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The role of the hospital environment as contributory to healthcare acquisition of multidrug-resistant organisms (MDROs) is increasingly recognized. Ultraviolet light decontamination can minimize the environmental bioburden, thereby potentially reducing healthcare acquisition. This effect has been demonstrated for typical environmental MDROs, e.g. meticillin-resistant Staphylococcus aureus, vancomycin-resistant entero-cocci, and Clostridioides difficile; however, its role in reducing carbapenem-resistant Enterobacterales (CRE) incidence rates is unclear.To evaluate the impact of continuous ultraviolet light (C-UV) on healthcare acquisition rates of CRE.A 26-month pragmatic, prospective interventional study with addition of C-UV decontamination to standard cleaning was conducted in units at high risk for CRE acquisition. Introduction of C-UV followed a 12 month baseline period, with a two-month wash-in period. Implementation included terminal decontamination at discharge and a novel in-use protocol, whereby rooms occupied for ≥48 h were decontaminated during the course of the patients' in-hospital stay. Incidence density rates of CRE during the intervention period were compared to the baseline period using interrupted time series regression. Rates were adjusted for ward/admission prevalence and analysed according to C-UV protocol.The in-use C-UV protocol demonstrated a significant negative association with the incidence density rate of CRE when adjusting for CRE admission rate (P = 0.0069). CRE incidence density rates decreased significantly during the intervention period (P = 0.042). Non-intervention units demonstrated no change in incidence density rates when adjusting for ward and/or admission prevalence.C-UV decontamination can potentially reduce healthcare acquisition of CRE when implemented with an in-use protocol.

The cell wall contains mannans and glucans that are recognized by the host immune system. In this chapter, we will describe the methods to isolate mannans and glucans from the C. albicans cell wall. In addition, we describe how to determine purity, molecular size, and structure of the mannans and glucans. We also detail how to prepare the carbohydrates for in vitro, ex vivo, or in vivo use by describing endotoxin removal (depyrogenation), derivatization, and labeling and evaluation of bioactivity.

Candida auris, a recently emergent fungal pathogen, has caused invasive infections in health care settings worldwide. Mortality rates approach 60% and hospital spread poses a public health threat. Compared to other Candida spp., C. auris avoids triggering the antifungal activity of neutrophils, innate immune cells that are critical for responding to many invasive fungal infections, including candidiasis. However, the mechanism underpinning this immune evasion has been largely unknown. Here, we show that C. auris cell wall mannosylation contributes to the evasion of neutrophils ex vivo and in a zebrafish infection model. Genetic disruption of mannosylation pathways (PMR1 and VAN1) diminishes the outer cell wall mannan, unmasks immunostimulatory components, and promotes neutrophil engagement, phagocytosis, and killing. Upon examination of these pathways in other Candida spp. (Candida albicans and Candida glabrata), we did not find an impact on neutrophil interactions. These studies show how C. auris mannosylation contributes to neutrophil evasion though pathways distinct from other common Candida spp. The findings shed light on innate immune evasion for this emerging pathogen. IMPORTANCE The emerging fungal pathogen Candida auris presents a global public health threat. Therapeutic options are often limited for this frequently drug-resistant pathogen, and mortality rates for invasive disease are high. Previous study has demonstrated that neutrophils, leukocytes critical for the antifungal host defense, do not efficiently recognize and kill C. auris. Here, we show how the outer cell wall of C. auris promotes immune evasion. Disruption of this mannan polysaccharide layer renders C. auris susceptible to neutrophil killing ex vivo and in a zebrafish model of invasive candidiasis. The role of these mannosylation pathways for neutrophil evasion appears divergent from other common Candida species.

The fungal cell wall serves as the interface between the organism and its environment. Complex carbohydrates are a major component of the Candida albicans cell wall, i.e., glucan, mannan and chitin. β-Glucan is a pathogen associated molecular pattern (PAMP) composed of β-(1 → 3,1 → 6)-linked glucopyranosyl repeat units. This PAMP plays a key role in fungal structural integrity and immune recognition. Glycogen is an α-(1 → 4,1 → 6)-linked glucan that is an intracellular energy storage carbohydrate. We observed that glycogen was co-extracted during the isolation of β-glucan from C. albicans SC5314. We hypothesized that glucan and glycogen may form a macromolecular species that links intracellular glycogen with cell wall β-(1 → 3,1 → 6)-glucan. To test this hypothesis, we examined glucan-glycogen extracts by multi-dimensional NMR to ascertain if glycogen and β-glucan were interconnected. 1H NMR analyses confirmed the presence of glycogen and β-glucan in the macromolecule. Diffusion Ordered SpectroscopY (DOSY) confirmed that the β-glucan and glycogen co-diffuse, which indicates a linkage between the two polymers. We determined that the linkage is not via peptides and/or small proteins. Our data indicate that glycogen is covalently linked to β-(1 → 3,1 → 6) glucan via the β -(1 → 6)-linked side chain. We also found that the glucan-glycogen complex was present in C. dublinensis, C. haemulonii and C. auris, but was not present in C. glabrata or C. albicans hyphal glucan. These data demonstrate that glucan and glycogen form a novel macromolecular complex in the cell wall of C. albicans and other Candida species. This new and unique structure expands our understanding of the cell wall in Candida species.

Biomarkers like procalcitonin (PCT) are an important antimicrobial stewardship tool for critically ill patients. There is little evidence regarding the use of PCT-guided antibiotic algorithms in developing countries. Evidence is also lacking for PCT-based antibiotic algorithms in surgical trauma patients admitted to the intensive care unit (ICU).A prospective, two period cross-over study was conducted in a surgical trauma intensive care unit in South Africa. In the first period, 40 patients were recruited into the control group and antibiotics were discontinued as per standard of care. In the second period, 40 patients were recruited into the procalcitonin group and antibiotics were discontinued if the PCT decreased by ≥ 80% from the peak PCT level, or to an absolute value of less than 0.5 μg/L. Antibiotic duration of treatment was the primary outcome. Patients were followed up for 28 days from the first sepsis event.For the first sepsis event the PCT group had a mean antibiotic duration of 9.3 days while the control group had a mean duration of 10.9 days (There was no significant difference in duration of antibiotic treatment between the two groups. However, the PCT group had more antibiotic free days alive and lower in-hospital mortality compared to the control group.

Background: Biomarkers like procalcitonin (PCT) are an important antimicrobial stewardship tool for critically ill patients. There is little evidence regarding the use of PCT-guided antibiotic algorithms in developing countries. Evidence is also lacking for PCT-based antibiotic algorithms in surgical trauma patients admitted to the intensive care unit (ICU). Methods: A prospective, two period cross-over study was conducted in a surgical trauma intensive care unit in South Africa. In the first period, 40 patients were recruited into the control group and antibiotics were discontinued as per standard of care. In the second period, 40 patients were recruited into the procalcitonin group and antibiotics were discontinued if the PCT decreased by ≥ 80% from the peak PCT level, or to an absolute value of less than 0.5 µg/L. Antibiotic duration of treatment was the primary outcome. Patients were followed up for 28 days from the first sepsis event. Results: For the first sepsis event the PCT group had a mean antibiotic duration of 9.3 days while the control group had a mean duration of 10.9 days (p = 0.10). Patients in the intervention group had higher mean (SD) antibiotic free days alive of 7.7 (6.57) days compared to the control group mean (SD) of 3.8 (5.22) days, (p = 0.004). In-hospital mortality rate was lower in the intervention group (15%) compared to the control group (30%) and was statistically significant (p = 0.045). Conclusion: There was no significant difference in duration of antibiotic treatment between the two groups. However, the PCT group had more antibiotic free days alive and lower in-hospital mortality compared to the control group.&nbsp

Invasive aspergillosis is a serious threat to immunodeficient and critically ill patients caused mainly by the fungus

Bacterial biofilms represent a challenge to the healthcare system because of their resilience against antimicrobials and immune attack. Biofilms consist of bacterial aggregates embedded in an extracellular polymeric substance (EPS) composed of carbohydrate polymers, nucleic acids and proteins. Carbohydrates within P. aeruginosa biofilms include neutral and mannose-rich Psl, and cationic Pel composed of N-acetyl-galactosamine and N-acetyl-glucosamine. Here we show that P. aeruginosa biofilms display ligands for the C-type lectin receptors mannose receptor (MR, CD206) and Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209). Binding of MR and DC-SIGN to P. aeruginosa biofilms is carbohydrate-and calcium-dependent and extends to biofilms formed by clinical isolates. Confocal analysis of P. aeruginosa biofilms shows abundant DC-SIGN ligands among bacteria aggregates while MR ligands concentrate into discrete clusters. DC-SIGN ligands are also detected in planktonic P. aeruginosa cultures and depend on the presence of the common polysaccharide antigen. Carbohydrates purified from P. aeruginosa biofilms are recognised by DC-SIGN and MR; both receptors preferentially bind the high molecular weight fraction (HMW; >132,000Da) with KDs in the nM range. HMW preparations contain 74.9-80.9% mannose, display α-mannan segments and alter the morphology of human dendritic cells without causing obvious changes in cytokine responses. Finally, HMW interferes with the endocytic activity of cell-associated MR and DC-SIGN. This work identifies MR and DC-SIGN as receptors for bacterial biofilms and highlights the potential for biofilm-associated carbohydrates as immunomodulators through engagement of C-type lectin receptors.Author SummarySelective engagement of pattern recognition receptors during infection guides the decision-making process during induction of immune responses. This work identifies mannose-rich carbohydrates within bacterial biofilms as novel molecular patterns associated with bacterial infections. P. aeruginosa biofilms and biofilm-derived carbohydrates bind two important lectin receptors, MR (CD206) and DC-SIGN (CD209), involved in recognition of self and immune evasion. Abundance of MR and DC-SIGN ligands in the context of P. aeruginosa biofilms could impact immune responses and promote chronic infection.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.

Ligation of Dectin-1 by fungal glucans elicits a Th17 response that is necessary for clearing many fungal pathogens. Laminarin is a (1→3, 1→6)-β-glucan that is widely reported to be a Dectin-1 antagonist, however, there are reports that laminarin is also a Dectin-1 agonist. To address this controversy, we assessed the physical properties, structure, purity, Dectin-1 binding, and biological activity of five different laminarin preparations from three different commercial sources. The proton nuclear magnetic resonance analysis indicated that all of the preparations contained laminarin although their molecular mass varied considerably (4400–34,400 Da). Two of the laminarins contained substantial quantities of very low m.w. compounds, some of which were not laminarin. These low m.w. moieties could be significantly reduced by extensive dialysis. All of the laminarin preparations were bound by recombinant human Dectin-1 and mouse Dectin-1, but the affinity varied considerably, and binding affinity did not correlate with Dectin-1 agonism, antagonism, or potency. In both human and mouse cells, two laminarins were Dectin-1 antagonists and two were Dectin-1 agonists. The remaining laminarin was a Dectin-1 antagonist, but when the low m.w. moieties were removed, it became an agonist. We were able to identify a laminarin that is a Dectin-1 agonist and a laminarin that is Dectin-1 antagonist, both of which are relatively pure preparations. These laminarins may be useful in elucidating the structure and activity relationships of glucan/Dectin-1 interactions. Our data demonstrate that laminarin can be either a Dectin-1 antagonist or agonist, depending on the physicochemical properties, purity, and structure of the laminarin preparation employed.

The fungal cell wall serves as the interface between the organism and its environment. Complex carbohydrates are a major component of the Candida albicans cell wall, i.e., glucan, mannan and chitin. β-Glucan is a pathogen associated molecular pattern (PAMP) composed of β-(1 → 3,1 → 6)-linked glucopyranosyl repeat units. This PAMP plays a key role in fungal structural integrity and immune recognition. Glycogen is an α-(1 → 4,1 → 6)-linked glucan that is an intracellular energy storage carbohydrate. We observed that glycogen was co-extracted during the isolation of β-glucan from C. albicans SC5314. We hypothesized that glucan and glycogen may form a macromolecular species that links intracellular glycogen with cell wall β-(1 → 3,1 → 6)-glucan. To test this hypothesis, we examined glucan-glycogen extracts by multi-dimensional NMR to ascertain if glycogen and β-glucan were interconnected. 1H NMR analyses confirmed the presence of glycogen and β-glucan in the macromolecule. Diffusion Ordered SpectroscopY (DOSY) confirmed that the β-glucan and glycogen co-diffuse, which indicates a linkage between the two polymers. We determined that the linkage is not via peptides and/or small proteins. Our data indicate that glycogen is covalently linked to β-(1 → 3,1 → 6) glucan via the β -(1 → 6)-linked side chain. We also found that the glucan-glycogen complex was present in C. dublinensis, C. haemulonii and C. auris, but was not present in C. glabrata or C. albicans hyphal glucan. These data demonstrate that glucan and glycogen form a novel macromolecular complex in the cell wall of C. albicans and other Candida species. This new and unique structure expands our understanding of the cell wall in Candida species.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate long-lasting adaptive immunity. While it is known that PRRs detect unique chemical patterns associated with invading microorganism, if and how the physical properties of PRR ligands influence development of the immune response is largely overlooked. Through the study of fungal mannans we present data that put the physical form of PRR ligands at the center of the process that determines the outcome of the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both periphery and dLN. When combined with SARS-CoV-2 Spike, this formulation elicits neutralizing anti-SARS-CoV-2 antibodies that cross-react with pathogenic coronaviruses. Thus, the physical properties of fungal ligands can be harnessed for rational adjuvant design and vaccine development.

The global problem of resistance to antimicrobials has resulted in a co-ordinated drive to use antimicrobial agents more responsibly. At a clinical level this is promoted through antimicrobial stewardship which demands appropriate use through optimal drug selection. Many factors play a role in this process of selection, antimicrobial susceptibility and the pharmacodynamics of the drug being two key determinants. Yet the detail provided by current diagnostic antimicrobial susceptibility testing is suboptimal and does not allow for adequate dose optimisation. The minimum inhibitory concentration (MIC) which underlies all antimicrobial susceptibility testing is largely ignored in the decision-making process of optimal drug selection. Understanding and application of MIC-guided antimicrobial therapy is desperately needed if antimicrobial stewardship is to truly fulfil its mandate.

N-linked mannans (N-mannans) in the cell wall of Candida albicans are thought to mask β-(1,3)-glucan from recognition by Dectin-1, contributing to innate immune evasion. Lateral cell wall exposures of glucan on Candida albicans are predominantly single receptor-ligand interaction sites and are restricted to nanoscale geometries. Candida species exhibit a range of basal glucan exposures and their mannans also vary in size and complexity at the molecular level. We used super resolution fluorescence imaging and a series of protein mannosylation mutants in C. albicans and C. glabrata to investigate the role of specific N-mannan features in regulating the nanoscale geometry of glucan exposure. Decreasing acid labile mannan abundance and α-(1,6)-mannan backbone length correlated most strongly with increased density and nanoscopic size of glucan exposures in C. albicans and C. glabrata, respectively. Additionally, a C. albicans clinical isolate with high glucan exposure produced similarly perturbed N-mannan structures and exhibited similar changes to nanoscopic glucan exposure geometry. We conclude that acid labile N-mannan controls glucan exposure geometry at the nanoscale. Furthermore, variations in glucan nanoexposure characteristics are clinically relevant and are likely to impact the nature of the pathogenic surface presented to innate immunocytes at dimensions relevant to receptor engagement, aggregation and signaling.

Contains fulltext : 136409.pdf (Publisher’s version ) (Open Access) The innate immune system differentially recognizes Candida albicans yeast and hyphae. It is not clear how the innate immune system effectively discriminates between yeast and hyphal forms of C. albicans. Glucans are major components of the fungal cell wall and key fungal pathogen-associated molecular patterns. C. albicans yeast glucan has been characterized; however, little is known about glucan structure in C. albicans hyphae. Using an extraction procedure that minimizes degradation of the native structure, we extracted glucans from C. albicans hyphal cell walls. (1)H NMR data analysis revealed that, when compared with reference (1-->3,1-->6) beta-linked glucans and C. albicans yeast glucan, hyphal glucan has a unique cyclical or "closed chain" structure that is not found in yeast glucan. GC/MS analyses showed a high abundance of 3- and 6-linked glucose units when compared with yeast beta-glucan. In addition to the expected (1-->3), (1-->6), and 3,6 linkages, we also identified a 2,3 linkage that has not been reported previously in C. albicans. Hyphal glucan induced robust immune responses in human peripheral blood mononuclear cells and macrophages via a Dectin-1-dependent mechanism. In contrast, C. albicans yeast glucan was a much less potent stimulus. We also demonstrated the capacity of C. albicans hyphal glucan, but not yeast glucan, to induce IL-1beta processing and secretion. This finding provides important evidence for understanding the immune discrimination between colonization and invasion at the mucosal level. When taken together, these data provide a structural basis for differential innate immune recognition of C. albicans yeast versus hyphae.

We aimed to describe regional differences in therapy for bloodstream infection (BSI) caused by extended-spectrum ?-lactamase-producing Enterobacteriaceae (ESBL-E) or carbapenemase-producing Enterobacteriaceae (CPE). 1,482 patients in 12 countries were included from an observational study of BSI caused by ESBL-E or CPE. Multivariate logistic regression was used to calculate adjusted odds ratios (aORs) for the influence of country of recruitment on empirical use of ?-lactam/?-lactamase inhibitors (BLBLI) or carbapenems, targeted use of BLBLI for ESBL-E and use of targeted combination therapy for CPE. The use of BLBLI for empirical therapy was least likely in sites from Israel (aOR 0.34, 95% CI 0.14-0.81), Greece (aOR 0.49, 95% CI 0.26-0.94) and Canada (aOR 0.31, 95% CI 0.11-0.88) but more likely in Italy (aOR 1.58, 95% CI 1.11-2.2) and Turkey (aOR 2.09, 95% CI 1.14-3.81), compared to Spain as a reference. Empirical carbapenems were more likely to be used in sites from Taiwan (aOR 1.73, 95% CI 1.03-2.92) and USA (aOR 1.89; 95% CI 1.05-3.39), and less likely in Italy (aOR 0.44, 95% CI 0.28-0.69) and Canada (aOR 0.10, 95% CI 0.01-0.74). Targeted BLBLI for ESBL-E was more likely in sites from Italy. Treatment at sites within Israel, Taiwan, Turkey and Brazil was associated with less combination therapy for CPE. Although this study does not provide precise data on the relative prevalence of ESBL-E or CPE, significant variation in therapy exists across countries even after adjustment for patient factors. A better understanding of what influences therapeutic choices for these infections will aid antimicrobial stewardship efforts. PH is supported by an Australian Postgraduate Award from the University of Queensland. The study was funded by the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III - co-financed by European Development Regional Fund "A way to achieve Europe" ERDF, Spanish Network for the Research in Infectious Diseases (REIPI RD12/0015). BGG, JRB, APH and YC also received funds from the COMBACTE-CARE project (grant agreement 115620), Innovative Medicines Initiative (IMI), the European Union's Seventh Framework Programme (FP7/2007-2013) and in-kind contributions from EFPIA companies.

The cell wall of Candida albicans is central to the yeasts ability to withstand osmotic challenge, to adhere to host cells, to interact with the innate immune system and ultimately to the virulence of the organism. Little is known about the effect of culture conditions on the cell wall structure and composition of C. albicans. We examined the effect of different media and culture temperatures on the molecular weight (Mw), polymer distribution and composition of cell wall mannan and mannoprotein complex. Strain SC5314 was inoculated from frozen stock onto yeast peptone dextrose (YPD), blood or 5% serum agar media at 30 or 37°C prior to mannan/mannoprotein extraction. Cultivation of the yeast in blood or serum at physiologic temperature resulted in an additive effect on Mw, however, cultivation media had the greatest impact on Mw. Mannan from a yeast grown on blood or serum at 30°C showed a 38.9 and 28.6% increase in Mw, when compared with mannan from YPD-grown yeast at 30°C. Mannan from the yeast pregrown on blood or serum at 37°C showed increased Mw (8.8 and 26.3%) when compared with YPD mannan at 37°C. The changes in Mw over the entire polymer distribution were due to an increase in the amount of mannoprotein (23.8–100%) and a decrease in cell wall mannan (5.7–17.3%). We conclude that C. albicans alters the composition of its cell wall, and thus its phenotype, in response to cultivation in blood, serum and/or physiologic temperature by increasing the amount of the mannoprotein and decreasing the amount of the mannan in the cell wall.

The immunostimulatory property of glucan isolates from three marine yeasts (Debaryomyces hansenii S8, Debaryomyces hansenii S169 and Candida tropicalis S186) and one Baker's yeast (Saccharomyces cerevisiae S36) as examined for potential application as immunostimulants in Penaeus monodon postlarvae against White Spot Syndrome Virus (WSSV) infection. Structural characterization of the glucan component in the isolates by proton nuclear magnetic resonance (NMR) indicated similar structures containing (1-3)-linked anhydroglucose repeat units (AGRUs) in the backbone with (1-6)-linked AGRUs in side chains that are (1-6)-linked to the backbone AGRUs. Glucan from C. tropicalis (S186) with the highest molecular weight and the lowest level of branching supported maximum survival (69%) followed by the other two marine yeast (S169 and S8) glucans of 27% and 23% respectively while glucan from Baker's yeast, S. cerevisiae S36 with the lowest molecular weight and the highest level of branching exhibited poor survival (4%) in P. monodon post challenge WSSV. The present study showed that the glucan isolate from marine yeast with a higher molecular weight and a lower degree of branching acts as better immunostimulants in P. monodon postlarvae than did the glucan isolate from S. cerevisiae.

Summary Cell wall mannans of Candida albicans mask β-(1,3)-glucan from recognition by Dectin-1, contributing to innate immune evasion. Glucan exposures are predominantly single receptor-ligand interaction sites of nanoscale dimensions. Candida species vary in basal glucan exposure and molecular complexity of mannans. We used super-resolution fluorescence imaging and a series of protein mannosylation mutants in C. albicans and C. glabrata to investigate the role of specific N-mannan features in regulating the nanoscale geometry of glucan exposure. Decreasing acid labile mannan abundance and α-(1,6)-mannan backbone length correlated most strongly with increased density and nanoscopic size of glucan exposures in C. albicans and C. glabrata, respectively. Additionally, a C. albicans clinical isolate with high glucan exposure produced similarly perturbed N-mannan structures and elevated glucan exposure geometry. Thus, acid labile mannan structure influences the nanoscale features of glucan exposure, impacting the nature of the pathogenic surface that triggers immunoreceptor engagement, aggregation, and signaling.

Several published functions associated with the CHK1 histidine kinase of Candida albicans resemble those of the MAPK Cek1p and its cognate receptor Sho1p (SSU81). To explore this further, we have compared mutants lacking the proteins mentioned above and have constructed a double sho1/chk1Delta null mutant to determine relationships among these proteins. We observed that the sensitivity to Congo red (CR), calcofluor white (CW), as well as clumping of cells, was slightly increased in the double mutant compared to the single chk1Delta or sho1Delta mutants. However, Cek1p phosphorylation via Sho1p, which occurs during log phase growth in the presence or absence of CR in Wt cells, does not require Chk1p. These data suggest that Chk1p and Sho1p are components of parallel but independent signal pathways. In addition, bulk mannan of strains was analyzed by GLC/MS and GPC MALLS and NMR. Compared to Wt and a CHK1 gene-reconstituted strain (CHK23) that contained high, intermediate and low Mw mannan species, we found that the mannan of strains CHK21 (chk1Delta null), the cek1Delta null, and the double mutant consisted only of low Mw mannan. The sho1Delta null mutant only demonstrated a reduced intermediate type of mannan. Alcian blue binding was lower in cek1Delta, chk1Delta, and the double sho1/chk1Delta null mutant lacking high and intermediate Mw mannan than in the sho1Delta null which had a partial loss of intermediate Mw mannan only. We conclude that the Chk1p HK is part of a functionally similar but parallel pathway to the Sho1p-Cek1p pathway that confers resistance to the cell wall inhibitors CR and CW. However, a functional relationship in mannan biosynthesis of Chk1p and Cek1p exists that only partially requires Sho1p.

A comparative study was performed to evaluate the immunostimulatory effect of yeast and yeast-derived glucan in white prawn Fenneropenaeus indicus (sub-adults of ∼20 gm). Feed with a whole cell biomass of marine yeast Candida sake S165 (CSY) at a concentration of 10% (w/w) and another feed with 0.2% glucan of C. sake S165 (CSG) were used in the study. Fenneropenaeus indicus were fed with these diets for 40 days and subsequently challenged with the white spot syndrome virus (WSSV). Haematological parameters such as the total haemocyte count, phenoloxidase activity, superoxide anion (O2−) level, haemolymph peroxidase level and post-challenge survival against WSSV infection were determined to assess the immune status. In the present experiment, a higher immunity index and post-challenge survival were recorded in shrimps fed with the whole cell yeast diet. The better immunostimulatory performance of the whole cell yeast diet compared with the glucan diet could be attributed to the cellular constituents of yeast including the cell wall glucan, nucleotides, carotenoid pigments and vitamins. Here we observed that whole cell yeast performed better as an immunostimulant than the extracted cell wall glucans. Therefore, the use of yeast biomass in diets, rather than the yeast cell wall extract, glucan, would confer better protection against microbial infection besides reducing the cost of shrimp production.

Eiect of an extraction method on the structure of glucan and its immunostimulatory response in Fenneropenaeus indicus was investigated. Here we extracted alkali insoluble glucan (AIG) and alkali soluble glucan (ASG) from a ¢lamentous fungi Acremonium diospyri following alkali^acid hydrolysis and the sodium hypochlorite oxidation and dimethyl sulphoxide extraction method respectively. Structural analysis showed that 85% of glucan in AIG was a (1 ! 3)-b-D-glucan and it increased the prophenoloxidase and reactive oxygen intermediate activity when administered to F. indicus . On the other hand, ASG, which contained 93% (1 ! 3)-a-glucan, did not induce signi¢cant immune response in shrimp. Here we report that the diierence in immunostimulatory potential between AIG and ASG is due to the diierence in the percentage of (1 ! 3)-b-D-glucans present in each preparation, which varies with the method of extraction employed. Also our observations suggest that glucan can be used as a potential immunostimulant to shrimp, provided it contains (1 ! 3)-b-D-glucan as the major fraction.

The Goa1p of Candida albicans regulates mitochondrial Complex I (CI) activities in its role as a putative CI accessory protein. Transcriptional profiling of goa1∆ revealed a down regulation of genes encoding β-oligomannosyl transferases. Herein, we present data on cell wall phenotypes of goa1∆ (strain GOA31). We used transmission electron microscopy (TEM), GPC/MALLS, and NMR to compare GOA31 to a gene-reconstituted strain (GOA32) and parental cells. We note by TEM a reduction in outer wall fibrils, increased inner wall transparency, and the loss of a defined wall layer close to the plasma membrane. GPC-MALLS revealed a reduction in high and intermediate Mw mannan by 85% in GOA31. A reduction of β-mannosyl but not α-mannosyl linkages was noted in GOA31 cells. β-(1,6)-linked glucan side chains were branched about twice as often but were shorter in length for GOA31. We conclude that mitochondrial CI energy production is highly integrated with cell wall formation. Our data also suggest that not all cell wall biosynthetic processes are dependent upon Goa1p even though it provides high levels of ATP to cells. The availability of both broadly conserved and fungal-specific mutants lacking CI subunit proteins should be useful in assessing functions of fungal-specific functions subunit proteins.

The human pathogen Candida albicans encodes at least three putative two-component histidine kinase signal transduction proteins, including Chk1p and a response regulator protein (Cssk1p). Strains deleted in CHK1 are avirulent in a murine model of hematogenously disseminated disease. The specific function of Chk1p has not been established, but hyphae of the chk1 mutant exhibit extensive flocculation while yeast forms are less adherent to reconstituted human esophageal tissue, indicating that this protein may regulate cell surface properties. Herein, we analyze glucan, mannan and chitin profiles in strains deleted in chk1 (CHK21) compared to a gene-reconstituted strain (CHK23) and a parental strain CAF2. Total alkali-soluble hexose from the cell wall of the chk1 mutant (strain CHK21) was significantly reduced. Western blots of cell wall extracts from CHK21, CHK23 and CAF2 reacted with a Mab to the acid-stable mannan fraction revealed extensive staining of lower molecular mass species in strain CHK21 only. FACE (fluorophore assisted carbohydrate electrophoresis) was used to characterize the oligosaccharide side chains of beta-eliminated (O-linked), acid-hydrolyzed (acid-labile phosphomannan) and acetolysis (acid-stable mannan) extracted fractions of total mannan. The profiles of O-linked as well as the acid-labile oligosaccharides were similar in both CAF2 and CHK21, but the acid-stable oligosaccharide side chains were significantly truncated. We also characterized the beta-glucan from each strain using NMR, and found that both the degree of polymerization and the ratio of (1-3)/(1-6) linkages was lower in CHK21 relative to wild-type cells. The sensitivity of CHK21 to antifungal drugs and inhibitors was unaffected. In summary, our data have identified a new function for a histidine kinase two-component signal protein in a human pathogenic fungus.

The publisher regrets that in the above-mentioned article the title and authors were printed wrong. The correct version is reprinted here. “The role of the Candida albicans histidine kinase ( CHK1 ) gene in the regulation of cell wall mannan …

We have examined the synthesis of hydroxybutenyl cyclomaltooligosaccharides (cyclodextrins) and the ability of these cyclodextrin ethers to form guest–host complexes with guest molecules. The hydroxybutenyl cyclodextrin ethers were prepared by a base-catalyzed reaction of 3,4-epoxy-1-butene with the parent cyclodextrins in an aqueous medium. Reaction byproducts were removed by nanofiltration before the hydroxybutenyl cyclodextrins were isolated by co-evaporation of water–EtOH. Hydroxybutenyl cyclodextrins containing no unsubstituted parent cyclodextrin typically have a degree of substitution of 2–4 and a molar substitution of 4–7. These hydroxybutenyl cyclodextrins are randomly substituted, amorphous solids. The hydroxybutenyl cyclodextrin ethers were found to be highly water soluble. Complexes of HBen-β-CD with glibenclamide and ibuprofen were prepared and isolated. In both cases, the guest content of the complexes was large, and a significant increase in the solubility of the free drug was observed. Dissolution of the complexes in pH 1.4 water was very rapid, and significant increases in the solubility of the free drugs were observed. Significantly, after reaching equilibrium concentration, a decrease in the drug concentration over time was not observed.

Health benefits of the polysaccharide (1-->3)-beta-D-glucan, reported to induce immunobiological, hypocholesterolemic, and hypoglycemic effects in humans and animals, have made the isolation, characterization, and assay of a viable glucan product critical. A new analytical method, based on internal standard proton NMR analysis, for the assay of solvent-wet samples containing (1-->3)-beta-D-glucan is presented. The method enables glucan identification, provides a solvent-free assay, and improves upon the previous multistep extraction and lyophilization procedure by reducing the 1-2 day analysis time to 1-2 h. NMR offers a rapid method for quantifying the glucan in commercial samples, such as nutraceuticals, as well as industrial samples enabling better evaluation of the efficacy of these carbohydrates in health-related applications.

Glucans are fungal cell wall polysaccharides which stimulate innate immune responses. We determined the minimum unit ligand that would bind to glucan receptors on human U937 cells using laminarin-derived pentaose, hexaose, and heptaose glucan polymers. When U937 membranes were pretreated with the oligosaccharides and passed over a glucan surface, only the heptasaccharide inhibited the interaction of glucan with membrane receptors at a Kd of 31 µM (95% CI 20-48 µM) and 100% inhibition. However, the glucan heptasaccharide did not stimulate U937 monocyte NFjB signaling, nor did it increase survival in a murine model of polymicrobial sepsis. Laminarin, a larger and more complex glucan polymer (M w = 7 700 g/mol), only partially inhibited binding (61 ± 4%) at a K d of 2.6 µM (99% CI 1.7-4.2 µM) with characteristics of a single binding site. These results indicate that a heptasaccharide is the smallest unit ligand recognized by macrophage glucan receptors. The data also indicate the presence of at least two glucan-binding sites on U937 cells and that the binding sites on human monocyte/macrophages can discriminate between glucan polymers. The heptasaccharide and laminarin were receptor antagonists, but they were not receptor agonists with respect to activation of NFjB-dependent signaling pathways or protection against experimental sepsis. © 2001 Editions scientifiques et medicales Elsevier SAS glucan / receptors / heptasaccharide / NFjB / macrophage / monocyte / sepsis

In December 1994, a steep slope on sidelong ground above an attenuated stretch of the A5 trunk road failed after a period of exceptionally heavy rainfall and obstructed the highway. A change in the water regime behind a retaining wall and the lack of drainage facilities within the slope created the preconditions for landslide. Site investigation characterised the ground conditions but identified only shallow slip surfaces. Although emergency works achieved temporary relief, enabling traffic to continue to use the road, the design and construction of permanent repairs were severely constrained by the absence of a practicable alternative route and the need to keep the highway open. The permanent remedial measures adopted proved to be of considerable practical interest, involving innovative techniques to cope with the unusually restricted working space. A mini-pile wall was constructed by single-pass pile emplacement into Silurian mudstone and siltstone bedrock using a compact drilling rig (total weight 1·5 t) which was positioned on a scaffolding platform. Single-pass pile installation was achieved using an air-flush ODEX drilling system. This technique decreased the time required for installation of the piles and reduced the number of concurrent activities involved in the operations. The final mini-pile wall incorporated drainage provision to prevent the future build-up of pore water pressures behind the wall. En décembre 1994, après une période de précipitations anormalement élevées, une pente abrupte sur un sol situé à l'oblique au-dessus d'un tronçon atténué de la route A5 s'est effondrée et a envahi la voie. Le changement du régime des eaux derrière le mur de soutènement et l'absence de systèmes de drainage à l'intérieur de la pente ont créé les conditions favorables à un glissement de terrain. L'investigation menée sur le site a analysé l'état du sol mais n'a identifié que des surfaces de glissement peu profondes. Bien que les travaux entrepris d'urgence aient apporté une solution temporaire et aient permis la reprise du trafic routier, la conception et la réalisation des réparations permanentes ont été sévèrement gênées car, en l'absence d'une autre route praticable, il fallait garder cette voie de circulation ouverte. Les mesures de réfection permanentes qui ont été adoptées se sont révélées être très intéressantes sur le plan pratique, faisant intervenir des techniques novatrices pour compenser les graves restrictions de place. Un mur à mini-pile a été construit en implantant une pile en une seule passe dans une argilite silurienne et une roche de fond en siltstone au moyen d'un appareil de forage compact (poids total 1,5 t) placé sur un échafaudage. La mise en place de cette pile en une seule passe a été effectuée en utilisant un système de forage ODEX à flux d'air. Cette technique a permis de réduire le temps nécessaire à l'installation des piles ainsi que le nombre d'opérations concourantes entraânées par ce genre d'activité. Le mur final à mini-pile prévoit des systèmes de drainage qui empêcheront que la pression d'eau de pore ne s'amplifie derrière lui.

Summary Background Screening for carriage of carbapenemase-producing Enterobacteriaceae (CPE) is considered an important infection prevention and control strategy. To date, screening has relied primarily on culture although polymerase chain reaction (PCR)-based screening is gaining momentum. Currently there is no gold standard screening method and consequently it is important to consider the implications of different diagnostic strategies used in active surveillance. Aim To assess the utility of a multiplex PCR screening strategy, as a component of active surveillance, for detection of CPE in patients admitted to various hospitals. Methods A single rectal swab was collected from patients at various hospitals, considered to be at risk of colonization with CPE. Comparison of a modified US Centers for Disease Control and Prevention culture protocol with a PCR-based assay for the detection of the bla NDM , bla KPC , bla OxA-48-like , bla VIM , bla IMP , and bla GES genes was performed. Findings Of the 251 consecutive rectal swabs collected, 30 were PCR positive for one or more carbapenemase genes. Fifteen (50%) were culture positive and CPE only accounted for six isolates. PCR demonstrated excellent sensitivity (100%), specificity (89.8%), and negative predictive value (100%) for detection of CPE, but a positive predictive value of only 46.6% and 16.6% for detection of carbapenemase-producing Gram-negatives and CPE, respectively. Conclusion The apparent excellent performance characteristics of PCR for detection of CPE from rectal swabs must be tempered by knowledge of CPE prevalence and be interpreted within a defined epidemiological context. Further comparative research with culture, evaluating the clinical utility of PCR-based assays as a screening tool, is needed.

Glucans are (1 → 3)-β-linked glucose polymers which have immune-stimulating capability. The extraction of water-insoluble (1 → 3)-β- d - glucan form Saccharomyces cerevisiae employs hydrochloric acid. Hydrochloric acid is difficult to employ in the large-scale pharmaceutical extraction of glucans due to its corrosive nature and toxicity. To address these concerns, we determined whether acetic, formic or phosphoric acid can be substituted for hydrochloric acid in the process for the isolation of (1 → 3)-β- d - glucan . The resulting microparticulate glucans were employed as the starting material for the production of (1 → 3)-β- d - glucan phosphate. 13C NMR analysis of the glucan phosphates derived from the acetic, formic or phosphoric acid-extracted microparticulate glucan show excellent correspondence to hydrochloric acid extracted glucan and laminarin, a (1 → 3)-β- d -glucan standard, indicating that the primary structure is not altered by the acid used for extraction. Glucan phosphate prepared from hydrochloric acid had a Mw of 7.2 × 104 g/mol, rmsz of 17.7 nm, of 1.50 and (η) of 49.0 mL/g. Glucan phosphate prepared from acetic acid had a primary polymer peak with a Mw of 1.4 × 106 g/mol, rmsz, of 23.6 nm, I of 1.93 and (η) of 62.4 mL/g. Glucan phosphate prepared from formic acid had a main polymer peak with a Mw of 1.2 × 106 g/mol, rmsz, 27.1 nm, I of 1.56 and (η) of 89.0 mL/g. Glucan phosphate prepared from phosphoric acid had a primary polymer peak with a Mw of 6.6 × 105 g/mol, rmsz, of 32.3 nm, I of 2.70 and (η) of 91.3 mL/g. These data indicate that the molecular mass, size, polydispersity and intrinsic viscosity of the glucan phosphate obtained is influenced by the pKa of protic acid employed to extract the microparticulate glucan. However, the primary structure and side-chain branching are not substantially altered regardless of the acid employed. (1 → 3)-β- d -Glucans that are known as biological response modifiers were extracted from Saccharomyces cerevisiae with HOAc, HO(C=O)H, and H3PO4. By analysis of the glucan phosphates derived from the (1 → 3)-β- d -glucans, it is determined that primary structure is not affected by type of acid; however, molecular mass, size, polydispersity, and intrinsic viscosity of the glycan phosphate vary with pKa of the acid employed.

Background: Candida glabrata virulence is poorly understood at the molecular level. Results: Inactivation of components of the C. glabrata glycosylation machinery results in changes in fungal mannan structure and altered virulence. Conclusion: Changes in C. glabrata cell wall architecture impact the host-pathogen interactions. Significance: Greater understanding of C. glabrata virulence will provide insights that can be adopted for development of novel diagnostic and therapeutic interventions., The fungus Candida glabrata is an important and increasingly common pathogen of humans, particularly in immunocompromised hosts. Despite this, little is known about the attributes that allow this organism to cause disease or its interaction with the host immune system. However, in common with other fungi, the cell wall of C. glabrata is the initial point of contact between the host and pathogen, and as such, it is likely to play an important role in mediating interactions and hence virulence. Here, we show both through genetic complementation and polysaccharide structural analyses that C. glabrata ANP1, MNN2, and MNN11 encode functional orthologues of the respective Saccharomyces cerevisiae mannosyltransferases. Furthermore, we show that deletion of the C. glabrata Anp1, Mnn2, and Mnn11 mannosyltransferases directly affects the structure of the fungal N-linked mannan, in line with their predicted functions, and this has implications for cell wall integrity and consequently virulence. C. glabrata anp1 and mnn2 mutants showed increased virulence, compared with wild-type (and mnn11) cells. This is in contrast to Candida albicans where inactivation of genes involved in mannan biosynthesis has usually been linked to an attenuation of virulence. In the long term, a better understanding of the attributes that allow C. glabrata to cause disease will provide insights that can be adopted for the development of novel therapeutic and diagnostic approaches.

The fungal cell wall is the first point of interaction between an invading fungal pathogen and the host immune system. The outer layer of the cell wall is comprised of GPI anchored proteins, which are post-translationally modified by both N- and O-linked glycans. These glycans are important pathogen associated molecular patterns (PAMPs) recognised by the innate immune system. Glycan synthesis is mediated by a series of glycosyl transferases, located in the endoplasmic reticulum and Golgi apparatus. Mnn2 is responsible for the addition of the initial α1,2-mannose residue onto the α1,6-mannose backbone, forming the N-mannan outer chain branches. In Candida albicans, the MNN2 gene family is comprised of six members (MNN2, MNN21, MNN22, MNN23, MNN24 and MNN26). Using a series of single, double, triple, quintuple and sextuple mutants, we show, for the first time, that addition of α1,2-mannose is required for stabilisation of the α1,6-mannose backbone and hence regulates mannan fibril length. Sequential deletion of members of the MNN2 gene family resulted in the synthesis of lower molecular weight, less complex and more uniform N-glycans, with the sextuple mutant displaying only un-substituted α1,6-mannose. TEM images confirmed that the sextuple mutant was completely devoid of the outer mannan fibril layer, while deletion of two MNN2 orthologues resulted in short mannan fibrils. These changes in cell wall architecture correlated with decreased proinflammatory cytokine induction from monocytes and a decrease in fungal virulence in two animal models. Therefore, α1,2-mannose of N-mannan is important for both immune recognition and virulence of C. albicans., Author Summary Candida albicans is an opportunistic fungal pathogen of humans, which can cause superficial infections of the oral and genital epithelium or fatal systemic disease in immune compromised individuals. The cell wall of C. albicans consists of an inner layer of chitin and β-glucan and an outer fibrillar layer of highly glycosylated mannoproteins. These mannose-rich carbohydrates play crucial roles in fungal immune recognition. Deletion of genes encoding the Mnn2 family of α1,2-mannosyltransferases, which are involved in the biosynthesis of N-mannan outer chains, demonstrated that the addition of mannan side chains is necessary for the stabilisation and extension of the N-mannan backbone. Mutants of the MNN2 gene family had marked alterations in mannan microfibril length. Reductions in the degree of N-mannan branching resulted in decreased immune recognition by monocytes, whilst complete removal of the branched outer chains increased β-glucan exposure, and correlated with an attenuated virulence. Therefore, the Mnn2 mannosyltransferase family is critical for microfibril structure, immune recognition and virulence.

Cellulose acetoacetates with and without other ester groups were prepared directly from cellulose by reaction with diketene or tert-butyl acetoacetate (and, where appropriate, a carboxylic anhydride) in N,N-dimethylacetamide (DMAC)/LiCl or 1-methyl-2-pyrrolidinone (NMP)/LiCl solution. This is the only method yet described for the direct synthesis of these polymers from cellulose and, except for the triester, the only method available for synthesis of cellulose acetoacetates which do not contain another ester group. The products span the entire degree of substitution (DS) range, are amorphous, and are readily soluble in various solvents depending on DS. Cellulose acetoacetates of low DS are soluble in water. Some of these water-soluble materials can be formulated into cross-linkable coatings which have outstanding solvent resistance. Methods have been developed for the determination ofDS by proton NMR spectroscopy. Proton and carbon-13 NMR resonance assignments for cellulose Iris(acetoacetate) are also provided.

β-Glucan is a (1→3)-β-linked glucose polymer with (1→6)-β-linked side chains and a major component of fungal cell walls. β-Glucans provide structural integrity to the fungal cell wall. The nature of the (1-6)-β-linked side chain structure of fungal (1→3,1→6)-β-D-glucans has been very difficult to elucidate. Herein, we report the first detailed structural characterization of the (1→6)-β-linked side chains of Candida glabrata using high-field NMR. The (1→6)-β-linked side chains have an average length of 4 to 5 repeat units spaced every 21 repeat units along the (1→3)-linked polymer backbone. Computer modeling suggests that the side chains have a bent curve structure that allows for a flexible interconnection with parallel (1→3)-β-D-glucan polymers, and/or as a point of attachment for proteins. Based on these observations we propose new approaches to how (1→6)-β-linked side chains interconnect with neighboring glucan polymers in a manner that maximizes fungal cell wall strength, while also allowing for flexibility, or plasticity.

The Candida albicans cell wall provides an architecture that allows for the organism to survive environmental stress as well as interaction with host tissues. Previous work has focused on growing C. albicans on media such as Sabouraud or YPD at 30 °C. Because C. albicans normally colonizes a host, we hypothesized that cultivation on blood or serum at 37 °C would result in structural changes in cell wall mannan. C. albicans SC5314 was inoculated onto YPD, 5% blood, or 5% serum agar media three successive times at 30 °C and 37 °C, then cultivated overnight at 30 °C in YPD. The mannan was extracted and characterized using 1D and 2D 1H NMR techniques. At 30 °C cells grown in blood and serum contain less acid-stable terminal β-(1→2)-linked d -mannose and α-(1→2)-linked d -mannose-containing side chains, while the acid-labile side chains of mannan grown in blood and serum contain fewer β-Man-(1→2)-α-Man-(1→ side chains. The decrement in acid-stable mannan side chains is greater at 37 °C than at 30 °C. Cells grown on blood at 37 °C show fewer →6)-α-Man-(1→ structural motifs in the acid-stable polymer backbone. The data indicate that C. albicans, grown on media containing host-derived components, produces less complex mannan. This is accentuated when the cells are cultured at 37 °C. This study demonstrates that the C. albicans cell wall is a dynamic and adaptive organelle, which alters its structural phenotype in response to growth in host-derived media at physiological temperature.

The reaction of diiodobenzene and sulfur in air at elevated temperatures (230-300°C), has been investigated by high-temperature EPR spectroscopy. The first direct observation of radicals under conditions of poly(p-phenylene sulfide/disulfide) synthesis is reported. Radicals observed include both a sulfur radical and a carbon radical. EPR experiments with monomers indicate the importance of polysulfide linkages, such as disulfide, with and without iodine or iodo substitution for the formation of these radical species

Members of the genus Malassezia are commensal fungi found on the skin of both human and domestic animals and are associated with skin diseases including dandruff/seborrheic dermatitis, pityriasis versicolor, and atopic eczema (AE) in humans. In this study we have characterized the cell-wall carbohydrates of Malassezia sympodialis, one of the species most frequently isolated from both AE patients and healthy individuals. Cells were grown in liquid Dixon media at 32 degrees C, harvested, and processed using a standard Fehling's precipitation methodology for the isolation of mannan and a standard base/acid extraction for (1--3)-beta-D-glucans. Using these classic extraction methods we were unable to isolate precipitable mannan or insoluble (1--3)-beta-D-glucan. However, acidification and addition of methanol to the remaining Fehling's-treated sample resulted in a very clean precipitate. This material was characterized by GPC-MALLS, 1D and 2D NMR, and GC-MS for monomer-type and linkage-type composition. We determined that trace amounts of both mannan and branched (1--3, 1--6)-beta-D-glucan were present in the recovered precipitate, but not linear (1--3)-beta-D-glucan. Surprisingly, NMR analysis indicated that (1--6)-beta-D-glucan was the major carbohydrate component isolated from M. sympodialis cell wall. GC-MS linkage analysis confirmed the (1--6)-beta-D-glucan structure. Based on these studies we have determined that the M. sympodialis cell wall contains (1--6)-beta-D-glucan as the major carbohydrate component along with trace amounts of mannan and (1--3, 1--6)-beta-d-glucan. In addition, these data indicate that modification of the classic mannan isolation methodology may be useful in the simultaneous isolation of both mannan and (1--6)-beta-D-glucan from other fungi.

The purpose of this Note is to propose a scheme for obtaining ZQC correlated 2D NMR spectra in a COSY-type format from pure-phase exchange spectroscopy (EXSY) spectra based upon a modified scheme for postacquisition processing of the EXSY data set

Glucans are structurally diverse fungal biopolymers that stimulate innate immunity and are fungal pathogen-associated molecular patterns. Dectin-1 is a C-type lectin-like pattern recognition receptor that binds glucans and induces innate immune responses to fungal pathogens. We examined the effect of glucan structure on recognition and binding by murine recombinant Dectin-1 with a library of natural product and synthetic (1-->3)-beta/(1-->6)-beta-glucans as well as nonglucan polymers. Dectin-1 is highly specific for glucans with a pure (1-->3)-beta-linked backbone structure. Although Dectin-1 is highly specific for (1-->3)-beta-d-glucans, it does not recognize all glucans equally. Dectin-1 differentially interacted with (1-->3)-beta-d-glucans over a very wide range of binding affinities (2.6 mM-2.2 pM). One of the most striking observations that emerged from this study was the remarkable high-affinity interaction of Dectin-1 with certain glucans (2.2 pM). These data also demonstrated that synthetic glucan ligands interact with Dectin-1 and that binding affinity increased in synthetic glucans containing a single glucose side-chain branch. We also observed differential recognition of glucans derived from saprophytes and pathogens. We found that glucan derived from a saprophytic yeast was recognized with higher affinity than glucan derived from the pathogen Candida albicans. Structural analysis demonstrated that glucan backbone chain length and (1-->6)-beta side-chain branching strongly influenced Dectin-1 binding affinity. These data demonstrate: 1) the specificity of Dectin-1 for glucans; 2) that Dectin-1 differentiates between glucan ligands based on structural determinants; and 3) that Dectin-1 can recognize and interact with both natural product and synthetic glucan ligands.