A Wettability Contrast SERS Droplet Assay for Multiplexed Analyte Detection.

Droplet assay platforms have emerged as a significant methodology, providing distinct advantages such as sample compartmentalization, high throughput, and minimal analyte consumption. However, inherent complexities, especially in multiplexed detection, remain a challenge. We demonstrate a novel strategy to fabricate a plasmonic droplet assay platform (PDAP) for multiplexed analyte detection, enabling surface-enhanced Raman spectroscopy (SERS). PDAP efficiently splits a microliter droplet into submicroliter to nanoliter droplets under gravity-driven flow by wettability contrast between two distinct regions. The desired hydrophobicity and adhesive contrast between the silicone oil-grafted nonadhesive hydrophilic zone with gold nanoparticles is attained through (3-aminopropyl) triethoxysilane (APTES) functionalization of gold nanoparticles (AuNPs) using a scotch-tape mask. The wettability contrast surface facilitates the splitting of aqueous droplets with various surface tensions (ranging from 39.08 to 72 mN/m) into ultralow volumes of nanoliters. The developed PDAP was used for the multiplexed detection of Rhodamine 6G (Rh6G) and Crystal Violet (CV) dyes. The limit of detection for 120 nL droplet using PDAP was found to be 134 pM and 10.1 nM for Rh6G and CV, respectively. These results align with those from previously reported platforms, highlighting the comparable sensitivity of the developed PDAP. We have also demonstrated the competence of PDAP by testing adulterant spiked milk and obtained very good sensitivity. Thus, PDAP has the potential to be used for the multiplexed screening of food adulterants.

Chiral nanomaterial-based approaches for diagnosis and treatment of protein-aggregated neurodiseases: current status and future opportunities.

Protein misfolding and its aggregation, known as amyloid aggregates (Aß), are some of the major causes of more than 20 diseases such as Parkinson's disease, Alzheimer's disease, and type 2 diabetes. The process of Aß formation involves an energy-driven oligomerization of Aß monomers, leading to polymerization and eventual aggregation into fibrils. Aß fibrils exhibit multilevel chirality arising from its amino acid residues and the arrangement of folded polypeptide chains; thus, a chirality-driven approach can be utilized for the detection and inhibition of Aß fibrils. In this regard, chiral nanomaterials have recently opened new possibilities for various biomedical applications owing to their stereoselective interaction with biological systems. Leveraging this chirality-driven approach with chiral nanomaterials against protein-aggregated diseases could yield promising results, particularly in the early detection of Aß forms and the inhibition of Aß aggregate formation via specific and strong "chiral-chiral interaction." Despite the advantages, the development of advanced theranostic systems using chiral nanomaterials against protein-aggregated diseases has received limited attention so far because of considerably limited formulations for chiral nanomaterials and lack of information of their chiroptical behavior. This review aims to present the current status of chiral nanomaterials explored for detecting and inhibiting Aß forms. This review covers the origin of chirality in amyloid fibrils and nanomaterials and different chiral detection methods; furthermore, different chiral nanosystems such as chiral plasmonic nanomaterials, chiral carbon-based nanomaterials, and chiral nanosurfaces, which have been used so far for different therapeutic applications against protein-aggregated diseases, are discussed in detail. The findings from this review may pave the way for the development of novel approaches using chiral nanomaterials to combat diseases resulting from protein misfolding and can further be extended to other disease forms.

Pilus gene pool variation and the virulence of Corynebacterium diphtheriae clinical isolates during infection of a nematode.

Toxigenic Corynebacterium diphtheriae strains cause diphtheria in humans. The toxigenic C. diphtheriae isolate NCTC13129 produces three distinct heterotrimeric pili that contain SpaA, SpaD, and SpaH, making up the shaft structure. The SpaA pili are known to mediate bacterial adherence to pharyngeal epithelial cells. However, to date little is known about the expression of different pili in various clinical isolates and their importance in bacterial pathogenesis. Here, we characterized a large collection of C. diphtheriae clinical isolates for their pilin gene pool by PCR and for the expression of the respective pilins by immunoblotting with antibodies against Spa pilins. Consistent with the role of a virulence factor, the SpaA-type pili were found to be prevalent among the isolates, and most significantly, corynebacterial adherence to pharyngeal epithelial cells was strictly correlated with isolates that were positive for the SpaA pili. By comparison, the isolates were heterogeneous for the presence of SpaD- and SpaH-type pili. Importantly, using Caenorhabditis elegans as a model host for infection, we show here that strain NCTC13129 rapidly killed the nematodes, the phenotype similar to isolates that were positive for toxin and all pilus types. In contrast, isogenic mutants of NCTC13129 lacking SpaA-type pili or devoid of toxin and SpaA pili exhibited delayed killing of nematodes with similar kinetics. Consistently, nontoxigenic or toxigenic isolates that lack one, two, or all three pilus types were also attenuated in virulence. This work signifies the important role of pili in corynebacterial pathogenesis and provides a simple host model to identify additional virulence factors.

Structure of Streptococcus agalactiae tip pilin GBS104: a model for GBS pili assembly and host interactions.

The crystal structure of a 75 kDa central fragment of GBS104, a tip pilin from the 2063V/R strain of Streptococcus agalactiae (group B streptococcus; GBS), is reported. In addition, a homology model of the remaining two domains of GBS104 was built and a model of full-length GBS104 was generated by combining the homology model (the N1 and N4 domains) and the crystal structure of the 75 kDa fragment (the N2 and N3 domains). This rod-shaped GBS104 model is constructed of three IgG-like domains (the N1, N2 and N4 domains) and one vWFA-like domain (the N3 domain). The N1 and N2 domains of GBS104 are assembled with distinct and remote segments contributed by the N- and C-termini. The metal-binding site in the N3 domain of GBS104 is in the closed/low-affinity conformation. Interestingly, this domain hosts two long arms that project away from the metal-binding site. Using site-directed mutagenesis, two cysteine residues that lock the N3 domain of GBS104 into the open/high-affinity conformation were introduced. Both wild-type and disulfide-locked recombinant proteins were tested for binding to extracellular matrix proteins such as collagen, fibronectin, fibrinogen and laminin, and an increase in fibronectin binding affinity was identified for the disulfide-locked N3 domain, suggesting that induced conformational changes may play a possible role in receptor binding.

Single-platform, Attomolar Detection of Multiple Biomarkers by a Flexible SERS Sensor.

Early detection of Alzheimer's disease (AD) is critical for better healthcare management. Herein, we demonstrate a Surface Enhanced Raman Spectroscopy (SERS) active sensor for highly sensitive and selective detection of ß-Amyloid Peptide (Aß1-42 ), a biomarker of Alzheimer's disease. Polyacrylonitrile (PAN) nanofiber mats, containing purine-based ligand (L; 0 mg (P1 ), 50 mg (P2 ), and 100 mg (P3 )) were prepared by electrospinning followed by functionalization with silver nanoparticles (AgNPs). The fabricated SERS sensors were employed for the detection of Rhodamine 6G (Rh-6G) dye for optimization and the highest sensitivity was achieved on P3 /AgNPs SERS sensor. The P3 /AgNPs sensor was chosen for the detection of Aß1-42 and human Insulin (HI). The limit of detection (LoD) was found to be 76×10-18 M and 26×10-18 M for Aß1-42 and HI, respectively. The sensitivity achieved is one order improved for Aß1-42 and four orders for HI when compared with reported values. Also, demonstrated the selectivity of the P3 /AgNPs sensor by testing a simulated cerebrospinal fluid (CSF) and achieved easily identifiable peaks of Aß1-42 among the noise of HI and bovine serum albumin should be written before the acronym of BSA. This approach could be extended to develop ultra-sensitive flexible SERS sensors for the facile detection of multiple biomarkers on a single platform with excellent sensitivity, selectivity and stability.

cis-Acting elements that control expression of the master virulence regulatory gene atxA in Bacillus anthracis.

Transcription of the Bacillus anthracis structural genes for the anthrax toxin proteins and biosynthetic operon for capsule is positively regulated by AtxA, a transcription regulator with unique properties. Consistent with the role of atxA in virulence factor expression, a B. anthracis atxA-null mutant is avirulent in a murine model for anthrax. In culture, multiple signals impact atxA transcript levels, and the timing and steady-state level of atxA expression are critical for optimal toxin and capsule synthesis. Despite the apparent complex control of atxA transcription, only one trans-acting protein, the transition state regulator AbrB, has been demonstrated to interact directly with the atxA promoter. Here we employ 5' and 3' deletion analysis and site-directed mutagenesis of the atxA control region to demonstrate that atxA transcription from the major start site P1 is dependent upon a consensus sequence for the housekeeping sigma factor SigA and an A+T-rich upstream element for RNA polymerase. We also show that an additional trans-acting protein(s) binds specifically to atxA promoter sequences located between -13 and +36 relative to P1 and negatively impacts transcription. Deletion of this region increases promoter activity up to 15-fold. Site-directed mutagenesis of a 9-bp palindromic sequence within the region prevents binding of the trans-acting protein(s), increasing promoter activity 7-fold and resulting in a corresponding increase in AtxA and anthrax toxin production. Notably, an atxA promoter mutant that produced elevated levels of AtxA and toxin proteins during culture was unaffected for virulence in a murine model for anthrax.

Two autonomous structural modules in the fimbrial shaft adhesin FimA mediate Actinomyces interactions with streptococci and host cells during oral biofilm development.

By combining X-ray crystallography and modelling, we describe here the atomic structure of distinct adhesive moieties of FimA, the shaft fimbrillin of Actinomyces type 2 fimbriae, which uniquely mediates the receptor-dependent intercellular interactions between Actinomyces and oral streptococci as well as host cells during the development of oral biofilms. The FimA adhesin is built with three IgG-like domains, each of which harbours an intramolecular isopeptide bond, previously described in several Gram-positive pilins. Genetic and biochemical studies demonstrate that although these isopeptide bonds are dispensable for fimbrial assembly, cell-cell interactions and biofilm formation, they contribute significantly to the proteolytic stability of FimA. Remarkably, FimA harbours two autonomous adhesive modules, which structurally resemble the Staphylococcus aureus Cna B domain. Each isolated module can bind the plasma glycoprotein asialofetuin as well as the polysaccharide receptors present on the surface of oral streptococci and epithelial cells. Thus, FimA should serve as an excellent paradigm for the development of therapeutic strategies and elucidating the precise molecular mechanisms underlying the interactions between cellular receptors and Gram-positive fimbriae.

Plasmonic Paper-Based Flexible SERS Biosensor for Highly Sensitive Detection of Lactic and Uric Acid.

Selective detection and quantification of biomarkers related to human diseases are essential for preventive healthcare. Surface-enhanced Raman scattering (SERS) spectroscopy is a powerful analytical tool offering high sensitivity. However, the success of this promising analytical tool relies on the ability to effectively fabricate SERS substrate. Herein we have demonstrated a plasmonic paper-based flexible substrate (PPFS) for SERS sensing. In situ growth of silver nanostructures (AgNS) on the paper-based substrate was achieved by using a simple one-step silver mirror reaction (SMR). FESEM and TEM results depicts that the increasing silver ion content influences the morphology (growth of multifacets), as well as size of AgNS. Further, the PPFS substrate was tested with Rhodamine-6G (Rh-6G) dye and an attomole sensitivity with a LOD of 4.54 × 10-18 M was achieved. Further, two biomarkers, lactic acid (LA) and uric acid (UA) were detected on the PPFS substrate, with [Formula: see text] and pM sensitivity, having LOD values of 0.6 × 10-6 and 0.3 × 10-12 M respectively. Above detection levels for UA on PPFS is two orders better than reported values, whereas for LA it is comparable with reported substrates. Finally, UA, LA and their mixtures were tested on PPFS and results compared with commercial substrate. The performance of PPFS were found better in all cases, thus, multifaceted AgNS paper based PPFS offers the potential to be used as a biosensor for detection of various biomarkers from body fluids, responsible for the detection of the critical disease for preventive health care.

Ultra-sensitive reusable SERS sensor for multiple hazardous materials detection on single platform.

We demonstrate the detection of dipicolinic acid, (DPA), a biomarker of bacterial spores for Bacillus anthracis, 2,4-Dinitrotoluene (DNT) and picric acid (PA) nitroaromatic hazardous chemicals on ultra-sensitive, reusable femtosecond laser textured Au nanostructures decorated with hierarchical AuNPs as a SERS substrate. The AuNPs were achieved by ablating an Au sheet using two different laser scan speeds (1 and 0.1 mm/s) in linear and crossed patterns. The morphological studies revealed dense hierarchical nanostructures decorated with spherical AuNPs possessing 30-40 nm in size in 0.1 mm/s laser scan. The limits of detection (LOD) of the sensor were determined from the detailed SERS measurements and were estimated to be 0.83 pg/L, 3.6 pg/L and 2.3 pg/L for DPA, DNT, and PA, respectively. To the best of our knowledge, the achieved sensitivity is nearly 2 orders improved for DPA when compared with the currently reported LODs using other techniques and 1 order in the case of SERS. Moreover, for DNT and PA the LODs were found to be either superior or comparable with recent reports. We have also demonstrated the competence of our SERS substrates by testing a few real samples (water spiked with these analytes) and again obtained very good sensitivity.

Purification, crystallization and halide phasing of a Streptococcus agalactiae backbone pilin GBS80 fragment.

The Gram-positive pathogen Streptococcus agalactiae or group B streptococcus (GBS) is the leading cause of bacterial septicemia, pneumonia and meningitis among neonates around the world. The pathogen assembles two types of pili on its surface, named PI-1 and PI-2, that mediate bacterial adherence to host cells. The GBS PI-1 pilus is formed by the major pilin GBS80, which forms the pilus shaft, and two minor pilins GBS104 and GBS52, which are incorporated into the pilus structure. While considerable structural information exists on Gram-negative pili, the structural study of Gram-positive pili is an emerging area of research. Here, the purification, crystallization and initial phasing of the 35 kDa major fragment of the backbone pilin GBS80 are reported. Crystals were obtained in two different space groups: P2(1) and C2. SAD data collected from an iodide-derivative crystal at the home source were used to obtain initial phases and interpretable electron-density maps.

Purine-blended nanofiber woven flexible nanomats for SERS-based analyte detection.

Flexible and free-standing polymeric fibers, coupled with metal nanoparticles, present an excellent opportunity for highly sensitive surface-enhanced Raman scattering (SERS) spectroscopy-based detection platforms. Such host matrices are prepared by only a few preferred methods, followed by induction of metal nanoparticle aggregation in a protected environment to afford functional SERS platforms. We provide an interesting advance in this area by choosing a thiol-modified purine (L)-polymer blend, obtained through the electrospinning process, for directed decoration with gold nanoparticles. The described SERS purine-nanomat substrate enables the detection of uric acid, an important indicator of gout, preeclampsia, cardiovascular and kidney diseases, in aqueous solution up to 100 nM, with good stability and high sensitivity, offering superiority over existing techniques in terms of sensitivity and cost-effectiveness.

Acyl enzyme intermediates in sortase-catalyzed pilus morphogenesis in gram-positive bacteria.

In gram-positive bacteria, covalently linked pilus polymers are assembled by a specific transpeptidase enzyme called pilus-specific sortase. This sortase is postulated to cleave the LPXTG motif of a pilin precursor between threonine and glycine and to form an acyl enzyme intermediate with the substrate. Pilus polymerization is believed to occur through the resolution of this intermediate upon specific nucleophilic attack by the conserved lysine located within the pilin motif of another pilin monomer, which joins two pilins with an isopeptide bond formed between threonine and lysine. Here, we present evidence for sortase reaction intermediates in Corynebacterium diphtheriae. We show that truncated SrtA mutants that are loosely bound to the cytoplasmic membrane form high-molecular-weight complexes with SpaA polymers secreted into the extracellular milieu. These complexes are not formed with SpaA pilin mutants that have alanine substitutions in place of threonine in the LPXTG motif or lysine in the pilin motif. The same phenotype is observed with alanine substitutions of either the conserved cysteine or histidine residue of SrtA known to be required for catalysis. Remarkably, the assembly of SpaA pili, or the formation of intermediates, is abolished with a SrtA mutant missing the membrane-anchoring domain. We infer that pilus polymerization involves the formation of covalent pilin-sortase intermediates, which occurs within a molecular platform on the exoplasmic face of the cytoplasmic membrane that brings together both sortase and its cognate substrates in close proximity to each other, likely surrounding a secretion apparatus. We present electron microscopic data in support of this picture.

Hierarchical Laser-Patterned Silver/Graphene Oxide Hybrid SERS Sensor for Explosive Detection.

We demonstrate an ultrafast laser-ablated hierarchically patterned silver nanoparticle/graphene oxide (AgNP/GO) hybrid surface-enhanced Raman scattering (SERS) substrate for highly sensitive and reproducible detection of an explosive marker 2,4-dinitrotoluene (2,4-DNT). A hierarchical laser-patterned silver sheet (Ag-S) is achieved by ultrafast laser ablation in air with pulse energies of 25, 50, and 100 µJ. Multiple laser pulses at a wavelength of 800 nm and a pulse repetition rate of 50 fs at 1 kHz are directly focused on Ag-S to produce and deposit AgNPs onto Ag-S. The surface morphology of ablated Ag-S was evaluated using atomic force microscopy, optical profilometry, and field emission scanning electron microscopy (FESEM). A rapid increase in the ablation rate with increasing laser energy was observed. Selected area Raman mapping is performed to understand the intensity and size distribution of AgNPs on Ag-S. Further, GO was spin-coated onto the AgNPs produced by ultrafast ablation on Ag-S. The hierarchical laser-patterned AgNP/GO hybrid structure was characterized using FESEM, high-resolution transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and Raman spectroscopy. Further, hierarchical laser-patterned AgNP/GO hybrid structures have been utilized as SERS-active substrates for the selective detection of 2,4-DNT, an explosive marker. The developed SERS-active sensor shows good stability and high sensitivity up to picomolar (pM) concentration range with a Raman intensity enhancement of ∼1010 for 2,4-DNT. The realized enhancement of SERS intensity is due to the cumulative effect of GO coated on Ag-S as a proactive layer and AgNPs produced by ultrafast ablation.

Infrared microlenses and gratings of chalcogenide: confined self-organization in solution processed thin liquid films.

This work demonstrates the fabrication of chalcogenide microstructures such as gratings, lenses and needles using a lithographically directed, evaporative self-organization of chalcogenide thin liquid films for the first time. Using a two-step annealing protocol, excess solvent of freshly coated ChG films is eliminated and then the liquid films are patterned using elastomeric masters with continuous or disconnected features during solvent evaporation. Although microcontact printing or capillary flow lithography has been proven to be useful to create continuous gratings and waveguide like structures in solid films, our method overcomes the limitation of structural continuity of the generated pattern and uses self-organization of solute ChG within the master's confinement to produce isolated microstructures. Fabrication of disjointed arrays of microlenses of various dimensions as well as conical shaped needles in ChG thin films has been demonstrated for relevant optical IR applications. This methodology establishes evaporative self-organization of ChG thin films as a viable alternative to creating microstructures in bulk ChG with hot-embossing, bypassing the need for ultra high temperature processing.

Large area IR microlens arrays of chalcogenide glass photoresists by grayscale maskless lithography.

The ability to use chalcogenide glass thin films as photoresists for one-step maskless grayscale lithographic patterning is demonstrated. It is shown that the chalcogenide photoresists can be used to fabricate grayscale patterns with smooth and continuous profiles such as arrays of cylindrical and spherical microlenses, which are useful as optical structures for IR applications. The etching and exposure parameters are optimized to obtain smooth reproducible lens arrays of 150 µm periodicity and up to ∼170 nm height on large areas (∼1 cm(2)). The roughness is found to increase as a function of the exposure dose and is attributed to the selective dissolution of the As-Se, As-As, and Se-Se bonds present in the nanodistributed phases and the presence of the oxide phase. Thus, a minimum exposure dose produces optimally patterned lens arrays. The focal length calculated for the smooth microlens array is ∼9.3 mm, indicating the suitability of the lens arrays for focusing applications in the IR region.

PECVD based silicon oxynitride thin films for nano photonic on chip interconnects applications.

Thin silicon oxynitride (SiO(x)N(y)) films were deposited by low temperature (~300°C) plasma enhanced chemical vapour deposition (PECVD), using SiH(4), N(2)O, NH(3) precursor of the flow rate 25, 100, 30 sccm and subjected to the post deposition annealing (PDA) treatment at 400°C and 600°C for nano optical/photonics on chip interconnects applications. AFM result reveals the variation of roughness from 60.9 Å to 23.4 Å after PDA treatment with respect to the as-deposited films, favourable surface topography for integrated waveguide applications. A model of decrease in island height with the effect of PDA treatment is proposed in support of AFM results. Raman spectroscopy and FTIR measurements are performed in order to define the change in crystallite and chemical bonding of as-deposited as well as PDA treated samples. These outcomes endorsed to the densification of SiO(x)N(y) thin films, due to decrease in Si-N and Si-O bonds strain, as well the O-H, N-H bonds with in oxynitride network. The increase in refractive index and PL intensity of as deposited SiO(x)N(y) thin films to the PDA treated films at 400°C and 600°C are observed. The significant shift of PL spectra peak positions indicate the change in cluster size as the result of PDA treatment, which influence the optical properties of thin films. It might be due to out diffusion of hydrogen containing species from silicon oxynitride films after PDA treatment. In this way, the structural and optical, feasibility of SiO(x)N(y) films are demonstrated in order to obtain high quality thin films for nano optical/photonics on chip interconnects applications.

A model for group B Streptococcus pilus type 1: the structure of a 35-kDa C-terminal fragment of the major pilin GBS80.

The Gram-positive pathogen Streptococcus agalactiae, known as group B Streptococcus (GBS), is the leading cause of bacterial septicemia, pneumonia, and meningitis among neonates. GBS assembles two types of pili-pilus islands (PIs) 1 and 2-on its surface to adhere to host cells and to initiate colonization for pathogenesis. The GBS PI-1 pilus is made of one major pilin, GBS80, which forms the pilus shaft, and two secondary pilins, GBS104 and GBS52, which are incorporated into the pilus at various places. We report here the crystal structure of the 35-kDa C-terminal fragment from GBS80, which is composed of two IgG-like domains (N2-N3). The structure was solved by single-wavelength anomalous dispersion using sodium-iodide-soaked crystals and diffraction data collected at the home source. The N2 domain exhibits a cnaA/DEv-IgG fold with two calcium-binding sites, while the N3 domain displays a cnaB/IgG-rev fold. We have built a model for full-length GBS80 (N1, N2, and N3) with the help of available homologous major pilin structures, and we propose a model for the GBS PI-1 pilus shaft. The N2 and N3 domains are arranged in tandem along the pilus shaft, whereas the respective N1 domain is tilted by approximately 20° away from the pilus axis. We have also identified a pilin-like motif in the minor pilin GBS52, which might aid its incorporation at the pilus base.

Role of Bcr1-activated genes Hwp1 and Hyr1 in Candida albicans oral mucosal biofilms and neutrophil evasion.

Candida albicans triggers recurrent infections of the oropharyngeal mucosa that result from biofilm growth. Prior studies have indicated that the transcription factor Bcr1 regulates biofilm formation in a catheter model, both in vitro and in vivo. We thus hypothesized that Bcr1 plays similar roles in the formation of oral mucosal biofilms and tested this hypothesis in a mouse model of oral infection. We found that a bcr1/bcr1 mutant did not form significant biofilm on the tongues of immunocompromised mice, in contrast to reference and reconstituted strains that formed pseudomembranes covering most of the tongue dorsal surface. Overexpression of HWP1, which specifies an epithelial adhesin that is under the transcriptional control of Bcr1, partly but significantly rescued the bcr1/bcr1 biofilm phenotype in vivo. Since HWP1 overexpression only partly reversed the biofilm phenotype, we investigated whether additional mechanisms, besides adhesin down-regulation, were responsible for the reduced virulence of this mutant. We discovered that the bcr1/bcr1 mutant was more susceptible to damage by human leukocytes when grown on plastic or on the surface of a human oral mucosa tissue analogue. Overexpression of HYR1, but not HWP1, significantly rescued this phenotype. Furthermore a hyr1/hyr1 mutant had significantly attenuated virulence in the mouse oral biofilm model of infection. These discoveries show that Bcr1 is critical for mucosal biofilm infection via regulation of epithelial cell adhesin and neutrophil function.

Characterization of mucosal Candida albicans biofilms.

C. albicans triggers recurrent infections of the alimentary tract mucosa that result from biofilm growth. Although the ability of C. albicans to form a biofilm on abiotic surfaces has been well documented in recent years, no information exists on biofilms that form directly on mucosal surfaces. The objectives of this study were to characterize the structure and composition of Candida biofilms forming on the oral mucosa. We found that oral Candida biofilms consist of yeast, hyphae, and commensal bacteria, with keratin dispersed in the intercellular spaces. Neutrophils migrate through the oral mucosa and form nests within the biofilm mass. The cell wall polysaccharide beta-glucan is exposed during mucosal biofilm growth and is more uniformly present on the surface of biofilm organisms invading the oral mucosa. We conclude that C. albicans forms complex mucosal biofilms consisting of both commensal bacterial flora and host components. These discoveries are important since they can prompt a shift of focus for current research in investigating the role of Candida-bacterial interactions in the pathogenesis of mucosal infections as well as the role of beta-glucan mediated signaling in the host response.