One-carbon metabolizing enzyme ALDH1L1 influences mitochondrial metabolism through 5-aminoimidazole-4-carboxamide ribonucleotide accumulation and serine depletion, contributing to tumor suppression.

Tumor cells generally require large amounts of nucleotides, and thus activate de novo purine synthesis (dnPS). In the dnPS reactions, 10-formyltetrahydorofolate (10-fTHF) supplied by one-carbon metabolism is utilized as a formyl group donor. We focused on aldehyde dehydrogenase 1 family member L1 (ALDH1L1), which metabolizes 10-fTHF to tetrahydrofolate and whose expression is often attenuated in hepatocellular carcinoma (HCC). We generated ALDH1L1-expressing HuH-7 cells to perform metabolome analysis and found that intracellular levels of serine were reduced and glycine was increased. In addition, 5-aminoimidazole-4-carboxamide ribonucleotide (ZMP), a dnPS intermediate, accumulated due to the consumption of 10-fTHF by ALDH1L1, which inhibited ZMP formylation. Importantly, ALDH1L1-expressing cells showed reduced ZMP sensitivity and higher mitochondrial activity. The suppression of mitochondrial serine catabolism by ALDH1L1 expression was speculated to be closely related to this phenotype. Gene set enrichment analysis utilizing The Cancer Genome Atlas data revealed that genes related to oxidative phosphorylation were enriched in HCC patients with high ALDH1L1 expression. Moreover, drug sensitivity data analysis demonstrated that HCC cell lines with low expression of ALDH1L1 were sensitive to ZMP and cordycepin, a structural analog of ZMP and AMP. Our study revealed that ZMP and AMP analogs might be effective in the pharmacotherapy of HCC patients with low expression of ALDH1L1.

A real-time gas monitoring system based on ion mobility spectrometry for workplace environmental measurements.

A volatile organic compounds (VOC) gas monitor was developed using an ion mobility spectrometer (IMS). It was designed for real-time monitoring in work environments, where gas chromatography/mass spectrometry (GC/MS) does not perform well. To evaluate the identification capability, response time, and quantitative accuracy of this device, experiments were conducted using methyl ethyl ketone. This is widely used in many factories, and its short-term exposure limit (STEL) has been set by the American Conference of Governmental Industrial Hygienists. Because the ionizable amount of methyl ethyl ketone has already been exceeded in STEL value of 300 ppm which belongs to the measurement range of interest in this study, this study estimated the peak shift amount rather than the peak intensity. Calibration curves with high accuracy were obtained in a range including 300 ppm which is the STEL of methyl ethyl ketone. The results of the experiment suggest that the device can be used for monitoring chemical substances in a work environment.

Adsorptive Removal of Heavy Metal Ions, Organic Dyes, and Pharmaceuticals by DNA-Chitosan Hydrogels.

DNA-chitosan (DNA-CS) hydrogel was prepared by in situ complexation between oppositely charged DNA and chitosan polyelectrolytes via electrostatic cross-linking to study its adsorption characteristics. The DNA-chitosan hydrogel matrix contains (i) cationic (NH3+) and anionic (PO4-) sites for electrostatic binding with ionic species, (ii) -OH and -NH2 groups and heteroaromatic DNA nucleobases for chelation of heavy metal ions, and (iii) DNA double-helix for recognition and binding to small organic molecules of various structures and polarities. DNA-CS hydrogels efficiently bind with Hg2+, Pb2+, Cd2+, and Cu2+ metal cations of significant environmental concern. Adsorption capacities of DNA-CS hydrogels for studied metal ions depend on hydrogel composition and pH of solution and reach ca. 50 mg/g at neutral pHs. Hydrogels with higher DNA contents show better adsorption characteristics and notably higher adsorption capacity to Hg2+ ions. Because of the co-existence of cationic and anionic macromolecules in the DNA-CS hydrogel, it demonstrates an affinity to both anionic (Congo Red) and cationic (Methylene Blue) dyes with moderate adsorption capacities of 12.6 mg/g and 29.0 mg/g, respectively. DNA-CS hydrogel can also be used for adsorptive removal of pharmaceuticals on conditions that their molecules are sufficiently hydrophobic and have ionogenic group(s). Facile preparation and multitarget adsorption characteristics of DNA-CS hydrogel coupled with sustainable and environmentally friendly characteristics render this system promising for environmental cleaning applications.

Cell wall N-glycan of Candida albicans ameliorates early hyper- and late hypo-immunoreactivity in sepsis.

Severe infection often causes a septic cytokine storm followed by immune exhaustion/paralysis. Not surprisingly, many pathogens are equipped with various anti-inflammatory mechanisms. Such mechanisms might be leveraged clinically to control septic cytokine storms. Here we show that N-glycan from pathogenic C. albicans ameliorates mouse sepsis through immunosuppressive cytokine IL-10. In a sepsis model using lipopolysaccharide (LPS), injection of the N-glycan upregulated serum IL-10, and suppressed pro-inflammatory IL-1ß, TNF-α and IFN-γ. The N-glycan also improved the survival of mice challenged by LPS. Analyses of structurally defined N-glycans from several yeast strains revealed that the mannose core is key to the upregulation of IL-10. Knocking out the C-type lectin Dectin-2 abrogated the N-glycan-mediated IL-10 augmentation. Furthermore, C. albicans N-glycan ameliorated immune exhaustion/immune paralysis after acute inflammation. Our results suggest a strategy where the immunosuppressive mechanism of one pathogen can be applied to attenuate a severe inflammation/cytokine storm caused by another pathogen.

Author Correction: Identification of Two Mannosyltransferases Contributing to Biosynthesis of the Fungal-type Galactomannan α-Core-Mannan Structure in Aspergillus fumigatus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Biosynthesis of ß-(1→5)-Galactofuranosyl Chains of Fungal-Type and O-Mannose-Type Galactomannans within the Invasive Pathogen Aspergillus fumigatus.

The pathogenic fungus Aspergillus fumigatus contains galactomannans localized on the surface layer of its cell walls, which are involved in various biological processes. Galactomannans comprise α-(1→2)-/α-(1→6)-mannan and ß-(1→5)-/ß-(1→6)-galactofuranosyl chains. We previously revealed that GfsA is a ß-galactofuranoside ß-(1→5)-galactofuranosyltransferase involved in the biosynthesis of ß-(1→5)-galactofuranosyl chains. In this study, we clarified the biosynthesis of ß-(1→5)-galactofuranosyl chains in A. fumigatus Two paralogs exist within A. fumigatus: GfsB and GfsC. We show that GfsB and GfsC, in addition to GfsA, are ß-galactofuranoside ß-(1→5)-galactofuranosyltransferases by biochemical and genetic analyses. GfsA, GfsB, and GfsC can synthesize ß-(1→5)-galactofuranosyl oligomers at up to lengths of 7, 3, and 5 galactofuranoses within an established in vitro highly efficient assay of galactofuranosyltransferase activity. Structural analyses of galactomannans extracted from ΔgfsB, ΔgfsC, ΔgfsAC, and ΔgfsABC strains revealed that GfsA and GfsC synthesized all ß-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans and that GfsB exhibited limited function in A. fumigatus The loss of ß-(1→5)-galactofuranosyl residues decreased the hyphal growth rate and conidium formation ability and increased the abnormal hyphal branching structure and cell surface hydrophobicity, but this loss is dispensable for sensitivity to antifungal agents and virulence toward immunocompromised mice.IMPORTANCE ß-(1→5)-Galactofuranosyl residues are widely distributed in the subphylum Pezizomycotina of the phylum Ascomycota. Pezizomycotina includes many plant and animal pathogens. Although the structure of ß-(1→5)-galactofuranosyl residues of galactomannans in filamentous fungi was discovered long ago, it remains unclear which enzyme is responsible for biosynthesis of this glycan. Fungal cell wall formation processes are complicated, and information concerning glycosyltransferases is essential for understanding them. In this study, we showed that GfsA and GfsC are responsible for the biosynthesis of all ß-(1→5)-galactofuranosyl residues of fungal-type and O-mannose-type galactomannans. The data presented here indicate that ß-(1→5)-galactofuranosyl residues are involved in cell growth, conidiation, polarity, and cell surface hydrophobicity. Our new understanding of ß-(1→5)-galactofuranosyl residue biosynthesis provides important novel insights into the formation of the complex cell wall structure and the virulence of the members of the subphylum Pezizomycotina.

Dectin-2-mediated signaling triggered by the cell wall polysaccharides of Cryptococcus neoformans.

Cryptococcus neoformans is rich in polysaccharides of the cell wall and capsule. Dectin-2 recognizes high-mannose polysaccharides and plays a central role in the immune response to fungal pathogens. Previously, we demonstrated Dectin-2 was involved in the activation of dendritic cells upon stimulation with C. neoformans, suggesting the existence of a ligand recognized by Dectin-2. In the present study, we examined the cell wall structures of C. neoformans contributing to the Dectin-2-mediated activation of immune cells. In a NFAT-GFP reporter assay of the reported cells expressing Dectin-2, the lysates, but not the whole yeast cells, of an acapsular strain of C. neoformans (Cap67) delivered Dectin-2-mediated signaling. This activity was detected in the supernatant of ß-glucanase-treated Cap67 and more strongly in the semi-purified polysaccharides of this supernatant using ConA-affinity chromatography (ConA-bound fraction), in which a large amount of saccharides, but not protein, were detected. Treatment of this supernatant with periodic acid and the addition of excessive mannose, but not glucose or galactose, strongly inhibited this activity. The ConA-bound fraction of the ß-glucanase-treated Cap67 supernatant was bound to Dectin-2-Fc fusion protein in a dose-dependent manner and strongly induced the production of interleukin-12p40 and tumour necrosis factor-α by dendritic cells; this was abrogated under the Dectin-2-deficient condition. Finally, 98 kDa mannoprotein (MP98) derived from C. neoformans showed activation of the reporter cells expressing Dectin-2. These results suggested that a ligand with mannose moieties may exist in the cell walls and play a critical role in the activation of dendritic cells during infection with C. neoformans.

Identification of Two Mannosyltransferases Contributing to Biosynthesis of the Fungal-type Galactomannan α-Core-Mannan Structure in Aspergillus fumigatus.

Fungal-type galactomannan (FTGM) is a polysaccharide composed of α-(1 → 2)-/α-(1 → 6)-mannosyl and ß-(1 → 5)-/ß-(1 → 6)-galactofuranosyl residues located at the outer cell wall of the human pathogenic fungus Aspergillus fumigatus. FTGM contains a linear α-mannan structure called core-mannan composed of 9 or 10 α-(1 → 2)-mannotetraose units jointed by α-(1 → 6)-linkages. However, the enzymes involved in core-mannan biosynthesis remain unknown. We speculated that two putative α-1,2-mannosyltransferase genes in A. fumigatus, Afu5g02740/AFUB_051270 (here termed core-mannan synthase A [CmsA]) and Afu5g12160/AFUB_059750 (CmsB) are involved in FTGM core-mannan biosynthesis. We constructed recombinant proteins for CmsA and detected robust mannosyltransferase activity using the chemically synthesized substrate p-nitrophenyl α-D-mannopyranoside as an acceptor. Analyses of CmsA enzymatic product revealed that CmsA possesses the capacity to transfer a mannopyranoside to the C-2 position of α-mannose. CmsA could also transfer a mannose residue to α-(1 → 2)-mannobiose and α-(1 → 6)-mannobiose and showed a 31-fold higher specific activity toward α-(1 → 6)-mannobiose than toward α-(1 → 2)-mannobiose. Proton nuclear magnetic resonance (1H-NMR) spectroscopy and gel filtration chromatography of isolated FTGM revealed that core-mannan structures were drastically altered and shortened in disruptant A. fumigatus strains ∆cmsA, ∆cmsB, and ∆cmsA∆cmsB. Disruption of cmsA or cmsB resulted in severely repressed hyphal extension, abnormal branching hyphae, formation of a balloon structure in hyphae, and decreased conidia formation. The normal wild type core-mannan structure and developmental phenotype were restored by the complementation of cmsA and cmsB in the corresponding disruptant strains. These findings indicate that both CmsA, an α-1,2-mannosyltransferase, and CmsB, a putative mannosyltransferase, are involved in FTGM biosynthesis.

Cooperation between ER stress and calcineurin signaling contributes to the maintenance of cell wall integrity in Candida glabrata.

Candida glabrata is the second most common source of Candida infections in humans. In this pathogen, the maintenance of cell wall integrity (CWI) frequently precludes effective pharmacological treatment by antifungal agents. In numerous fungi, cell wall modulation is reported to be controlled by endoplasmic reticulum (ER) stress, but how the latter affects CWI maintenance in C. glabrata is not clearly understood. Here, we characterized a C. glabrata strain harboring a mutation in the CNE1 gene, which encodes a molecular chaperone associated with nascent glycoprotein maturation in the ER. Disruption of cne1 induced ER stress and caused changes in the normal cell wall structure, specifically a reduction in the ß-1,6-glucan content and accumulation of chitin. Conversely, a treatment with the typical ER stress inducer tunicamycin up-regulated the production of cell wall chitin but did not affect ß-1,6-glucan content. Our results also indicated that C. glabrata features a uniquely evolved ER stress-mediated CWI pathway, which differs from that in the closely related species Saccharomyces cerevisiae. Furthermore, we demonstrated that ER stress-mediated CWI pathway in C. glabrata is also induced by the disruption of other genes encoding proteins that function in a correlated manner in the quality control of N-linked glycoproteins in the ER. These results suggest that calcineurin and ER quality control system act as a platform for maintaining CWI in C. glabrata.

Effect of shelf aging on vibration transmissibility of anti-vibration gloves.

Anti-vibration gloves have been used in real workplaces to reduce vibration transmitted through hand-held power tools to the hand. Generally materials used for vibration attenuation in gloves are resilient materials composed of certain synthetic and/or composite polymers. The mechanical characteristics of the resilient materials used in anti-vibration gloves are prone to be influenced by environmental conditions such as temperature, humidity, and photo-irradiation, which cause material degradation and aging. This study focused on the influence of shelf aging on the vibration attenuation performance of air-packaged anti-vibration gloves following 2 yr of shelf aging. Effects of shelf aging on the vibration attenuation performance of anti-vibration gloves were examined according to the Japan industrial standard JIS T8114 test protocol. The findings indicate that shelf aging induces the reduction of vibration attenuation performance in air-packaged anti-vibration gloves.

GfsA is a ß1,5-galactofuranosyltransferase involved in the biosynthesis of the galactofuran side chain of fungal-type galactomannan in Aspergillus fumigatus.

Previously, we reported that GfsA is a novel galactofuranosyltransferase involved in the biosynthesis of O-glycan, the proper maintenance of fungal morphology, the formation of conidia and anti-fungal resistance in Aspergillus nidulans and A. fumigatus (Komachi Y et al., 2013. GfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O-glycan in Aspergillus nidulans and Aspergillus fumigatus. Mol. Microbiol. 90:1054-1073). In the present paper, to gain an in depth-understanding of the enzymatic functions of GfsA in A. fumigatus (AfGfsA), we established an in vitro assay to measure galactofuranosyltransferase activity using purified AfGfsA, UDP-α-d-galactofuranose as a sugar donor, and p-nitrophenyl-ß-d-galactofuranoside as an acceptor substrate. LC/MS, 1H-NMR and methylation analyses of the enzymatic products of AfGfsA revealed that this protein has the ability to transfer galactofuranose to the C-5 position of the ß-galactofuranose residue via a ß-linkage. AfGfsA requires a divalent cation of manganese for maximal activity and consumes UDP-α-d-galactofuranose as a sugar donor. Its optimal pH range is 6.5-7.5 and its optimal temperature range is 20-30°C. 1H-NMR, 13C-NMR and methylation analyses of fungal-type galactomannan extracted from the ∆AfgfsA strain revealed that AfGfsA is responsible for the biosynthesis of ß1,5-galactofuranose in the galactofuran side chain of fungal-type galactomannan. Based on these results, we conclude that AfGfsA acts as a UDP-α-d-galactofuranose: ß-d-galactofuranoside ß1,5-galactofuranosyltransferase in the biosynthetic pathway of galactomannans.

KRE5 Suppression Induces Cell Wall Stress and Alternative ER Stress Response Required for Maintaining Cell Wall Integrity in Candida glabrata.

The maintenance of cell wall integrity in fungi is required for normal cell growth, division, hyphae formation, and antifungal tolerance. We observed that endoplasmic reticulum stress regulated cell wall integrity in Candida glabrata, which possesses uniquely evolved mechanisms for unfolded protein response mechanisms. Tetracycline-mediated suppression of KRE5, which encodes a predicted UDP-glucose:glycoprotein glucosyltransferase localized in the endoplasmic reticulum, significantly increased cell wall chitin content and decreased cell wall ß-1,6-glucan content. KRE5 repression induced endoplasmic reticulum stress-related gene expression and MAP kinase pathway activation, including Slt2p and Hog1p phosphorylation, through the cell wall integrity signaling pathway. Moreover, the calcineurin pathway negatively regulated cell wall integrity, but not the reduction of ß-1,6-glucan content. These results indicate that KRE5 is required for maintaining both endoplasmic reticulum homeostasis and cell wall integrity, and that the calcineurin pathway acts as a regulator of chitin-glucan balance in the cell wall and as an alternative mediator of endoplasmic reticulum stress in C. glabrata.

Heterologous Production of Hyaluronic Acid in an ε-Poly-L-Lysine Producer, Streptomyces albulus.

Hyaluronic acid (HA) is used in a wide range of medical applications, where its performance and therapeutic efficacy are highly dependent on its molecular weight. In the microbial production of HA, it has been suggested that a high level of intracellular ATP enhances the productivity and molecular weight of HA. Here, we report on heterologous HA production in an ε-poly-l-lysine producer, Streptomyces albulus, which has the potential to generate ATP at high level. The hasA gene from Streptococcus zooepidemicus, which encodes HA synthase, was refactored and expressed under the control of a late-log growth phase-operating promoter. The expression of the refactored hasA gene, along with genes coding for UDP-glucose dehydrogenase, UDP-N-acetylglucosamine pyrophosphorylase, and UDP-glucose pyrophosphorylase, which are involved in HA precursor sugar biosynthesis, resulted in efficient production of HA in the 2.0 MDa range, which is greater than typical bacterial HA, demonstrating that a sufficient amount of ATP was provided to support the biosynthesis of the precursor sugars, which in turn promoted HA production. In addition, unlike in the case of streptococcal HA, S. albulus-derived HA was not cell associated. Based on these findings, our heterologous production system appears to have several advantages for practical HA production. We propose that the present system could be applicable to the heterologous production of a wide variety of molecules other than HA in the case their biosynthesis pathways require ATP in vivo.

Significant structural change in both O- and N-linked carbohydrate moieties of the antigenic galactomannan from Aspergillus fumigatus grown under different culture conditions.

Invasive aspergillosis is an important cause of morbidity and mortality in immunocompromised patients. Diagnosis of this infection frequently employs detection of the circulating galactomannan in the patient serum using enzyme immunoassay (EIA), a highly sensitive and specific system. Although there are many structural studies of the galactomannan of Aspergillus fumigatus, some inconsistencies are present in these results. In this study, to clarify the relationship between the growth conditions and structure of the galactomannans, we cultured A. fumigatus using two distinct yeast/fungal cultivation media, i.e. the yeast extract-peptone-dextrose (YPD) medium and yeast nitrogen base (YNB) medium. Galactomannans prepared from the resulting culture supernatants were structurally characterized by (1)H and (13)C nuclear magnetic resonance, methylation analysis, acetolysis and α-mannosidase degradation. These assays revealed that the galactomannan from the YPD cultivation had short ß-1,5-linked galactofuranose (Galf) oligosaccharide chains in both the O- and N-linked carbohydrate moieties, while the galactomannan from the YNB cultivation incorporated long Galf oligosaccharide chains. The galactomannans derived from the two culture conditions significantly differed in reactivity based on the EIA diagnostic system. We also demonstrated the presence of a novel Galf-containing branched oligosaccharide in the O-linked moiety.

Mechanism of high-affinity abscisic acid binding to PYL9/RCAR1.

Arabidopsis receptors of abscisic acid (ABA), the key plant hormone for adaptation to water stress, comprise 14 PYR/PYLs/RCARs proteins classified into three subfamilies I, II, and III, which suggests functional differentiation. Although their monomer-dimer equilibria may be correlated with differences in their ABA-binding affinities, how the dimerization decreases the affinity is unclear. Comparative structural and binding studies between PYL9, which is a representative of high-affinity subfamily I, and low-affinity members of subfamily III reveals that the nonpolar triplet (Ile110, Val162, and Leu165) and Pro64 contribute to enhance ABA-binding affinity by inducing a shift of the ABA carboxyl group to form additional direct hydrogen bonds with conserved Asn169. Our mutation studies of PYL1 successfully produced a monomeric mutant PYL1 exhibiting low ABA affinity and also a dimeric mutant PYL1 exhibiting high ABA-binding affinity, suggesting that dimer formation of ABA receptors is not essential for their low ABA-binding affinity. Our study contributes toward establishing the structural basis for the higher ABA-binding affinity of the subfamily receptors and provides a clue for understanding the broad spectrum of hormone actions in plants manifested by the different hormone-binding affinity of multiple receptors.

Evaluation of interactions between metal ions and nonionic surfactants in high-concentration HCl using low-pressure high-performance liquid chromatography with low-flow-resistance polystyrene-based monolithic column.

A method for evaluating the interactions between metal ions and nonionic surfactants in aqueous solutions containing high-concentration HCl, using gas pressure-driven low-pressure high-performance liquid chromatography (LP-HPLC) as a highly acid-resistant HPLC system, was developed. To construct the LP-HPLC for this purpose, poly(styrene-co-divinylbenzene)-based low-flow-resistance monolithic columns tolerant to highly acidic conditions were prepared using low-conversion thermal polymerization. Thermal polymerization at 65 °C for 1.5 h (monomer conversions, 33% for styrene and 59% for divinylbenzene) allowed preparation of a column with both high separation efficiency (around 60,000 plates m(-1) for alkylbenzenes) and a quite low back pressure of 0.14 MPa at a linear flow rate of 1 mm s(-1) (2.8 × 10(-13) m(2) in permeability). The base column prepared under the above conditions was coated with a nonionic surfactant, polyoxyethylene nonylphenyl ether (PONPE, average oxyethylene unit numbers (n) = 3, 7.5, 15, and 20), and used for evaluation of the interactions between PONPEs and metal ions in 6 M HCl. The interactions between PONPEs and Au(III), Ga(III), Fe(III), Zn(II), and Cu(II) were successfully evaluated using both breakthrough and chromatographic methods. Furthermore, a study of the effect of the polyoxyethylene (POE) chain length revealed that the use of PONPE with the longer POE moiety enhanced the magnitude of the interaction together with the increase in the amount of oxyethylene (OE) units coated on the monolith. Moreover, the interactions of metal ions with a single OE unit were almost constant in the range of n = 7.5-20, whereas the suppression of the interaction between Au(III) with the shortest PONPE chain (n = 3) was also observed.

Identification of distinct ligands for the C-type lectin receptors Mincle and Dectin-2 in the pathogenic fungus Malassezia.

Various C-type lectin receptors (CLRs), including Mincle and Dectin-2, function as pattern recognition receptors and play a central role in immunity to fungal pathogens. However, the precise structures of the CLR ligands in various pathogenic fungi have yet to be completely defined. Here we report that Malassezia, an opportunistic skin fungal pathogen, is cooperatively recognized by Mincle and Dectin-2 through distinct ligands. Solvent-based fractionation revealed that Mincle and Dectin-2 recognize lipophilic and hydrophilic components of Malassezia, respectively. Mass spectrometry and nuclear magnetic resonance (NMR) revealed glyceroglycolipid and unique mannosyl fatty acids linked to mannitol as two Mincle ligands. An O-linked mannobiose-rich glycoprotein was identified as a Malassezia ligand for Dectin-2. Cytokine production in response to the Mincle ligands and the Dectin-2 ligand was abrogated in Mincle(-/-) and Dectin-2(-/-) dendritic cells, respectively. These results demonstrate that Mincle and Dectin-2 recognize distinct ligands in Malassezia to induce host immune responses.

Immunochemistry of pathogenic yeast, Candida species, focusing on mannan.

This review describes recent findings based on structural and immunochemical analyses of the cell wall mannan of Candida albicans, and other medically important Candida species. Mannan has been shown to consist of α-1,2-, α-1,3-, α-1,6-, and ß-1,2-linked mannopyranose units with few phosphate groups. Each Candida species has a unique mannan structure biosynthesized by sequential collaboration between species-specific mannosyltransferases. In particular, the ß-1,2-linked mannose units have been shown to comprise a characteristic oligomannosyl side chain that is strongly antigenic. For these pathogenic Candida species, cell-surface mannan was also found to participate in the adhesion to the epithelial cells, recognition by innate immune receptors and development of pathogenicity. Therefore, clarification of the precise chemical structure of Candida mannan is indispensable for understanding the mechanism of pathogenicity, and for development of new antifungal drugs and immunotherapeutic procedures.

Significant differences in the cell-wall mannans from three Candida glabrata strains correlate with antifungal drug sensitivity.

Candida glabrata is often the second or third most common cause of candidiasis after Candida albicans. C. glabrata infections are difficult to treat, often resistant to many azole antifungal agents and are associated with a high mortality rate in compromised patients. We determined the antigenic structure of the cell-wall mannoproteins from three C. glabrata strains, NBRC 0005, NBRC 0622 and NBRC 103857. (1)H NMR and methylation analyses of the acetolysis products of these mannoproteins showed a significant difference in the amount of the ß-1,2-linked mannose residue and side-chain structure. The C. glabrata NBRC 103857 strain contained up to the triose side chains and the nonreducing terminal of the triose was predominantly the ß-1,2-linked mannose residue. By contrast, the mannans of the two former strains possessed up to the tetraose side chains and the amount of the ß-1,2-linked mannose residue was very low. Larger oligosaccharides than tetraose in the acetolysis products of these mannans were identified as incomplete cleavage fragments by analyzing methylation, (1)H NMR spectra and the α1-2,3 mannosidase degradation reaction. Resistance to the antifungal drugs itraconazole and micafungin was significantly different in these strains. Interestingly, the NBRC 103857 strain, which involved a large amount of the ß-1,2-linked mannose residues, exhibited significant sensitivity to these antifungal drugs.

Difference in fine specificity to polysaccharides of Candida albicans mannoprotein between mouse SIGNR1 and human DC-SIGN.

C-type lectin SIGNR1 directly recognizes Candida albicans and zymosan and has been considered to share properties of polysaccharide recognition with human DC-SIGN (hDC-SIGN). However, the precise specificity of SIGNR1 and the difference from that of hDC-SIGN remain to be elucidated. We prepared soluble forms of SIGNR1 and hDC-SIGN and conducted experiments to examine their respective specificities. Soluble SIGNR1 (sSIGNR1) bound several types of live C. albicans clinical isolate strains in an EDTA-sensitive manner. Inhibition analyses of sSIGNR1 binding by glycans from various yeast strains demonstrated that SIGNR1 preferentially recognizes N-glycan α-mannose side chains in Candida mannoproteins, as reported in hDC-SIGN. Unlike shDC-SIGN, however, sSIGNR1 recognized not only Saccharomyces cerevisiae, but also C. albicans J-1012 glycan, even after α-mannosidase treatment that leaves only ß1,2-mannose-capped α-mannose side chains. In addition, glycomicroarray analyses showed that sSIGNR1 binds mannans from C. albicans and S. cerevisiae but does not recognize Lewis(a/b/x/y) antigen polysaccharides as in shDC-SIGN. Consistent with these results, RAW264.7 cells expressing hDC-SIGN in which the carbohydrate recognition domain (CRD) was replaced with that of SIGNR1 (RAW-chimera) produced comparable amounts of interleukin 10 (IL-10) in response to glycans from C. albicans and S. cerevisiae, but those expressing hDC-SIGN produced less IL-10 in response to S. cerevisiae than C. albicans. Furthermore, RAW-hDC-SIGN cells remarkably reduced IL-10 production after α-mannosidase treatment compared with RAW-chimera cells. These results indicate that SIGNR1 recognizes C. albicans/yeast through a specificity partly distinct from that of its homologue hDC-SIGN.