The human transmembrane mucin MUC17 responds to TNFα by increased presentation at the plasma membrane.

Transmembrane mucin MUC17 is an integral part of the glycocalyx as it covers the brush border membrane of small intestinal enterocytes and presents an extended O-glycosylated mucin domain to the intestinal lumen. Here, we identified two unknown phosphorylated serine residues, S4428 and S4492, in the cytoplasmic tail of human MUC17. We have previously demonstrated that MUC17 is anchored to the apical membrane domain via an interaction with the scaffolding protein PDZK1. S4492, localized in the C-terminal PDZ binding motif of MUC17, was mutated to generate phosphomimetic and phosphodeficient variants of MUC17. Using Caco-2 cells as a model system, we found that induction of an inflammatory state by long-term stimulation with the proinflammatory cytokine TNFα resulted in an increase of MUC17 protein levels and enhanced insertion of MUC17 and its two phospho-variants into apical membranes. Up-regulation and apical insertion of MUC17 was followed by shedding of MUC17-containing vesicles. Transmembrane mucins have previously been shown to play a role in the prevention of bacterial colonization by acting as sheddable decoys for encroaching bacteria. Overexpression and increased presentation at the plasma membrane of wild-type MUC17 and its phosphodeficient variant MUC17 S-4492A protected Caco-2 cells against adhesion of enteropathogenic Escherichia coli, indicating that C-terminal phosphorylation of MUC17 may play a functional role in epithelial cell protection. We propose a new function for MUC17 in inflammation, where MUC17 acts as a second line of defense by preventing attachment of bacteria to the epithelial cell glycocalyx in the small intestine.

The invasive cell coat at the microsporidian Trachipleistophora hominis-host cell interface contains secreted hexokinases.

Microsporidia are obligate intracellular parasites causing significant disease in humans and economically important animals. In parallel to their extreme genetic reduction, Microsporidia have evolved novel mechanisms for exploiting host metabolism. A number of microsporidians confer secretion of otherwise cytosolic proteins by coding for signal peptides that direct entry into the endoplasmic reticulum. The human pathogen Trachipleistophora hominis encodes for four hexokinases, three of which have signal peptides at the N-terminus. Here, we localized hexokinase 2 and hexokinase 3 through developmental stages of T. hominis using light and electron microscopy. Both proteins were concentrated in an extracellular coat previously termed the plaque matrix (PQM). The PQM (containing hexokinases) was morphologically dynamic, infiltrating the host cytoplasm predominantly during replicative stages. Throughout development the PQM interacted closely with endoplasmic reticulum that was demonstrated to be active in membrane protein biosynthesis and export. The impact of hexokinase on the host metabolism was probed using the fluorescent analog of glucose, 2-NBDG, which displayed spatially restricted increases in signal intensity at the parasite/vacuole surface, coincident with hexokinase/PQM distribution. Gross metabolic aberrations, measured using metabolic profiling with the Seahorse XF Analyzer, were not detectable in mixed stage cocultures. Overall, these results highlight a role for the extended cell coat of T. hominis in host-parasite interactions, within which secreted hexokinases may work as part of a metabolic machine to increase glycolytic capacity or ATP generation close to the parasite surface.

Preventing biofilm formation and associated occlusion by biomimetic glycocalyxlike polymer in central venous catheters.

The use of catheters and other implanted devices is constantly increasing in modern medicine. Although catheters improve patients' healthcare, the hydrophobic nature of their surface material promotes protein adsorption and cell adhesion. Catheters are therefore prone to complications, such as colonization by bacterial and fungal biofilms, associated infections, and thrombosis. Here we describe the in vivo efficacy of biologically inspired glycocalyxlike antiadhesive coatings to inhibit Staphylococcus aureus and Pseudomonas aeruginosa colonization on commercial totally implantable venous access ports (TIVAPs) in a clinically relevant rat model of biofilm infection. Although noncoated TIVAPs implanted in rats were heavily colonized by the 2 biofilm-forming pathogens with a high percentage of occlusion, coating TIVAPs reduced their initial adherence and subsequently led to 4-log reduction in biofilm formation and reduced occlusion. Our antiadhesive approach is a simple and generalizable strategy that could be used to minimize clinical complications associated with the use of implantable medical devices.

Quorum sensing detected by atomic force microscopy imaging of corrals surrounding multicellular arrangement of bacteria.

Connectivity of the glycocalyx covering of small communities of Acidithiobacillus ferrooxidans bacteria deposited on hydrophilic mica plates was imaged by atomic force microscopy. When part of the coverage was removed by water rinsing, an insoluble structure formed by corrals surrounding each individual bacterium was observed. A collective ring structure with clustered bacteria (>or=3) was observed, which indicates that the bacteria perceived the neighborhood in order to grow a protective structure that results in smaller production of exopolysaccharides material. The most surprising aspect of these collective corral structures was that they occur at a low bacterial cell density. The deposited layers were also analyzed by confocal Raman microscopy and shown to contain polysaccharides, protein, and glucoronic acid.

Characterization of mucosal biofilms on human adenoid tissues.

OBJECTIVES: To demonstrate the presence of mucosal biofilm in adenoid tissue using double staining for visualization of both the bacterial matrix and the bacterial cells. To identify bacterial species present on the surface of the studied adenoids. STUDY DESIGN: Prospective study. METHODS: A total of 39 specimens of adenoidectomy were removed from children with chronic and/or recurrent otitis media. The specimens were prepared for light microscopy using Gram staining, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Double staining was performed with CLSM to visualize both the bacteria and the glycocalyx matrix. Nine adenoids on which bacterial biofilms were visualized with CLSM were used for identification of bacterial species by 16S-DNA polymerase chain reaction (PCR) amplification and homology analysis. RESULTS: Of the 39 adenoids investigated, 22 (54%) showed evidence of mucosal biofilms. Gram staining, SEM and CLSM showed the presence of bacterial cells, organized in bacterial microcolonies. CLSM with double staining demonstrated mucosal biofilms by showing the presence of both bacteria and the glycocalyx. The use of 16S-DNA polymerase chain reaction (PCR) amplification and subsequent sequence analyses identified the presence of Corynebacterium argentoratense, Streptococcus salivarius, Micrococcus luteus, and Staphylococcus aureus. CONCLUSIONS: This study demonstrates that adenoid tissue in children with chronic or/and recurrent otitis media contains mucosal biofilms in 54% of the cases. The existence of living bacteria has been demonstrated. Further studies are required to describe the panel of bacteria that can be harbored in the biofilms present in adenoids and the mechanisms involved in the physiopathology of otitis prone children.

Demonstration of bacterial cells and glycocalyx in biofilms on human tonsils.

OBJECTIVES: To demonstrate mucosal biofilms in human tissue by direct visualization of bacteria and glycocalyx using confocal laser scanning microscopy with double fluorescent staining on tonsils and to compare the findings with the results of scanning electron microscopy analysis. DESIGN: Prospective study. SETTING: Tertiary university-based referral center. PATIENTS: Twenty-four tonsils were obtained from children with chronic or recurrent tonsillitis. INTERVENTIONS: Tonsils were prepared for analysis by scanning electronic microscopy and by confocal laser scanning microscopy. MAIN OUTCOME MEASURES: Double fluorescent staining for confocal laser scanning microscopy consisted of propidium iodide staining to detect bacterial cells and fluorescein isothiocyanate concanavalin A staining to detect the glycocalyx matrix. Images were analyzed for characteristic biofilm morphologic features by 3 investigators who evaluated the images independently in a blinded retrospective manner. Consensus of all observers was required to demonstrate the presence of a biofilm in a specimen. RESULTS: Findings from analyses using scanning electronic microscopy suggested the presence of biofilm formations on tonsils by showing bacterial cells in microcolonies. Double-staining technique using confocal laser scanning microscopy showed bacterial cells and the glycocalyx matrix, providing visual evidence for the presence of biofilms on tonsils. CONCLUSION: Using a novel visualization approach in single sections of human mucosal tissue, the presence of biofilms was demonstrated on tonsils in most (17/24 [70.8%]) patients with tonsillitis.

Ultrastructure of the adhesion of bacteria to the epithelial cell membrane of three-day postnatal rat tongue mucosa: a transmission and high-resolution scanning electron microscopic study.

Togue mucosa surface of 3-day postnatal rats was examined under transmission electron microscopy (TEM) and high-resolution scanning electron microscopy (HRSEM). For HRSEM analysis, the specimens were fixed in the same solution for 24 h, postfixed in 2% osmiun tetroxide, critical-point dried and coated with platinum-palladium. For TEM analysis, the specimens were fixed using modified Karnovsky solution and embedded in Spurr resin. The results revealed the presence of numerous microplicae in the membrane surface of keratinized epithelial cells to which groups of bacteria were attached. These bacteria were staphylococcus and coccus organized either in rows or at random, which were visualized in three-dimensional HRSEM images. At high magnification, the TEM images revealed the adhesion of bacteria to the cell membrane through numerous filamentous structures comprising the glycocalyx. The fine fibrillar structures rising from each bacterium and from cell membrane were clearly seen. These characteristics on bacteria structure may be used for future control or prevention of bacterial diseases and for installation of the oral native flora.

Ultrastructure of the adhesion of bacteria to the epithelial cell membrane of three-day postnatal rat tongue mucosa: a transmission and high-resolution scanning electron microscopic study

Togue mucosa surface of 3-day postnatal rats was examined under transmission electron microscopy (TEM) and high-resolution scanning electron microscopy (HRSEM). For HRSEM analysis, the specimens were fixed in the same solution for 24 h, postfixed in 2 percent osmiun tetroxide, critical-point dried and coated with platinum-palladium. For TEM analysis, the specimens were fixed using modified Karnovsky solution and embedded in Spurr resin. The results revealed the presence of numerous microplicae in the membrane surface of keratinized epithelial cells to which groups of bacteria were attached. These bacteria were staphylococcus and coccus organized either in rows or at random, which were visualized in three-dimensional HRSEM images. At high magnification, the TEM images revealed the adhesion of bacteria to the cell membrane through numerous filamentous structures comprising the glycocalyx. The fine fibrillar structures rising from each bacterium and from cell membrane were clearly seen. These characteristics on bacteria structure may be used for future control or prevention of bacterial diseases and for installation of the oral native flora.

A superfície lingual de ratos de três dias de idade foi examinada em microscópia eletrônica de transmissão (MET) e em microscópia eletrônica varredura de alta resolução (MEVAR). Para o método de MEVAR, os espécimes foram fixados na mesma solução por 24 h, pós fixados em solução de tetróxido de ósmio a 2 por cento, secos em ponto crítico e cobertos com platina- paládio. Para análise em MET, os espécimes foram fixados utilizando-se solução de Karnovsky modificada e emblocadas em resina Spurr. Os resultados mostraram a presença de numerosas micropregas na membrana superficial das células epiteliais queratinizadas, nas quais estavam aderidos grupos de bactérias. Estas bactérias eram estafilococos e cocos, organizados em fileiras ou a esmo, e puderam ser observadas em imagens tri-dimensionais em MEVAR. Em maiores aumentos, as imagens em MET revelaram a adesão de bactérias nas células por meio de numerosas estruturas filamentares compondo o glicocálice. As delicadas estruturas filamentares na periferia das bactérias e das células foram nitidamente identificadas. Estas características da estrutura bacteriana podem ser utilizadas, no futuro, para controle e prevenção de doenças bacteriana, bem como para a instalação da flora oral nativa.

Alterations in the intestinal glycocalyx and bacterial flora in response to oral indomethacin.

Nonsteroidal anti-inflammatory drugs (NSAIDs), used extensively in clinical medicine, tend to cause adverse effects in the gastrointestinal tract. Earlier work has shown that oral administration of indomethacin produced oxidative damage in the small intestine and attenuation of the glycocalyx layer of the mucosa. The present study assessed, in greater detail, the alterations produced in the glycocalyx of rat small intestinal mucosa in response to indomethacin, with specific reference to surfactant-like particles (SLP) and brush border membranes (BBM). Changes in gut flora in response to the drug were also studied, as it has been shown that luminal bacteria play a role in the pathogenesis of NSAID-induced intestinal damage. The levels of sugars such as sialic acid, fucose, hexose and hexosamine were increased in SLP and decreased in the BBM following indomethacin treatment, with the effects being maximal 24h after the administration of the drug. The composition of lipids in the SLP was also found to be altered. There was a significant increase in the number of bacteria in the luminal contents of the small intestine and caecum in these animals, as compared with controls. The number of bacteria adherent to the intestinal mucosa was also significantly higher in the drug-treated group. In vitro studies revealed that there was an increased tendency for bacteria to adhere to SLP isolated from indomethacin-treated rats. These results suggest that alterations in glycosylation of SLP and BBM in response to indomethacin, along with qualitative and quantitative changes in the luminal bacterial flora, may facilitate translocation of bacteria into the mucosa. These changes may contribute to the enteropathy observed as a result of NSAID treatment.

Carcinoembryonic antigen (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1), apically expressed on human colonic M cells, are potential receptors for microbial adhesion.

In the human gut mucosa, specialized M cells deliver intact foreign macromolecules and commensal bacteria from the lumen to organized mucosal lymphoid tissues triggering immune responses. M cells are also major sites of adhesion and invasion for enteric pathogens. The molecular features of M cell apical surfaces that promote microbial normal attachment are still largely unknown. We have demonstrated previously that in the human colonic epithelium, carcinoembryonic antigen (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1) are integral components of the apical glycocalyx which participate in epithelial-microbial interactions. In this study, based on the reactivity of specific monoclonal antibodies and on immunoelectron microscopy, we show that M cells of human colonic solitary lymphoid follicles express CEA and CEACAM1 on the apical surface. Recently these highly glycosylated molecules have been characterized as protein receptors for different bacteria. This leads us to propose a role for CEA and CEACAM1 in the adherence of enteric bacteria to the apical membrane of colonic M cells. We also hypothesize that, unlike colonic enterocytes, M cells lack the defense mechanism that eliminates CEA and CEACAM1 upon microbial binding and which is based on vesiculation of microvillus plasma membrane.

Confocal laser scanning microscopic observation of glycocalyx production by Staphylococcus aureus in mouse skin: does S. aureus generally produce a biofilm on damaged skin?

BACKGROUND: Bacteria that adhere to damaged tissues encase themselves in a hydrated matrix of polysaccharides, forming a slimy layer known as a biofilm. This is the first report of detection of glycocalyx production by Staphylococcus aureus using confocal laser scanning microscopy (CLSM) on damaged skin tissues. OBJECTIVES: To analyse glycocalyx production by S. aureus cells on damaged skin tissues and the influence of polymorphonuclear leucocytes (PMNs) and various antimicrobial agents on its production using CLSM in cyclophosphamide (Cy)-treated (neutropenic) or non-Cy-treated (normal) mice. METHODS: S. aureus cells were inoculated on damaged skin tissues in neutropenic or normal mice with or without topical application of antimicrobial agents. S. aureus cells were stained with safranine, and positive staining with fluorescein isothiocyanate-conjugated concanavalin A was considered to indicate the presence of glycocalyx. RESULTS: All S. aureus cells tested on damaged skin tissues formed microcolonies encircled by glycocalyx. The colony counts of S. aureus cells on croton oil dermatitis in normal mice treated with 2% fusidic acid ointment were about 100 times lower than those in neutropenic mice (control). CONCLUSIONS: As S. aureus cells can generally produce a biofilm on damaged skin tissues, antimicrobial agents may not eradicate S. aureus cells without the help of PMNs. S. aureus glycocalyx may play a crucial role in colonization and adherence to damaged skin tissues.

Participation of bacterial biofilms in refractory and chronic periapical periodontitis.

The aim of this study was to examine morphologically the participation of extraradicular biofilm in refractory periapical periodontitis. Six teeth and five extruded root filling gutta-percha points associated with refractory periapical periodontitis were investigated by scanning electron microscope. In nine of 11 samples examined, bacterial biofilms were seen at the extraradicular area. The gutta-percha surface was covered with glycocalyx-like structures, and filaments, long rods, and spirochete-shaped bacteria were predominant in the extraradicular sites. Planktonic cells, which were filaments and spirochete-shaped bacteria, emigrated from the glycocalyx structures in some spots. In the extracted teeth, biofilm consisting of both bacteria and glycocalyx-like structures were observed on the periapical root surfaces. Next to the residual periodontal ligament, a few filaments, rods, and fusiforms were attached on the healthy cementum surface. The present findings suggested that bacterial biofilms formed in the extraradicular areas were related to refractory periapical periodontitis.

Identification of periodontal disease-associated bacteria in the "plaque-free zone".

BACKGROUND: Subgingival plaque bacteria live within a biofilm covered with glycocalyx, and little is known of the bacterial species associated with biofilm formation at the bottom of human periodontal pockets, the so-called "plaque-free zone"(PFZ). METHODS: Seventy-seven extracted teeth from 56 patients with severe advanced adult periodontitis were examined. Porphyromonas gingivalis, Campylobacter rectus, Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans, Treponema denticola, Prevotella nigrescens, and Actinomyces viscosus were examined by scanning immunoelectron microscopic techniques, using both secondary and back-scattered imaging, with rabbit antibodies specific for each bacteria. RESULTS: Secondary electron images showed that rods, filaments, and spirochete-shaped bacteria formed small aggregates in the PFZ. Some of the bacteria were covered with an amorphous film-like structure. By back-scattered electron imaging, positive reactions with anti-P. gingivalis were found in 8 of 13 samples examined, and film-like structures coated several cells of 6 positive samples examined. Labeled cells with anti-C. rectus, anti-T. denticola and anti-P. nigrescens were detected in 3 of 11, 5 of 10, and 1 of 8 samples examined. A. viscosus were found in 6 of 11 of the samples. A. viscosus tended to overlay the amorphous capsula and aggregate. F. nucleatum and A. actinomycetemcomitans were not detected in any samples examined. CONCLUSIONS: These findings indicated that P. gingivalis, C. rectus, T. denticola, P. nigrescens, and A. viscosus were present in the PFZ, and that some specified bacteria were possibly related to plaque-biofilm formation of subgingival plaque.

Ruthenium red and the bacterial glycocalyx.

Ruthenium red, a promising cationic reagent for electron microscopy (EM), has long been an important tool in histology. The reagent was initially used by botanists as a semispecific stain for pectic substances, but it has gradually been embraced by investigators in microbiology and the animal sciences as a stain for anionic glycosylated polymeric substances. Luft developed a reliable method and demonstrated that ruthenium red was a useful reagent for visualizing ultrastructural detail. Many investigators, using modifications of Luft's approach, have identified numerous applications for this important reagent. Ruthenium red has been used to show the ultrastructural detail of bacterial glycocalyces. Strong, sharp and consistent observations of this ultrastructural component of the bacterial cell have given a better understanding its fibrous anionic matrix. Any variations in staining owing to artifactual alteration of the fine delicate ultrastructural features have been overcome by incorporation of diamine lysine into ruthenium red methods, thus providing flexible processing times under less than ideal laboratory sampling conditions. Ruthenium red has broad utility in the biological sciences, and in combination with lysine, it is an excellent EM stain for enhanced visualization of bacterial glycocalyx from culture or from clinical specimens.

The gut's role in metabolism, mucosal barrier function, and gut immunology.

The gastrointestinal tract functions not only to absorb nutrients, it also plays an important immunologic role during health and critical illness. Under experimental and certain clinical conditions, stimulating the gut attentuates the stress response and avoids mucosal atrophy and increases permeability. Gut stimulation prevents atrophy of the gut-associated lymphoid tissue, the body's major defender of moist mucosal surfaces. A better understanding of gut function and improved nutrient delivery has clinical implications in the treatment of critically ill patients.

Visualization of bacterial glycocalyx with a scanning electron microscope.

Using a new technique for scanning electron microscopic preparation, bacterial glycocalyx is seen in its natural, highly hydrated state for the first time. Visual images of the glycocalyx obtained from these preparations are a marked departure from the visual images of glycocalyx obtained previously with conventional scanning electron microscopic analysis. The dominating presence of glycocalyx visualized in its naturally hydrated state gives credence to the role of bacterial glycocalyx as a mechanical barrier to host defenses and antibiotics and supports the role of glycocalyx as a significant factor in bacterial virulence.