifungal material. The polymer showed antifungal properties against AmB-sensitive strains of Candida albicans, Fusarium keratoplasticum and Aspergillus flavus (minimal inhibitory concentration ranged from 5 to 500 µg ml-1) but was not effective against an AmB resistant strain of C. albicans nor against Candida tropicalis. The polymer end groups bound to the AmB target, ergosterol, and the fluorescence spectrum of a dye used as a solvatochromic probe, Nile red, was blue shifted indicating that segments of the polymer became desolvated on binding. The polymer was less toxic to corneal and renal epithelial cells and explanted corneal tissue than the free drug. Also, the polymer did not induce reactive oxygen species release from peripheral blood mononuclear cells, nor did it cause a substantial release of the proinflammatory cytokines, tumour necrosis factor-α and interleukin-1ß (at 0.5 mg ml-1).

Semi-interpenetrating Polyurethane Network Foams Containing Highly Branched Poly(N-isopropyl acrylamide) with Vancomycin Functionality.

Highly branched poly(N-isopropylacrylamide) (HB-PNIPAM), functionalized with vancomycin at the chain ends, acted as a bacterial adhesive and was incorporated into polyurethane foams to form semi-interpenetrating networks. PNIPAM was labeled with a solvatochromic dye, Nile red. It was found that the thermal response of the polymer was dependent on the architecture, and temperature-dependent color changes were observed within the foam. The foams had open pore structures, and the presence of HB-PNIPAM substantially reduced the shrinkage of the foam as the temperature was increased up to 20 °C. The foams were selectively adhesive for Staphylococcus aureus (Gram-positive bacteria) compared to Pseudomonas aeruginosa (Gram-negative bacteria), and the presence of S. aureus was indicated by increased fluorescence intensity (590-800 nm).

Porphyromonas gingivalis lipopolysaccharide rapidly activates trigeminal sensory neurons and may contribute to pulpal pain.

AIM: To determine whether Porphyromonas gingivalis lipopolysaccharide (LPS) can directly activate trigeminal neurons, to identify which receptors are involved and to establish whether activation leads to secretion of the neuropeptide calcitonin gene-related peptide (CGRP) and/or the translocation of NF-κB. METHODOLOGY: Mouse trigeminal ganglion (TG) cells were cultured in vitro for 2 days. The effect of P. gingivalis LPS (20 µg mL-1 ) on calcium signalling was assessed (by calcium imaging using Cal-520 AM) in comparison with the transient receptor potential channel A1 (TRPA1) agonist cinnamaldehyde (CA; 100 µmol L-1 ), the TRP channel V1 (TRPV1) agonist capsaicin (CAP; 1 µmol L-1 ) and high potassium (60 mmol L-1 KCl). TG cultures were pre-treated with either 1 µmol L-1 CLI-095 to block Toll-like receptor 4 (TLR4) signalling or with 3 µmol L-1 HC-030031 to block TRPA1 signalling. CGRP release was determined using ELISA, and nuclear translocation of NF-κB was investigated using immunocytochemistry. Data were analysed by one-way analysis of variance, followed by Bonferroni's post hoc test as appropriate. RESULTS: Porphyromonas gingivalis LPS directly exerted a rapid excitatory response on sensory neurons and non-neuronal cells (P < 0.001 to P < 0.05). The effects on neurons appear to be mediated via TLR4- and TRPA1-dependent pathways. The responses were accompanied by an increased release of CGRP (P < 0.001) and by NF-κB nuclear translocation (P < 0.01). CONCLUSIONS: Porphyromonas gingivalis LPS directly activated trigeminal sensory neurons (via TLR4 and TRPA1 receptors) and non-neuronal cells, resulting in CGRP release and NF-κB nuclear translocation. This indicates that P. gingivalis can directly influence activity in trigeminal sensory neurons and this may contribute to acute and chronic inflammatory pain.

Identification of Novel Bacteriophages with Therapeutic Potential That Target Enterococcus faecalis.

The Gram-positive opportunistic pathogen Enterococcus faecalis is frequently responsible for nosocomial infections in humans and represents one of the most common bacteria isolated from recalcitrant endodontic (root canal) infections. E. faecalis is intrinsically resistant to several antibiotics routinely used in clinical settings (such as cephalosporins and aminoglycosides) and can acquire resistance to vancomycin (vancomycin-resistant enterococci). The resistance of E. faecalis to several classes of antibiotics and its capacity to form biofilms cause serious therapeutic problems. Here, we report the isolation of several bacteriophages that target E. faecalis strains isolated from the oral cavity of patients suffering root canal infections. All phages isolated were Siphoviridae with similar tail lengths (200 to 250 nm) and icosahedral heads. The genome sequences of three isolated phages were highly conserved with the exception of predicted tail protein genes that diverge in sequence, potentially reflecting the host range. The properties of the phage with the broadest host range (SHEF2) were further characterized. We show that this phage requires interaction with components of the major and variant region enterococcal polysaccharide antigen to engage in lytic infection. Finally, we explored the therapeutic potential of this phage and show that it can eradicate E. faecalis biofilms formed in vitro on a standard polystyrene surface but also on a cross-sectional tooth slice model of endodontic infection. We also show that SHEF2 cleared a lethal infection of zebrafish when applied in the circulation. We therefore propose that the phage described here could be used to treat a broad range of antibiotic-resistant E. faecalis infections.

Antibiotic functionalised polymers reduce bacterial biofilm and bioburden in a simulated infection of the cornea.

Microbial keratitis can arise from penetrating injuries to the cornea. Corneal trauma promotes bacterial attachment and biofilm growth, which decrease the effectiveness of antimicrobials against microbial keratitis. Improved therapeutic efficacy can be achieved by reducing microbial burden prior to antimicrobial therapy. This paper assesses a highly-branched poly(N-isopropyl acrylamide) with vancomycin end groups (HB-PNIPAM-van), for reducing bacterial attachment and biofilm formation. The polymer lacked antimicrobial activity against Staphylococcus aureus, but significantly inhibited biofilm formation (p = 0.0008) on plastic. Furthermore, pre-incubation of S. aureus cells with HB-PNIPAM-van reduced cell attachment by 50% and application of HB-PNIPAM-van to infected ex vivo rabbit corneas caused a 1-log reduction in bacterial recovery, compared to controls (p = 0.002). In conclusion, HB-PNIPAM-van may be a useful adjunct to antimicrobial therapy in the treatment of corneal infections.

Binding of Bacteria to Poly(N-isopropylacrylamide) Modified with Vancomycin: Comparison of Behavior of Linear and Highly Branched Polymers.

The behavior of a linear copolymer of N-isopropylacrylamide with pendant vancomycin functionality was compared to an analogous highly branched copolymer with vancomycin functionality at the chain ends. Highly branched poly(N-isopropylacrylamide) modified with vancomycin (HB-PNIPAM-van) was synthesized by functionalization of the HB-PNIPAM, prepared using reversible addition-fragmentation chain transfer polymerization. Linear PNIPAM with pendant vancomycin functionality (L-PNIPAM-van) was synthesized by functionalization of poly(N-isopropylacrylamide-co-vinyl benzoic acid). HB-PNIPAM-van aggregated S. aureus effectively, whereas the L-PNIPAM-van polymer did not. It was found that when the HB-PNIPAM-van was incubated with S. aureus the resultant phase transition provided an increase in the intensity of fluorescence of a solvatochromic dye, nile red, added to the system. In contrast, a significantly lower increase in fluorescence intensity was obtained when L-PNIPAM-van was incubated with S. aureus. These data showed that the degree of desolvation of HB-PNIPAM-van was much greater than the desolvation of the linear version. Using microcalorimetry, it was shown that there were no significant differences in the affinities of the polymer ligands for d-Ala-d-Ala and therefore differences in the interactions with bacteria were associated with changes in the probability of access of the polymer bound ligands to the d-Ala-d-Ala dipeptide. The data support the hypothesis that generation of polymer systems that respond to cellular targets, for applications such as cell targeting, detection of pathogens etc., requires the use of branched polymers with ligands situated at the chain ends.

Role of OmpA2 surface regions of Porphyromonas gingivalis in host-pathogen interactions with oral epithelial cells.

Outer membrane protein A (OmpA) is a key outer membrane protein found in Gram-negative bacteria that contributes to several crucial processes in bacterial virulence. In Porphyromonas gingivalis, OmpA is predicted as a heterotrimer of OmpA1 and OmpA2 subunits encoded by adjacent genes. Here we describe the role of OmpA and its individual subunits in the interaction of P. gingivalis with oral cells. Using knockout mutagenesis, we show that OmpA2 plays a significant role in biofilm formation and interaction with human epithelial cells. We used protein structure prediction software to identify extracellular loops of OmpA2, and determined these are involved in interactions with epithelial cells as evidenced by inhibition of adherence and invasion of P. gingivalis by synthetic extracellular loop peptides and the ability of the peptides to mediate interaction of latex beads with human cells. In particular, we observe that OmpA2-loop 4 plays an important role in the interaction with host cells. These data demonstrate for the first time the important role of P. gingivalis OmpA2 extracellular loops in interaction with epithelial cells, which may help design novel peptide-based antimicrobial therapies for periodontal disease.

Antimicrobial Graft Copolymer Gels.

In view of the growing worldwide rise in microbial resistance, there is considerable interest in designing new antimicrobial copolymers. The aim of the current study was to investigate the relationship between antimicrobial activity and copolymer composition/architecture to gain a better understanding of their mechanism of action. Specifically, the antibacterial activity of several copolymers based on 2-(methacryloyloxy)ethyl phosphorylcholine [MPC] and 2-hydroxypropyl methacrylate (HPMA) toward Staphylococcus aureus was examined. Both block and graft copolymers were synthesized using either atom transfer radical polymerization or reversible addition-fragmentation chain transfer polymerization and characterized via (1)H NMR, gel permeation chromatography, rheology, and surface tensiometry. Antimicrobial activity was assessed using a range of well-known assays, including direct contact, live/dead staining, and the release of lactate dehydrogenase (LDH), while transmission electron microscopy was used to study the morphology of the bacteria before and after the addition of various copolymers. As expected, PMPC homopolymer was biocompatible but possessed no discernible antimicrobial activity. PMPC-based graft copolymers comprising PHPMA side chains (i.e. PMPC-g-PHPMA) significantly reduced both bacterial growth and viability. In contrast, a PMPC-PHPMA diblock copolymer comprising a PMPC stabilizer block and a hydrophobic core-forming PHPMA block did not exhibit any antimicrobial activity, although it did form a biocompatible worm gel. Surface tensiometry studies and LDH release assays suggest that the PMPC-g-PHPMA graft copolymer exhibits surfactant-like activity. Thus, the observed antimicrobial activity is likely to be the result of the weakly hydrophobic PHPMA chains penetrating (and hence rupturing) the bacterial membrane.

The Periodontal Pathogen Porphyromonas gingivalis Preferentially Interacts with Oral Epithelial Cells in S Phase of the Cell Cycle.

Porphyromonas gingivalis, a key periodontal pathogen, is capable of invading a variety of cells, including oral keratinocytes, by exploiting host cell receptors, including alpha-5 beta-1 (α5ß1) integrin. Previous studies have shown that P. gingivalis accelerates the cell cycle and prevents apoptosis of host cells, but it is not known whether the cell cycle phases influence bacterium-cell interactions. The cell cycle distribution of oral keratinocytes was characterized by flow cytometry and BrdU (5-bromo-2-deoxyuridine) staining following synchronization of cultures by serum starvation. The effect of cell cycle phases on P. gingivalis invasion was measured by using antibiotic protection assays and flow cytometry, and these results were correlated with gene and surface expression levels of α5 integrin and urokinase plasminogen activator receptor (uPAR). There was a positive correlation (R = 0.98) between the number of cells in S phase and P. gingivalis invasion, the organism was more highly associated with cells in S phase than with cells in G2 and G1 phases, and S-phase cells contained 10 times more bacteria than did cells that were not in S phase. Our findings also show that α5 integrin, but not uPAR, was positively correlated with cells in S phase, which is consistent with previous reports indicating that P. gingivalis invasion of cells is mediated by α5 integrin. This study shows for the first time that P. gingivalis preferentially associates with and invades cells in the S phase of the cell cycle. The mechanism of targeting stable dividing cells may have implications for the treatment of periodontal diseases and may partly explain the persistence of this organism at subgingival sites.

Non-conventional therapeutics for oral infections.

As our knowledge of host-microbial interactions within the oral cavity increases, future treatments are likely to be more targeted. For example, efforts to target a single species or key virulence factors that they produce, while maintaining the natural balance of the resident oral microbiota that acts to modulate the host immune response would be an advantage. Targeted approaches may be directed at the black-pigmented anaerobes, Porphyromonas gingivalis and Prevotella intermedia, associated with periodontitis. Such pigments provide an opportunity for targeted phototherapy with high-intensity monochromatic light. Functional inhibition approaches, including the use of enzyme inhibitors, are also being explored to control periodontitis. More general disruption of dental plaque through the use of enzymes and detergents, alone and in combination, shows much promise. The use of probiotics and prebiotics to improve gastrointestinal health has now led to an interest in using these approaches to control oral disease. More recently the potential of antimicrobial peptides and nanotechnology, through the application of nanoparticles with biocidal, anti-adhesive and delivery capabilities, has been explored. The aim of this review is to consider the current status as regards non-conventional treatment approaches for oral infections with particular emphasis on the plaque-related diseases.

Physiological adaptations of key oral bacteria.

Oral colonising bacteria are highly adapted to the various environmental niches harboured within the mouth, whether that means while contributing to one of the major oral diseases of caries, pulp infections, or gingival/periodontal disease or as part of a commensal lifestyle. Key to these infections is the ability to adhere to surfaces via a range of specialised adhesins targeted at both salivary and epithelial proteins, their glycans and to form biofilm. They must also resist the various physical stressors they are subjected to, including pH and oxidative stress. Possibly most strikingly, they have developed the ability to harvest both nutrient sources provided by the diet and those derived from the host, such as protein and surface glycans. We have attempted to review recent developments that have revealed much about the molecular mechanisms at work in shaping the physiology of oral bacteria and how we might use this information to design and implement new treatment strategies.

Förster resonance energy transfer confirms the bacterial-induced conformational transition in highly-branched poly(N-isopropyl acrylamide with vancomycin end groups on binding to Staphylococcus aureus.

We describe a series of experiments designed to investigate the conformational transition that highly-branched polymers with ligands undergo when interacting with bacteria, a process that may provide a new sensing mechanism for bacterial detection. Fluorescent highly-branched poly(N-isopropyl acrylamide)s (HB-PNIPAM) were prepared by sequential self-condensing radical copolymerizations, using anthrylmethyl methacrylate (AMMA) and fluorescein-O-acrylate (FA) as fluorescent comonomers and 4-vinylbenzyl pyrrole carbodithioate as a branch forming monomer. Differences in reactivity necessitated to first copolymerize AMMA then react with FA in a separate sequential monomer feed step. Modifications of the chain ends produced vancomycin-functional derivatives (HB-PNIPAM-Van). The AMMA and FA labels allow probing of the conformational behaviour of the polymers in solution via Förster resonance energy transfer experiments. It was shown that interaction of this polymer's end groups with Staphylococcus aureus induced a macromolecular collapse. The data thus provide conclusive evidence for a conformational transition that is driven by binding to a bacterium.

Characterisation and optimisation of organotypic oral mucosal models to study Porphyromonas gingivalis invasion.

Porphyromonas gingivalis is a Gram-negative, keystone pathogen in periodontitis that leads to tissue destruction and ultimately tooth loss. The organism is able to infect oral epithelial cells and two-dimensional (monolayer) cultures have been used to investigate this process. However, recently there has been interest in the use of three-dimensional, organotypic mucosal models to analyse infection. These models are composed of collagen-embedded fibroblasts overlain with multilayers of oral epithelial cells. In this study we report for the first time significant differences in the response of oral mucosal models to P. gingivalis infection when compared to monolayer cultures of oral epithelial cells. Intracellular survival (3-fold) and bacterial release (4-fold) of P. gingivalis was significantly increased in mucosal models compared with monolayer cultures, which may be due to the multi-layered nature and exfoliation of epithelial cells in these organotypic models. Furthermore, marked differences in the cytokine profile between infected organotypic models and monolayer cultures were observed, particularly for CXCL8 and IL6, which suggested that degradation of cytokines by P. gingivalis may be less pronounced in organotypic compared to monolayer cultures. These data suggest that use of oral mucosal models may provide a greater understanding of the host responses to P. gingivalis invasion than simple monolayer cultures.

Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species.

Many human-dwelling bacteria acquire sialic acid for growth or surface display. We identified previously a sialic acid utilization operon in Tannerella forsythia that includes a novel outer membrane sialic acid-transport system (NanOU), where NanO (neuraminate outer membrane permease) is a putative TonB-dependent receptor and NanU (extracellular neuraminate uptake protein) is a predicted SusD family protein. Using heterologous complementation of nanOU genes into an Escherichia coli strain devoid of outer membrane sialic acid permeases, we show that the nanOU system from the gut bacterium Bacteroides fragilis is functional and demonstrate its dependence on TonB for function. We also show that nanU is required for maximal function of the transport system and that it is expressed in a sialic acid-responsive manner. We also show its cellular localization to the outer membrane using fractionation and immunofluorescence experiments. Ligand-binding studies revealed high-affinity binding of sialic acid to NanU (Kd ~400 nM) from two Bacteroidetes species as well as binding of a range of sialic acid analogues. Determination of the crystal structure of NanU revealed a monomeric SusD-like structure containing a novel motif characterized by an extended kinked helix that might determine sugar-binding specificity. The results of the present study characterize the first bacterial extracellular sialic acid-binding protein and define a sialic acid-specific PUL (polysaccharide utilization locus).

Polymersome-mediated intracellular delivery of antibiotics to treat Porphyromonas gingivalis-infected oral epithelial cells.

The gram-negative anaerobe Porphyromonas gingivalis colonizes the gingival crevice and is etiologically associated with periodontal disease that can lead to alveolar bone damage and resorption, promoting tooth loss. Although susceptible to antibiotics, P. gingivalis can evade antibiotic killing by residing within gingival keratinocytes. This provides a reservoir of organisms that may recolonize the gingival crevice once antibiotic therapy is complete. Polymersomes are nanosized amphiphilic block copolymer vesicles that can encapsulate drugs. Cells internalize polymersomes by endocytosis into early endosomes, where they are disassembled by the low pH, causing intracellular release of their drug load. In this study, polymersomes were used as vehicles to deliver antibiotics in an attempt to kill intracellular P. gingivalis within monolayers of keratinocytes and organotypic oral mucosal models. Polymersome-encapsulated metronidazole or doxycycline, free metronidazole, or doxycycline, or polymersomes alone as controls, were used, and the number of surviving intracellular P. gingivalis was quantified after host cell lysis. Polymersome-encapsulated metronidazole or doxycycline significantly (P<0.05) reduced the number of intracellular P. gingivalis in both monolayer and organotypic cultures compared to free antibiotic or polymersome alone controls. Polymersomes are effective delivery vehicles for antibiotics that do not normally gain entry to host cells. This approach could be used to treat recurrent periodontitis or other diseases caused by intracellular-dwelling organisms.

Gingipain-dependent degradation of mammalian target of rapamycin pathway proteins by the periodontal pathogen Porphyromonas gingivalis during invasion.

Porphyromonas gingivalis and Tannerella forsythia are gram-negative pathogens strongly associated with periodontitis. Their abilities to interact, invade and persist within host cells are considered crucial to their pathogenicity, but the mechanisms by which they subvert host defences are not well understood. In this study, we set out to investigate whether P. gingivalis and T. forsythia directly target key signalling molecules that may modulate the host cell phenotype to favour invasion and persistence. Our data identify, for the first time, that P. gingivalis, but not T. forsythia, reduces levels of intracellular mammalian target of rapamycin (mTOR) in oral epithelial cells following invasion over a 4-h time course, via the action of gingipains. The ability of cytochalasin D to abrogate P. gingivalis-mediated mTOR degradation suggests that this effect is dependent upon cellular invasion. We also show that levels of several other proteins in the mTOR signalling pathway are modulated by gingipains, either directly or as a consequence of mTOR degradation including p-4E-BP1. Taken together, our data suggest that P. gingivalis manipulates the mTOR pathway, providing evidence for a potentially novel mechanism by which P. gingivalis mediates its effects on host cell responses to infection.

Glycoprotein Ibα and FcγRIIa play key roles in platelet activation by the colonizing bacterium, Streptococcus oralis.

BACKGROUND: Infective endocarditis (IE) is characterized by thrombus formation on a cardiac valve. The oral bacterium, Streptococcus oralis, is recognized for its ability to colonize damaged heart valves and is frequently isolated from patients with IE. Platelet interaction with S. oralis leads to the development of a thrombotic vegetation on heart valves, which results in valvular incompetence and congestive heart failure. OBJECTIVE: To investigate the mechanism through which platelets become activated upon binding S. oralis. PATIENTS AND METHODS: Platelet interactions with immobilized bacteria under shear conditions were assessed using a parallel flow chamber. S. oralis-inducible platelet reactivity was determined using light transmission aggregometry. Dense granule secretion was measured by luminometry using a luciferin/luciferase assay. RESULTS: Using shear rates that mimic physiological conditions, we demonstrated that S. oralis was able to support platelet adhesion under venous (50-200 s(-1) ) and arterial shear conditions (800 s(-1) ). Platelets rolled along immobilized S. oralis through an interaction with GPIbα. Following rolling, platelet microaggregate formation was observed on immobilized S. oralis. Aggregate formation was dependent on S. oralis binding IgG, which cross-links to platelet FcγRIIa. This interaction led to phosphorylation of the ITAM domain on FcγRIIa, resulting in dense granule secretion, amplification through the ADP receptor and activation of RAP1, culminating in platelet microaggregate formation. CONCLUSIONS: These results suggest a model of interaction between S. oralis and platelets that leads to the formation of a stable septic vegetation on damaged heart valves.

Sterilizable gels from thermoresponsive block copolymer worms.

Biocompatible hydrogels have many applications, ranging from contact lenses to tissue engineering scaffolds. In most cases, rigorous sterilization is essential. Herein we show that a biocompatible diblock copolymer forms wormlike micelles via polymerization-induced self-assembly in aqueous solution. At a copolymer concentration of 10.0 w/w %, interworm entanglements lead to the formation of a free-standing physical hydrogel at 21 °C. Gel dissolution occurs on cooling to 4 °C due to an unusual worm-to-sphere order-order transition, as confirmed by rheology, electron microscopy, variable temperature (1)H NMR spectroscopy, and scattering studies. Moreover, this thermo-reversible behavior allows the facile preparation of sterile gels, since ultrafiltration of the diblock copolymer nanoparticles in their low-viscosity spherical form at 4 °C efficiently removes micrometer-sized bacteria; regelation occurs at 21 °C as the copolymer chains regain their wormlike morphology. Biocompatibility tests indicate good cell viabilities for these worm gels, which suggest potential biomedical applications.

Beta-hexosaminidase activity of the oral pathogen Tannerella forsythia influences biofilm formation on glycoprotein substrates.

Tannerella forsythia is an important pathogen in periodontal disease. Previously, we showed that its sialidase activity is key to utilization of sialic acid from a range of human glycoproteins for biofilm growth and initial adhesion. Removal of terminal sialic acid residues often exposes ß-linked glucosamine or galactosamine, which may also be important adhesive molecules. In turn, these residues are often removed by a group of enzymes known as ß-hexosaminidases. We show here that T. forsythia has the ability to cleave glucosamine and galactosamine from model substrates and that this activity can be inhibited by the hexosaminidase inhibitor PugNAc (O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenyl carbamate). We now demonstrate for the first time that ß-hexosaminidase activity plays a role in biofilm growth on glycoprotein-coated surfaces because biofilm growth and initial cell adhesion are inhibited by PugNAc. In contrast, adhesion to siallo-glycoprotein-coated surfaces is unaltered by PugNAc in the absence of sialidase activity (using a sialidase-deficient mutant) or surprisingly on the clinically relevant substrates saliva or serum. These data indicate that ß-hexosaminidase activity has a significant role in biofilm formation in combination with sialidase activity in the biofilm lifestyle of T. forsythia.

Role of sialidase in glycoprotein utilization by Tannerella forsythia.

The major bacterial pathogens associated with periodontitis include Tannerella forsythia. We previously discovered that sialic acid stimulates biofilm growth of T. forsythia, and that sialidase activity is key to utilization of sialoconjugate sugars and is involved in host-pathogen interactions in vitro. The aim of this work was to assess the influence of the NanH sialidase on initial biofilm adhesion and growth in experiments where the only source of sialic acid was sialoglycoproteins or human oral secretions. After showing that T. forsythia can utilize sialoglycoproteins for biofilm growth, we showed that growth and initial adhesion with sialylated mucin and fetuin were inhibited two- to threefold by the sialidase inhibitor oseltamivir. A similar reduction (three- to fourfold) was observed with a nanH mutant compared with the wild-type. Importantly, these data were replicated using clinically relevant serum and saliva samples as substrates. In addition, the ability of the nanH mutant to form biofilms on glycoprotein-coated surfaces could be restored by the addition of purified NanH, which we show is able to cleave sialic acid from the model glycoprotein fetuin and, much less efficiently, 9-O-acetylated bovine submaxillary mucin. These data show for the first time that glycoprotein-associated sialic acid is likely to be a key in vivo nutrient source for T. forsythia when growing in a biofilm, and suggest that sialidase inhibitors might be useful adjuncts in periodontal therapy.