Glycoconjugates for the recognition of Bacillus spores

Carbohydrates act as ligands in many biological processes, including the folding and secretion of proteins, cell-cell recognition, adhesion, and sporulation in the Bacillus genus. Fluorescent-labeled disaccharide glycoconjugates have been applied to evaluate binding to bacterial spores assuming that the spore surface is covered with carbohydrates. This study has shown that specific recognition of bacterial spores is based on interactions between disaccharide glycoconjugates acting as ligands and monosaccharide units expressed on the exterior of bacterial spores. Using fluorophore-assisted carbohydrate electrophoresis (FACE), carbohydrates that are expressed on the exterior of the spores were enumerated. The findings have an impact on how to improve ligand selection, essential for sensor development. In addition, the findings provide new information for inhibition of bacterial spores, and in general, demonstrate how carbohydrates function as recognition signals in nature.     

Lactobacillus johnsonii La1 shares carbohydrate-binding specificities with several enteropathogenic bacteria

The carbohydrate-binding specificities of the probiotic lactic acid bacterium Lactobacillus johnsonii La1 (a health-beneficial bacterial strain able to be incorporated into the human intestinal microflora) were investigated in vitro. First various soluble complex carbohydrates were tested as potential inhibitors of the strain adhesion onto Caco-2 intestinal epithelial cells, and then bacterial binding to glycolipids immobilized on TLC plates was probed. Two major carbohydrate-binding specificities of Lactobacillus johnsonii La1 were identified. A first one for an Endo-H treated yeast cell wall mannoprotein carrying mainly O:-linked oligomannosides, and a second one for the gangliotri- and gangliotetra-osylceramides (asialo-GM1). Similar carbohydrate-binding specificities are known to be expressed on cell surface adhesins of several enteropathogens, enabling them to adhere to the host gut mucosa. These findings corroborate the hypothesis that selected probiotic bacterial strains could be able to compete with enteropathogens for the same carbohydrate receptors in the gut.     

Blocking adhesion of cancer cells to endothelial cell types by S. agalactiae type-specific polysaccharides

By using immortalized and normal endothelial cells, we were able to detect inhibitory effects of type specific polysaccharides from Streptococcus agalactiae on adhesion of cancer cells to endothelial cells, which is an essential step of cancer metastasis. The inhibition was probably due to specific structures of the bacterial polysaccharides, since the structures of the saccharides are very similar to those of cancer specific sialyl Lewis carbohydrates (sialyl Le^a and Le^x) which bind to ELAM-1 of endothelial cells. This result indicated that the bacterial polysaccharides from S. agalactiae could be very useful and hopeful as cancer metastasis inhibitors.Abbreviations HUVECs human umbilical cord vein endothelial cells - ELAM-1 endothelial leukocyte adhesion molecule-1     

Specific cell behavior of human fibroblast onto carbohydrate surface detected by glycoblotting films

We synthesized an aminooxyl polymer that is reactive with the reduced end of carbohydrates using our sugar-displaying approach. The carbohydrates were easily immobilized on the polymer film (glycoblotting film) by simple immersion in a in sugar solution through stable oxime bond. The in vitro behaviors of human fibroblasts on the carbohydrate-coated surface were investigated. The adhesion of human fibroblasts on the cellobiose- and cellotriose-coated surfaces was much greater than on the other coated surfaces and the noncoated surface. This result indicated that simple structural differences in carbohydrates induced biological changes in human cells, especially cell adhesion. Our approach provides a high-throughput assay system for carbohydrate-related cell adhesion and proliferation.     

Characteristics of the adhesion of PCC Lactobacillus fermentum VRI 003 to Peyer's patches

The characteristics of the adhesion of PCC Lactobacillus fermentum VRI 003 to Peyer's patches was studied in vitro. The adhesion of L. fermentum 003 was strongly inhibited in the presence of d-mannose and methyl-alpha-d-mannoside although other carbohydrates tested, such as N-acetyl-glucosamine, d-galactose, d-glucose and l-fucose, did not affect the adhesion. Lactobacillus fermentum 003 was shown to strongly attach to mannose immobilized on a surface using BSA, suggesting that L. fermentum 003 specifically adhered to mannose-containing molecule(s). Pretreatment of L. fermentum 003 with proteinase K and trypsin decreased the adhesive capacity and bacterial surface extracts diminished adhesion of L. fermentum 003 indicating that cell surface proteins are involved in adhesion to Peyer's patches. It was concluded that a mannose-specific protein mediated adhesion of L. fermentum 003 to the Peyer's patches.     

Human milk glycoproteins inhibit the adherence of Salmonella typhimurium to HeLa cells

The ability of human milk, as well as its protein fractions, to inhibit the adhesion and invasion of Salmonella typhimurium to HeLa cells was investigated. The results revealed that milk secretory immunoglobulin A (sIgA) inhibited neither the adherence nor the bacterial invasion; however, free secretory component and lactoferrin inhibited the bacterial adhesion and interacted with several bacterial proteins. Our data indicated that glycoproteins such as free secretory component and lactoferrin could act as protective compounds against infant enteric diseases, possibly binding to bacterial surface and blocking adhesion, the primordial step of S. typhimurium infection.     

Carbohydrate Coating Reduces Adhesion of Biofilm-Forming Bacillus subtilis to Gold Surfaces

The growth of bacterial biofilms in pipes and food tanks causes severe problems in industry. Biofilms growing on medical implants or catheters are of great concern, as they can cause serious infections and decrease the functionality of the medical device. The prevention of bacterial adhesion—the first step in colonization and biofilm formation—is therefore very important. Current research comprises alterations in surface properties, the prevention of adhesin biosynthesis, inhibition with receptor analogs, or the development of anti-adhesive vaccines. We present a new approach that allows us to study bacterial adhesion with high sensitivity in real-time while testing several different surfaces in parallel. Using the cantilever-array technique we demonstrate that coating of gold surfaces with mono- or disaccharides results in a reduction of the bacterial adhesion of the biofilm-forming bacterium Bacillus subtilis NCIB 3610 to these gold surfaces. This reduction in bacterial adhesion is independent of the studied carbohydrate. Using several mutant strains, we investigate the underlying molecular interactions, and our results suggest that adhesion to gold surfaces is mediated by thiol groups present in proteins of the bacterial cell membrane or biofilm matrix proteins expressed at low levels by the wild-type strain. Furthermore, our data indicate that the adhesion of B. subtilis NCIB 3610 to carbohydrate-coated gold surfaces is facilitated by interactions between carbohydrates installed on the cantilever gold surface and an exopolysaccharide expressed by this strain. Understanding general and specific contributions of molecular interactions mediating bacterial adhesion will enable its prevention in the future.     

Influence of carp intestinal mucus molecular size and glycosylation on bacterial adhesion

The first step of the pathogenesis of many infectious diseases is the colonisation of the mucosal surface by the pathogen. Bacterial colonisation of the mucosal surface is promoted by adherence to high molecular weight mucus glycoproteins. We examined the effect of carp intestinal mucus glycoproteins on the adhesion of different bacteria. The bacteria used were 3 strains of Aeromonas hydrophila, and A. salmonicida, Edwardsiella tarda and Yersinia ruckeri. All bacteria adhered to mucus, but at varying intensities. All tested bacteria adhered best to molecules of 670 to 2000 kDa in size, less to molecules larger than 2000 kDa and weakest to molecules of 30 to 670 kDa. In general, bacteria that showed a stronger adhesion to intestinal mucus were cytotoxic to cells in vitro, and bacteria that showed a weaker adhesion to intestinal mucus did not lead to alterations of monolayers of EPC-cells. Furthermore, the involvement of glycan side chains of the glycoproteins for bacterial adhesion was analysed for one A. hydrophila strain. After cleavage of terminal sugar residues by treatment of mucus glycoproteins with different glycosidases, binding of bacteria was modulated. When mannose was cleaved off, adhesion significantly increased. Blocking of glycan receptors by incubation of bacteria with different oligosaccharides had no clear effect on bacterial binding to mucus glycoproteins. Our results suggest that bacteria interact with carbohydrate side chains of mucus glycoproteins, and that the carbohydrates of the core region are involved in bacterial binding.     

Statistical analysis of the Bacterial Carbohydrate Structure Data Base (BCSDB): Characteristics and diversity of bacterial carbohydrates in comparison with mammalian glycans

Background  

There are considerable differences between bacterial and mammalian glycans. In contrast to most eukaryotic carbohydrates, bacterial glycans are often composed of repeating units with diverse functions ranging from structural reinforcement to adhesion, colonization and camouflage. Since bacterial glycans are typically displayed at the cell surface, they can interact with the environment and, therefore, have significant biomedical importance.     

Biochemical composition of the marine conditioning film: implications for bacterial adhesion

The conditioning film formed on glass panels was analysed for total carbohydrates (CFCHO), total proteins (CFP) and total uronic acids (CFURA). The influence of these compounds on the adhesion of three marine bacterial cultures, Pseudomonas sp. CE-2, Pseudomonas sp. CE-10 and Bacillus sp. SS-10 was also evaluated. One-way analysis of variance suggested a significant increase in the attachment of all three cultures to conditioned glass panels. Moreover, CE-2 (r = 0.874) and CE-10 (r = 0.879) showed a significant positive correlation with CFCHO. Conversely, SS-10 (r = -0.69) showed a significant negative correlation with CFCHO. Backward multiple linear regression analysis indicated that CFCHO were the most predictive component of the conditioning film in explaining bacterial adhesion to the conditioned glass panels.     

Functional dissection of the phosphotransferase system provides insight into the prevalence of Faecalibacterium prausnitzii in the host intestinal environment

Faecalibacterium prausnitzii is a dominant member of healthy human colon microbiota, regarded as a beneficial gut bacterium due to its ability to produce anti-inflammatory substances. However, little is known about how F. prausnitzii utilizes the nutrients present in the human gut, influencing its prevalence in the host intestinal environment. The phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) is a widely distributed and highly efficient carbohydrate transport system found in most bacterial species that catalyses the simultaneous phosphorylation and import of cognate carbohydrates; its components play physiological roles through interaction with other regulatory proteins. Here, we performed a systematic analysis of the 16 genes encoding putative PTS components (2 enzyme I, 2 HPr, and 12 enzyme II components) in F. prausnitzii A2-165. We identified the general PTS components responsible for the PEP-dependent phosphotransfer reaction and the sugar-specific PTS components involved in the transport of two carbohydrates, N-acetylglucosamine and fructose, among five enzyme II complexes. We suggest that the dissection of the functional PTS in F. prausnitzii may help to understand how this species outcompetes other bacterial species in the human intestine.     

Heparan sulfate-like proteoglycans mediate adhesion of human malignant melanoma A375 cells to P-selectin under flow

Selectins, a family of cell adhesion molecules, bind to sialylated and fucosylated carbohydrates, such as sialyl Lewisx (SLex) and its derivatives, as their minimal recognition motif. Here we report that P-selectin bound to human malignant melanoma A375 cells and mediated their adhesion under flow. However, probing with a specific Ab failed to detect any apparent expression of SLex. This finding was bolstered by reduced expression of alpha-1,3-fucosyltransferase VII mRNA and by absence of the cell surface expression of P-selectin glycoprotein ligand-1. Instead, they expressed heparan sulfate-like proteoglycans on their cell surfaces. Treatment with beta-d -xyloside (a proteoglycan biosynthesis inhibitor) or heparinases could reduce the binding of these cells to P-selectin. In the competition assays, heparin, but not other proteoglycans, could abolish the P-selectin recognition. Further, we found that P-selectin could bind specifically to human tongue squamous cancer Tca-8113 cells, which had negative staining of SLex but positive staining of heparan sulfates. Both beta-d -xyloside and heparinases could reduce the binding of P-selectin to Tca-8113 cells. Our results thus indicate that heparan sulfate-like proteoglycans can mediate adhesion of certain types of non-blood borne, "epithelial-like" human cancer cells to P-selectin.     

Human Protein C inhibits selectin-mediated cell adhesion: role of unique fucosylated oligosaccharide

The human anticoagulant factor, Protein C, is a plasma glycoproteinthat has reported anti-ischaemic and anti-inflammatory properties.To explore potential mechanisms for these reported activities,we examined the effect of Protein C on the process of cell adhesionto vascular endothelial cells, which plays a critical role duringinflammatory responses. We show that both human plasma-derivedand human cell-produced recombinant Protein C inhibit E-selectin-mediatedcell adhesion. This effect was not mediated through the serineprotease activity of Protein C, but through its carbohydrates.Using oligosaccharides isolated from human cell-produced ProteinC, we have defined a polylactosamine structural determinantthat inhibits adhesion. This uncharged detenminant appears tobe a more potent ligand for E-selectin than the sialylated LewisX antigen. Our data suggest a potential mechanism for the reportedanti-inflammatory effects of Protein C and describe a new ligandfor selectin-mediated adhesion. cell adhesion fucosylated oligosaccharide human Protein C/PC-293 determinant selectin     

Disrupting the transmission of a vector-borne plant pathogen

Approaches to control vector-borne diseases rarely focus on the interface between vector and microbial pathogen, but strategies aimed at disrupting the interactions required for transmission may lead to reductions in disease spread. We tested if the vector transmission of the plant-pathogenic bacterium Xylella fastidiosa was affected by three groups of molecules: lectins, carbohydrates, and antibodies. Although not comprehensively characterized, it is known that X. fastidiosa adhesins bind to carbohydrates, and that these interactions are important for initial cell attachment to vectors, which is required for bacterial transmission from host to host. Lectins with affinity to substrates expected to occur on the cuticular surface of vectors colonized by X. fastidiosa, such as wheat germ agglutinin, resulted in statistically significant reductions in transmission rate, as did carbohydrates with N-acetylglucosamine residues. Presumably, lectins bound to receptors on the vector required for cell adhesion/colonization, while carbohydrate-saturated adhesins on X. fastidiosa's cell surface. Furthermore, antibodies against X. fastidiosa whole cells, gum, and afimbrial adhesins also resulted in transmission blockage. However, no treatment resulted in the complete abolishment of transmission, suggesting that this is a complex biological process. This work illustrates the potential to block the transmission of vector-borne pathogens without directly affecting either organism.     

Prevention of Pseudomonas aeruginosa adhesion by electric currents

The process of controlling bacterial adhesion using an electric current deserves attention because of its ease of automation and environmentally friendly nature. This study investigated the role of electric currents (negative, positive, alternating) for preventing adhesion of Pseudomonas aeruginosa and achieving bacterial inactivation. Indium tin oxide (ITO) film was used as a working electrode to observe adhesion and inactivation under electric polarization. Electric current types were classified into negative, positive, and alternating current. The working electrode acted as a cathode or anode by applying a negative or positive current, and an alternating current indicates that the negative current was combined sequentially with the positive current. The numbers of adhered cells were compared under a flow condition, and the in situ behavior of the bacterial cells and the extent of their inactivation were also investigated using time-lapse recording and live/dead staining, respectively. The application of a negative current prevented bacterial adhesion significantly (～81% at 15.0 μA cm(-2)). The positive current did not significantly inhibit adhesion (<20% at 15.0 μA cm(-2)), compared to the nonpolarized case. The alternating current had a similar effect as the negative current on preventing bacterial adhesion, but it also exhibited bactericidal effects, making it the most suitable method for bacterial adhesion control.     

Archaea and Fungi of the Human Gut Microbiome: Correlations with Diet and Bacterial Residents

Diet influences health as a source of nutrients and toxins, and by shaping the composition of resident microbial populations. Previous studies have begun to map out associations between diet and the bacteria and viruses of the human gut microbiome. Here we investigate associations of diet with fungal and archaeal populations, taking advantage of samples from 98 well-characterized individuals. Diet was quantified using inventories scoring both long-term and recent diet, and archaea and fungi were characterized by deep sequencing of marker genes in DNA purified from stool. For fungi, we found 66 genera, with generally mutually exclusive presence of either the phyla Ascomycota or Basiodiomycota. For archaea, Methanobrevibacter was the most prevalent genus, present in 30% of samples. Several other archaeal genera were detected in lower abundance and frequency. Myriad associations were detected for fungi and archaea with diet, with each other, and with bacterial lineages. Methanobrevibacter and Candida were positively associated with diets high in carbohydrates, but negatively with diets high in amino acids, protein, and fatty acids. A previous study emphasized that bacterial population structure was associated primarily with long-term diet, but high Candida abundance was most strongly associated with the recent consumption of carbohydrates. Methobrevibacter abundance was associated with both long term and recent consumption of carbohydrates. These results confirm earlier targeted studies and provide a host of new associations to consider in modeling the effects of diet on the gut microbiome and human health.     

Safe as mother's milk: Carbohydrates as future anti-adhesion drugs for bacterial diseases

The majority of infectious diseases are initiated by adhesion of pathogenic organisms to the tissues of the host. In many cases, this adhesion is mediated by lectins present on the surface of the infectious organism that bind to complementary carbohydrates on the surface of the host tissues. Lectin-deficient mutants often lack ability to initiate infection. Soluble carbohydrates recognized by the bacterial lectins block the adhesion of the bacteria to animal cells in vitro. Moreover, they have also been shown to protect against experimental infection by lectin-carrying bacteria in different organs of mammals such as mice, rabbits, calves and monkeys.In a phase II clinical trial, a pentasaccharide shown to have anti-adhesive activity against Streptococcus pneumoniae and Hemophilus influenzae in vitro failed to protect young children from nasopharyngeal colonization with these organisms and from developing otitis media. This could be because insufficient drug was delivered via nasal spray, because bacteria express multiple specificities, the inhibition of which may require a cocktail of oligosaccharides, or because children have different carbohydrate receptors from those of adults. The results of a clinical trial in which N-acetylneuraminyl(2-3)lactose was administered orally to Helicobacter pylori positive patients in an effort to reduce or eradicate bacterial colonization, are awaited with interest.Although the high cost of production of the required oligosaccharides is falling with the recent introduction of enzymatic methods of synthesis, new technologies, in particular the use of engineered bacteria, promise to lower it even further. Attachment of the oligosaccharides to soluble polymeric carriers will increase greatly their effectiveness as anti-adhesion agents. There is no doubt that anti-adhesive oligosaccharides will in the near future join the arsenal of drugs for the therapy of bacterial diseases.     

Fimbriae mediated nonspecific adhesion of Salmonella typhimurium to mineral particles

The adhesion of cells of Salmonella typhimurium to albite, biotite, felspar, magnetite and quartz was correlated to the presence of fimbriae and degree of hydrophobicity and charge of the bacterial surface. It was found that the presence of fimbriae resulted in a higher degree of adhesion compared to adhesion of nonfimbriated cells. The significance of the physico-chemical characteristics of fimbriae was shown by a direct linearity between high hydrophobicity of fimbriated cells and degree of adhesion to the mineral particles. Fimbriated cells exhibited higher negative as well as positive surface charge as compared to nonfimbriated cells. Adhesion to several of the minerals was shown to be independent of the extent of negative charges on the bacterial surfaces. A high degree of adhesion to biotite, possibly due to a combination of characteristics of the particles, was not related to either bacterial fimbriation or a physico-chemical characteristic of the bacterial surface. The results of the nonspecific adhesion observed are discussed in terms of available binding sites and distribution of physico-chemical characteristics on the bacterial cell surface structures.     

Anti-adhesive glycoproteins in echinoderm mucus secretions

Marine invertebrates produce a large variety of mucus secretions which are rich in glycoproteins. As part of our studies of natural antifouling mechanisms, mucus secretions from the starfish Marthasterias glacialis and Porania pulvillus and the brittlestar Ophiocomina nigra have been used to characterise the structure and function of some of the glycoproteins present in these secretions. Mucus was collected from all three species and fractionated by size exclusion chromatography. A high molecular weight glycoprotein fraction was collected from each species. Monosaccharide analysis and FTIR demonstrated a composition consistent with a mucin-type glycoprotein. The mucin from M. glacialis and O. nigra inhibited in vitro bacterial adhesion in a dose-dependent manner. In contrast, the mucin from P. pulvillus promoted bacterial adhesion in a dose-dependent manner. All of the mucins inhibited the adhesion of human neutrophils to cultured human vascular endothelial cells (HUVECs) and had no anticoagulant activity. The mucins described here have adhesion-regulating functions that may have a role in the antifouling or feeding mechanisms of the organisms that produce them. These mucins may also be of therapeutic value through their ability to regulate human neutrophil adhesion or bacterial adhesion.     

Development of biosensor-based assays to identify anti-infective oligosaccharides

It is now well accepted that milk oligosaccharides can have a direct inhibitory effect on pathogenic microorganisms by interfering with their adhesion to human cells. Many free oligosaccharides from milk are considered to be soluble receptor analogs of epithelial cell surface carbohydrates and, thus, function as receptor decoys to which pathogens can bind instead of the host. In reality, there are few rapid methods to screen for such oligosaccharides, and much of the research in this area has centered on using human cell line models of infection that are time-consuming. Therefore, a quick and sensitive method is required for detecting the binding of microorganisms to milk oligosaccharides. Our study describes a number of biosensor-based methods to achieve these aims. Our approach involved the exposure of whole bacterial cells to the well-characterized human milk oligosaccharide, 2′-fucosyllactose, immobilized to a pretreated gold chip surface. The technique was validated by screening a range of pathogenic bacteria, including Campylobacter jejuni, to which 2′-fucosyllactose is known to bind. Where binding was detected, its specificity was confirmed by preincubation studies using unlabeled 2′-fucosyllactose. The techniques described represent a quick, cost-effective, and highly reproducible detection method for identifying anti-infective oligosaccharides.