Aspergillus nidulans UDP-glucose-4-epimerase UgeA has multiple roles in wall architecture,hyphal morphogenesis,and asexual development

Aspergillus nidulans UDP-glucose-4-epimerase UgeA interconverts UDP-glucose and UDP-galactose and participates in galactose metabolism. The sugar moiety of UDP-galactose is predominantly found as galactopyranose (Galp, the six-membered ring form), which is the substrate for UDP-galactopyranose mutase (encoded by ugmA) to generate UDP-galactofuranose (Galf, the five-membered ring form) that is found in fungal walls. In A. fumigatus, Galf residues appear to be important for virulence. The A. nidulans ugeAΔ strain is viable, and has defects including wide, slow growing, highly branched hyphae and reduced conidiation that resemble the ugmAΔ strain. As for the ugmAΔ strain, ugeAΔ colonies had substantially reduced sporulation but normal spore viability. Conidia of the ugeAΔ strain could not form colonies on galactose as a sole carbon source, however they produced short, multinucleate germlings suggesting they ceased to grow from starvation. UgeA purified from an expression plasmid had a relative molecular weight of 40.6 kDa, and showed in vitro UDP-glucose-4-epimerase activity. Transmission electron microscope cross-sections of wildtype, ugeAΔ, and ugmAΔ hyphae showed they had similar cytoplasmic contents but the walls of each strain were different in appearance and thickness. Both deletion strains showed increased substrate adhesion. Localization of UgeA-GFP and UgmA-GFP was cytoplasmic, and was similar on glucose and galactose. Neither gene product had a longitudinal polarized distribution. Localization of a UgmA-mRFP in a strain that resembled the ugmAΔ strain was cytoplasmic and lacked a longitudinal polarized distribution. The roles of UgeA in A. nidulans growth and morphogenesis are consistent with the importance of Galf, and are related but not identical to the roles of UgmA.     

The light-dependent regulator velvet A of <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> acts as a repressor of the penicillin biosynthesis

The biosynthesis of the β-lactam antibiotic penicillin in Aspergillus nidulans is catalysed by three enzymes that are encoded by the genes acvA, ipnA and aatA. Several studies have indicated that these genes are controlled by a complex regulatory network, including a variety of cis-acting DNA elements and regulatory factors. Until now, however, relatively little information is available on external signals and their transmission influencing the expression of the structural genes. Here, we show that the light-dependent regulator velvet A (VeA) acts as a repressor on the penicillin biosynthesis, mainly via repression of the acvA gene. Expression of a regulatable alcAp-veA gene fusion in an A. nidulans strain carrying, in addition, acvAp-uidA and ipnAp-lacZ gene fusions indicated that under alcAp-inducing conditions, penicillin titres and expression of acvAp-uidA were drastically reduced compared with untransformed wild-type strains. The same level of repression was found irrespective of whether the alcAp-veA gene fusion was expressed in a veA1 or ΔveA background, with or without light. The expression of the ipnAp-lacZ gene fusion was only moderately affected indicating a less prominent effect. These findings were confirmed by the analysis of a regulatable niiAp-veA gene fusion. Under niiAp-inducing conditions, penicillin titres and acvAp-uidA expression were much lower than in untransformed wild-type strains.     

gfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O‐glycan in Aspergillus nidulans and Aspergillus fumigatus

The cells walls of filamentous fungi in the genus Aspergillus have galactofuranose (Galf)‐containing polysaccharides and glycoconjugates, including O‐glycans, N‐glycans, fungal‐type galactomannan and glycosylinositolphosphoceramide, which are important for cell wall integrity. Here, we attempted to identify galactofuranosyltransferases that couple Galf monomers onto other wall components in Aspergillus nidulans. Using reverse‐genetic and biochemical approaches, we identified that the AN8677 gene encoded a galactofuranosyltransferase, which we called GfsA, involved in Galf antigen biosynthesis. Disruption of gfsA reduced binding of β‐Galf‐specific antibody EB‐A2 to O‐glycosylated WscA protein and galactomannoproteins. The results of an in‐vitro Galf antigen synthase assay revealed that GfsA has β1,5‐ or β1,6‐galactofuranosyltransferase activity for O‐glycans in glycoproteins, uses UDP‐d ‐Galf as a sugar donor, and requires a divalent manganese cation for activity. GfsA was found to be localized at the Golgi apparatus based on cellular fractionation experiments. ΔgfsA cells exhibited an abnormal morphology characterized by poor hyphal extension, hyphal curvature and limited formation of conidia. Several gfsA orthologues were identified in members of the Pezizomycotina subphylum of Ascomycota, including the human pathogen Aspergillus fumigatus. To our knowledge, this is the first characterization of a fungal β‐galactofuranosyltransferase, which was shown to be involved in Galf antigen biosynthesis of O‐glycans in the Golgi.     

Functional Analysis of the α-1,3-Glucan Synthase Genes agsA and agsB in Aspergillus nidulans: AgsB Is the Major α-1,3-Glucan Synthase in This Fungus

Although α-1,3-glucan is one of the major cell wall polysaccharides in filamentous fungi, the physiological roles of α-1,3-glucan remain unclear. The model fungus Aspergillus nidulans possesses two α-1,3-glucan synthase (AGS) genes, agsA and agsB. For functional analysis of these genes, we constructed several mutant strains in A. nidulans: agsA disruption, agsB disruption, and double-disruption strains. We also constructed several CagsB strains in which agsB expression was controlled by the inducible alcA promoter, with or without the agsA-disrupting mutation. The agsA disruption strains did not show markedly different phenotypes from those of the wild-type strain. The agsB disruption strains formed dispersed hyphal cells under liquid culture conditions, regardless of the agsA genetic background. Dispersed hyphal cells were also observed in liquid culture of the CagsB strains when agsB expression was repressed, whereas these strains grew normally in plate culture even under the agsB-repressed conditions. Fractionation of the cell wall based on the alkali solubility of its components, quantification of sugars, and ¹³C-NMR spectroscopic analysis revealed that α-1,3-glucan was the main component of the alkali-soluble fraction in the wild-type and agsA disruption strains, but almost no α-1,3-glucan was found in the alkali-soluble fraction derived from either the agsB disruption strain or the CagsB strain under the agsB-repressed conditions, regardless of the agsA genetic background. Taken together, our data demonstrate that the two AGS genes are dispensable in A. nidulans, but that AgsB is required for normal growth characteristics under liquid culture conditions and is the major AGS in this species.     

Studies on (β,1→5) and (β,1→6) linked octyl Galf disaccharides as substrates for mycobacterial galactosyltransferase activity

The emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (MTB) and the continuing pandemic of tuberculosis emphasizes the urgent need for the development of new anti-tubercular agents with novel drug targets. The recent structural elucidation of the mycobacterial cell wall highlights a large variety of structurally unique components that may be a basis for new drug development. This publication describes the synthesis, characterization, and screening of several octyl Galf(β,1→5)Galf and octyl Galf(β,1→6)Galf derivatives. A cell-free assay system has been utilized for galactosyltransferase activity using UDP[¹⁴C]Galf as the glycosyl donor, and in vitro inhibitory activity has been determined in a colorimetric broth microdilution assay system against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC). Certain derivatives showed moderate activities against MTB and MAC. The biological evaluation of these disaccharides suggests that more hydrophobic analogues with a blocked reducing end showed better activity as compared to totally deprotected disaccharides that more closely resemble the natural substrates in cell wall biosynthesis.     

A Pericentrin-Related Protein Homolog in Aspergillus nidulans Plays Important Roles in Nucleus Positioning and Cell Polarity by Affecting Microtubule Organization

Pericentrin is a large coiled-coil protein in mammalian centrosomes that serves as a multifunctional scaffold for anchoring numerous proteins. Recent studies have linked numerous human disorders with mutated or elevated levels of pericentrin, suggesting unrecognized contributions of pericentrin-related proteins to the development of these disorders. In this study, we characterized AnPcpA, a putative homolog of pericentrin-related protein in the model filamentous fungus Aspergillus nidulans, and found that it is essential for conidial germination and hyphal development. Compared to the hyphal apex localization pattern of calmodulin (CaM), which has been identified as an interactive partner of the pericentrin homolog, GFP-AnPcpA fluorescence dots are associated mainly with nuclei, while the accumulation of CaM at the hyphal apex depends on the function of AnPcpA. In addition, the depletion of AnPcpA by an inducible alcA promoter repression results in severe growth defects and abnormal nuclear segregation. Most interestingly, in mature hyphal cells, knockdown of pericentrin was able to significantly induce changes in cell shape and cytoskeletal remodeling; it resulted in some enlarged compartments with condensed nuclei and anucleate small compartments as well. Moreover, defects in AnPcpA significantly disrupted the microtubule organization and nucleation, suggesting that AnPcpA may affect nucleus positioning by influencing microtubule organization.     

Depletion of Aspergillus nidulans cotA causes a severe polarity defect which is not suppressed by the nuclear migration mutation nudA2

The Aspergillus nidulans homologue of Neurospora crassa cot-1, cotA, encoding a member of the NDR protein kinase family, has been cloned and expressed under the control of the conditional alcA promoter. Depletion of CotA by repression of the alcA promoter led to a severe growth defect accompanied by loss of polarity. Germlings show greatly enlarged volume of the spores and hyphae, accompanied by an increase in number of nuclei per compartment, though the nucleus/volume ratio is not significantly altered. The depleted CotA phenotype was not suppressed by a nuclear migration mutation nudA2. Double mutants showed an additive, defective phenotype, unlike the suppression of the cot-1 ts mutation by ropy mutations seen in N. crassa, suggesting a different relationship between nuclear migration and the cot signalling pathway in A. nidulans. A functional CotA–GFP fusion protein was found in punctate regions of fluorescence similar to the distribution reported for human NDR2, and as a cap at the hyphal tip.     

The Aspergillus nidulans phsB4 mutation alters colonial growth and development of the mould at acidic pH

The phsB4 mutant of the mould Aspergillus nidulans, identified as showing increased sensitivity to acid pH, is mitotically unstable and its conidia swell and lyse, forming protoplasts during germination and early development in shaken liquid cultures. On solid medium, we observed balloon-shaped hyphal swellings, a phenotype also exhibited by the chitin synthase gene (chsD) disruptants. We also observed that lysis was osmotically remediable with 0.5 M NaCl, but the balloon-shaped hyphal swelling was remedied in a pH-dependent way i.e., this phenotype was remedied only at pH values above 6.5. Based on the nature of our mutant selection, the pH sensitive phenotype of the selected strains, the known occurrence of hyphal swelling in cell wall mutants of A. nidulans, and the transformation with cosmids that hybridize to chsD gene, the phsB and chsD genes are possibly alleles.     

Galactosyl transferases in mycobacterial cell wall synthesis

Two galactosyl transferases can apparently account for the full biosynthesis of the cell wall galactan of mycobacteria. Evidence is presented based on enzymatic incubations with purified natural and synthetic galactofuranose (Galf) acceptors that the recombinant galactofuranosyl transferase, GlfT1, from Mycobacterium smegmatis, the Mycobacterium tuberculosis Rv3782 ortholog known to be involved in the initial steps of galactan formation, harbors dual β-(1→4) and β-(1→5) Galf transferase activities and that the product of the enzyme, decaprenyl-P-P-GlcNAc-Rha-Galf-Galf, serves as a direct substrate for full polymerization catalyzed by another bifunctional Galf transferase, GlfT2, the Rv3808c enzyme.     

An extracellular β-galactofuranosidase from Aspergillus niger and its use as a tool for glycoconjugate analysis

Aspergillus niger produces an extracellular β-galactofuranosidase, which can specifically hydrolyse β-D-galactofuranose (Galf) from glycoconjugates. The production of this enzyme can be induced by the addition of a Galf-containing A. niger mycelial wall extract. However, on other carbon sources accumulation occurred only during the starvation conditions of the late stationary phase. Extracellular glucoamylases from this stage of cultivation possessed significantly lower levels of Galf than those from the earlier exponential growth phase when β-galactofuranosidase is absent, suggesting in situ β-galactofuranosidic hydrolysis. The β-galactofuranosidase responsible was subsequently purified to homogeneity and characterised. It is a glycoprotein of 90 kDa (determined by SDS–PAGE) with activity against β-linked Galf residues, with a K_m of 4 mM against p-nitrophenyl-β-D-galactofuranoside and a pH optimum of 3–4. The preparation did not contain other contaminating glycosidase activities; p-nitrophenyl-β-D- and -α-D-galactopyranose, and α-D-methyl-Galf were not hydrolysed. Results are presented to show that this enzyme could be employed as a useful tool for the analysis of glycoconjugates containing biologically important Galf components.     

Fungal surface remodelling visualized by atomic force microscopy

Most fungal growth is localized to the tips of hyphae, however, early stages of spore germination and the growth of certain morphological mutant strains exhibit non-polarized expansion. We used atomic force microscopy (AFM) to document changes in Aspergillus nidulans wall surfaces during non-polarized growth: spore germination, and growth in a strain containing the hypA1 temperature sensitive morphogenesis defect. We compared wall surface structures of both wild-type and mutant A. nidulans following growth at 28 ° and 42 °C, the latter being the restrictive temperature for hypA1. There was no appreciable difference in surface ultrastructure between wild-type and hypA1 spores, or hyphal walls grown at 28 °C. When dry mature A. nidulans conidia were wetted they lost their hydrophobin coat, indicating an intermediate stage between dormancy and swelling. The surface structure of hypA1 germlings grown at 42 °C was less organized than wild-type hyphae grown under the same conditions, and had a larger range of subunit sizes. AFM images of hyphal wall surface changes following a shift in growth temperature from restrictive (42 °C) to permissive (28 °C), showed a gradient of sizes for wall surface features similar to the trend observed for wild-type cells at branch points. Changes associated with the hyphal wall structure for A. nidulans hypA1 offer insight into the events associated with fungal germination, and wall remodelling.     

Synthesis and binding analysis of unique AG2 pentasaccharide to human Siglec-2 using NMR techniques

Siglec-2 is a mammalian sialic acid binding protein expressed on B-cell surfaces and is involved in the modulation of B-cell mediated immune response. We synthesized a unique starfish ganglioside, AG2 pentasaccharide Galfβ(1–3)Galpα(1–4)Neu5Acα(2–3)Galpβ(1–4)Glcp, and found that the synthetic pentasaccharide binds to human Siglec-2 by performing ¹H NMR experiments. Saturation transfer difference NMR experiments indicated that the C7–C9 side-chain and the acetamide moiety of the central sialic acid residue were located in the binding face of human Siglec-2. We determined the binding epitope of AG2 pentasaccharide to human Siglec-2, as the Galpα(1–4)Neu5Acα(2–3)Galp unit.     

Identification and Characterization of a Novel Galactofuranose-Specific β-D-Galactofuranosidase from Streptomyces Species

β-D-galactofuranose (Galf) is a component of polysaccharides and glycoconjugates and its transferase has been well analyzed. However, no β-D-galactofuranosidase (Galf-ase) gene has been identified in any organism. To search for a Galf-ase gene we screened soil samples and discovered a strain, identified as a Streptomyces species by the 16S ribosomal RNA gene analysis, that exhibits Galf-ase activity for 4-nitrophenyl β-D-galactofuranoside (pNP-β-D-Galf) in culture supernatants. By draft genome sequencing of the strain, named JHA19, we found four candidate genes encoding Galf-ases. Using recombinant proteins expressed in Escherichia coli, we found that three out of four candidates displayed the activity of not only Galf-ase but also α-L-arabinofuranosidase (Araf-ase), whereas the other one showed only the Galf-ase activity. This novel Galf-specific hydrolase is encoded by ORF1110 and has an optimum pH of 5.5 and a Km of 4.4 mM for the substrate pNP-β-D-Galf. In addition, this enzyme was able to release galactose residue from galactomannan prepared from the filamentous fungus Aspergillus fumigatus, suggesting that natural polysaccharides could be also substrates. By the BLAST search using the amino acid sequence of ORF1110 Galf-ase, we found that there are homolog genes in both prokaryotes and eukaryotes, indicating that Galf-specific Galf-ases widely exist in microorganisms.     

The Spt-Ada-Gcn5 Acetyltransferase (SAGA) Complex in Aspergillus nidulans

A mutation screen in Aspergillus nidulans uncovered mutations in the acdX gene that led to altered repression by acetate, but not by glucose. AcdX of A. nidulans is highly conserved with Spt8p of Saccharomyces cerevisiae, and since Spt8p is a component of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex, the SAGA complex may have a role in acetate repression in A. nidulans. We used a bioinformatic approach to identify genes encoding most members of the SAGA complex in A. nidulans, and a proteomic analysis to confirm that most protein components identified indeed exist as a complex in A. nidulans. No apparent compositional differences were detected in mycelia cultured in acetate compared to glucose medium. The methods used revealed apparent differences between Yeast and A. nidulans in the deubiquitination (DUB) module of the complex, which in S. cerevisiae consists of Sgf11p, Sus1p, and Ubp8p. Although a convincing homologue of S. cerevisiae Ubp8p was identified in the A. nidulans genome, there were no apparent homologues for Sus1p and Sgf11p. In addition, when the SAGA complex was purified from A. nidulans, members of the DUB module were not co-purified with the complex, indicating that functional homologues of Sus1p and Sgf11p were not part of the complex. Thus, deubiquitination of H2B-Ub in stress conditions is likely to be regulated differently in A. nidulans compared to S. cerevisiae.     

Transportation of Aspergillus nidulans Class III and V Chitin Synthases to the Hyphal Tips Depends on Conventional Kinesin

Cell wall formation and maintenance are crucial for hyphal morphogenesis. In many filamentous fungi, chitin is one of the main structural components of the cell wall. Aspergillus nidulans ChsB, a chitin synthase, and CsmA, a chitin synthase with a myosin motor-like domain (MMD) at its N-terminus, both localize predominantly at the hyphal tip regions and at forming septa. ChsB and CsmA play crucial roles in polarized hyphal growth in A. nidulans. In this study, we investigated the mechanism by which CsmA and ChsB accumulate at the hyphal tip in living hyphae. Deletion of kinA, a gene encoding conventional kinesin (kinesin-1), impaired the localization of GFP-CsmA and GFP-ChsB at the hyphal tips. The transport frequency of GFP-CsmA and GFP-ChsB in both anterograde and retrograde direction appeared lower in the kinA-deletion strain compared to wild type, although the velocities of the movements were comparable. Co-localization of GFP-ChsB and GFP-CsmA with mRFP1-KinA^rigor, a KinA mutant that binds to microtubules but does not move along them, was observed in the posterior of the hyphal tip regions. KinA co-immunoprecipitated with ChsB and CsmA. Co-localization and association of CsmA with KinA did not depend on the MMD. These findings indicate that ChsB and CsmA are transported along microtubules to the subapical region by KinA.     

Overlapping and Distinct Roles of Aspergillus fumigatus UDP-glucose 4-Epimerases in Galactose Metabolism and the Synthesis of Galactose-containing Cell Wall Polysaccharides

The cell wall of Aspergillus fumigatus contains two galactose-containing polysaccharides, galactomannan and galactosaminogalactan, whose biosynthetic pathways are not well understood. The A. fumigatus genome contains three genes encoding putative UDP-glucose 4-epimerases, uge3, uge4, and uge5. We undertook this study to elucidate the function of these epimerases. We found that uge4 is minimally expressed and is not required for the synthesis of galactose-containing exopolysaccharides or galactose metabolism. Uge5 is the dominant UDP-glucose 4-epimerase in A. fumigatus and is essential for normal growth in galactose-based medium. Uge5 is required for synthesis of the galactofuranose (Galf) component of galactomannan and contributes galactose to the synthesis of galactosaminogalactan. Uge3 can mediate production of both UDP-galactose and UDP-N-acetylgalactosamine (GalNAc) and is required for the production of galactosaminogalactan but not galactomannan. In the absence of Uge5, Uge3 activity is sufficient for growth on galactose and the synthesis of galactosaminogalactan containing lower levels of galactose but not the synthesis of Galf. A double deletion of uge5 and uge3 blocked growth on galactose and synthesis of both Galf and galactosaminogalactan. This study is the first survey of glucose epimerases in A. fumigatus and contributes to our understanding of the role of these enzymes in metabolism and cell wall synthesis.