Emp47 and Vip36 are required for polarized growth and protein trafficking between ER and Golgi apparatus in opportunistic fungal pathogen Aspergillus fumigatus.

In Aspergillus fumigatus, an opportunistic fungal pathogen causing fatal invasive aspergillosis, N-glycosylation is vital for polarized growth. To investigate its mechanism, two putative L-type lectin genes emp47 (AFUB_032470) and vip36 (AFUB_027870) were identified in A. fumigatus. Deletion of the emp47 or vip36 gene resulted in delayed germination and abnormal polarity. Also, the Δemp47 displayed an increased resistance to azoles whereas the Δvip36 showed an increased susceptibility to amphotericin B. Secretome analysis revealed that 205 proteins were differentially secreted in the Δemp47 and 145 of them were reduced, while 153 proteins displayed a differential secretion and 134 of them were increased in the Δvip36 as compared with that of the wild-type strain. Also, potential cargo glycoproteins of Emp47 and Vip36 were identified by comparative secretome analysis. Our results suggest that Emp47 is responsible for the transport of proteins from endoplasmic reticulum (ER) to Golgi, while Vip36 acts in protein retrieval from Golgi to ER.

Deficiency of GPI Glycan Modification by Ethanolamine Phosphate Results in Increased Adhesion and Immune Resistance of Aspergillus fumigatus.

Glycosylphosphatidylinositol (GPI)-anchored proteins play important roles in maintaining the function of the cell wall and participating in pathogenic processes. The addition and removal of phosphoethanolamine (EtN-P) on the second mannose residue in the GPI anchor are vital for maturation and sorting of GPI-anchored proteins. Previously, we have shown that deletion of the gpi7, the gene that encodes an EtN-P transferase responsible for the addition of EtN-P to the second mannose residue of the GPI anchor, leads to the mislocalization of GPI-anchored proteins, abnormal polarity, reduced conidiation, and fast germination in Aspergillus fumigatus. In this report, the adherence and virulence of the A. fumigatus gpi7 deletion mutant were further investigated. The germinating conidia of the mutant exhibited an increased adhesion and a higher exposure of cell wall polysaccharides. Although the virulence was not affected, an increased adherence and a stronger inflammation response of the mutant were documented in an immunocompromised mouse model. An in vitro assay confirmed that the Δgpi7 mutant induced a stronger immune response and was more resistant to killing. Our findings, for the first time, demonstrate that in A. fumigatus, GPI anchoring is required for proper organization of the conidial cell wall. The lack of Gpi7 leads to fast germination, stronger immune response, and resistance to macrophage killing.

Protein O-mannosylation affects protein secretion, cell wall integrity and morphogenesis in Trichoderma reesei.

Protein O-mannosyltransferases (PMTs) initiate O-mannosylation of proteins in the ER. Trichoderma reesei strains displayed a single representative of each PMT subfamily, Trpmt1, Trpmt2 and Trpmt4. In this work, two knockout strains ΔTrpmt1and ΔTrpmt4were obtained. Both mutants showed retarded growth, defective cell walls, reduced conidiation and decreased protein secretion. Additionally, the ΔTrpmt1strain displayed a thermosensitive growth phenotype, while the ΔTrpmt4 strain showed abnormal polarity. Meanwhile, OETrpmt2 strain, in which the Trpmt2 was over-expressed, exhibited increased conidiation, enhanced protein secretion and abnormal polarity. Using a lectin enrichment method and MS/MS analysis, 173 O-glycoproteins, 295 O-glycopeptides and 649 O-mannosylation sites were identified as the targets of PMTs in T. reesei. These identified O-mannoproteins are involved in various physiological processes such as protein folding, sorting, transport, quality control and secretion, as well as cell wall integrity and polarity. By comparing proteins identified in the mutants and its parent strain, the potential specific protein substrates of PMTs were identified. Based on our results, TrPMT1 is specifically involved inO-mannosylation of intracellular soluble proteins and secreted proteins, specially glycosidases. TrPMT2 is involved inO-mannosylation of secreted proteins and GPI-anchor proteins, and TrPMT4 mainly modifies multiple transmembrane proteins. The TrPMT1-TrPMT4 complex is responsible for O-mannosylation of proteins involved in cell wall integrity. Overexpression of TrPMT2 enhances protein secretion, which might be a new strategy to improve expression efficiency in T. reesei.

Aspergillus fumigatus phosphoethanolamine transferase gene gpi7 is required for proper transportation of the cell wall GPI-anchored proteins and polarized growth.

In fungi many proteins, which play important roles in maintaining the function of the cell wall and participating in pathogenic processes, are anchored to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. It has been known that modification and removal of phosphoethanolamine (EtN-P) on the second mannose residue in GPI anchors is important for maturation and sorting of GPI anchored proteins in yeast and mammalian cells, but is a step absent from some protist parasites. In Aspergillus fumigatus, an opportunistic fungal pathogen causing invasive aspergillosis in humans, GPI-anchored proteins are known to be involved in cell wall synthesis and virulence. In this report the gene encoding A. fumigatus EtN-P transferase GPI7 was investigated. By deletion of the gpi7 gene, we evaluated the effects of EtN-P modification on the morphogenesis of A. fumigatus and localization of GPI proteins. Our results showed that deletion of the gpi7 gene led to reduced cell membrane GPI anchored proteins, the mis-localization of the cell wall GPI anchored protein Mp1, abnormal polarity, and autophagy in A. fumigatus. Our results suggest that addition of EtN-P of the second mannose on the GPI anchor is essential for transportation and localization of the cell wall GPI-anchored proteins.

Aspergillus fumigatus Mnn9 is responsible for mannan synthesis and required for covalent linkage of mannoprotein to the cell wall.

Owing to the essential role in protection of the Aspergillus fumigatus cell against human defense reactions, its cell wall has long been taken as a promising antifungal target. The cell wall of A. fumigatus composed of chitin, glucan and galactomannan and mannoproteins. Although galactomannan has been used as a diagnostic target for a long time, its biosynthesis remains unknown in A. fumigatus. In this study, a putative α1,6-mannosyltransferase gene mnn9 was identified in A. fumigatus. Deletion of the mnn9 gene resulted in an increased sensitivity to calcofluor white, Congo red, or hygromycin B as well as in reduced cell wall components and abnormal polarity. Although there was no major effect on N-glycan synthesis, covalently-linked cell wall mannoprotein Mp1 was significantly reduced in the mutant. Based on our results, we propose that Mnn9p is a mannosyltransferase responsible for the formation of the α-mannan in cell wall mannoproteins, potentially via elongation of O-linked mannose chains.

Insight into Enzymatic Degradation of Corn, Wheat, and Soybean Cell Wall Cellulose Using Quantitative Secretome Analysis of Aspergillus fumigatus.

Lignocelluloses contained in animal forage cannot be digested by pigs or poultry with 100% efficiency. On contrary, Aspergillus fumigatus, a saprophytic filamentous fungus, is known to harbor 263 glycoside hydrolase encoding genes, suggesting that A. fumigatus is an efficient lignocellulose degrader. Hence the present study uses corn, wheat, or soybean as a sole carbon source to culture A. fumigatus under animal physiological condition to understand how cellulolytic enzymes work together to achieve an efficient degradation of lignocellulose. Our results showed that A. fumigatus produced different sets of enzymes to degrade lignocelluloses derived from corn, wheat, or soybean cell wall. In addition, the cellulolytic enzymes produced by A. fumigatus were stable under acidic condition or at higher temperatures. Using isobaric tags for a relative and absolute quantification (iTRAQ) approach, a total of ∼600 extracellular proteins were identified and quantified, in which ∼50 proteins were involved in lignocellulolysis, including cellulases, hemicellulases, lignin-degrading enzymes, and some hypothetical proteins. Data are available via ProteomeXchange with identifier PXD004670. On the basis of quantitative iTRAQ results, 14 genes were selected for further confirmation by RT-PCR. Taken together, our results indicated that the expression and regulation of lignocellulolytic proteins in the secretome of A. fumigatus were dependent on both nature and complexity of cellulose, thus suggesting that a different enzyme system is required for degradation of different lignocelluloses derived from plant cells. Although A. fumigatus is a pathogenic fungus and cannot be directly used as an enzyme source, as an efficient lignocellulose degrader its strategy to synergistically degrade various lignocelluloses with different enzymes can be used to design enzyme combination for optimal digestion and absorption of corn, wheat, or soybean that are used as forage of pig and poultry.

N-Glycosylation of Gel1 or Gel2 is vital for cell wall ß-glucan synthesis in Aspergillus fumigatus.

Fungal cell wall is a dynamic structure that communicates with and protects the cell from outside stress. In Aspergillus fumigatus, the cell wall ß-glucans are mainly elongated by ß-1,3-glucanosyltransferases Gels, which consist of seven family members (Gel1-7) utilizing ß-1,3-glucan chains as substrates. Previously, we have shown that the mutant deficient of N-glycan processing displays a reduction in the cell wall ß-glucans, suggesting that N-glycosylation is required for the proper function of ß-1,3-glucanosyltransferase. To verify this hypothesis, in this study, the gene encoding ß-1,3-glucanosyltransferase Gel1 or Gel2 was deleted in the Δcwh41 mutant to construct a double-mutant Δgel1Δcwh41 or Δgel2Δcwh41. The growth phenotypes of both double mutants were similar to the single-mutant Δcwh41, suggesting that Gel1 and Gel2 are proteins that are mainly affected by deficient N-glycan processing in Δcwh41. Furthermore, the mutant Δgel1(Gel1-NM) or Δgel2(Gel2-NM), in which all potential N-glycosylation sites on Gel1 or Gel2 were removed by site-directed mutagenesis, showed phenotypes similar to the single-mutant Δgel1 or Δgel2. Biochemical analysis revealed that N-glycosylation was essential for the function of Gel1 or Gel2 and thus required for ß-glucan synthesis in A. fumigatus.

One single basic amino acid at the ω-1 or ω-2 site is a signal that retains glycosylphosphatidylinositol-anchored protein in the plasma membrane of Aspergillus fumigatus.

Although the plasma membrane is the terminal destination for glycosylphosphatidylinositol (GPI) proteins in higher eukaryotes, cell wall-attached GPI proteins (GPI-CWPs) are found in many fungal species. In yeast, some of the cis-requirements directing localization of GPI proteins to the plasma membrane or cell wall are now understood. However, it remains to be determined how Aspergillus fumigatus, an opportunistic fungal pathogen, signals, and sorts GPI proteins to either the plasma membrane or the cell wall. In this study, chimeric green fluorescent proteins (GFPs) were constructed as fusions with putative C-terminal GPI signal sequences from A. fumigatus Mp1p, Gel1p, and Ecm33p, as well as site-directed mutations thereof. By analyzing cellular localization of chimeric GFPs using Western blotting, electron microscopy, and fluorescence microscopy, we showed that, in contrast to yeast, a single Lys residue at the ω-1 or ω-2 site alone could retain GPI-anchored GFP in the plasma membrane. Although the signal for cell wall distribution has not been identified yet, it appeared that the threonine/serine-rich region at the C-terminal half of AfMp1 was not required for cell wall distribution. Based on our results, the cis-requirements directing localization of GPI proteins in A. fumigatus are different from those in yeast.

[Construction of a cell-surface expression system in Trichoderma reesei].

OBJECTIVE: AfMp1p is a glycosylphosphatidylinositol (GPI) anchored cell wall protein (GPI-CWP) identified in filamentous fungus Aspergillus fumigatus, which contains a specific C terminal signal for cell wall localization. Trichoderma reesei is known as a safe fungal species (GRAS) and widely used in the industry. Thus, developing of the cell-surface expression systems in T. reesei is of industrial interest. METHODS: The GPI signal from the A. fumigatus AfMp1p was fused to the C-terminal of green fluorescent protein (GFP) and transformed into T. reesei. The optimization of T. reesei transformation and the expression profiles of the GFP were investigated in detail. The cellular location of the GFP fusion protein was detected. RESULTS: Fluorescent image analysis and Western blot analysis indicate that the GFP fusion protein locates on the cell wall of T. reesei. CONCLUSION: According to these results, the GPI signal from A. fumigatus can be recognized in T. reesei and the expression system constructed in this study can be used to express heterogeneous protein in the cell wall of T. reesei.

Transcriptome and biochemical analysis reveals that suppression of GPI-anchor synthesis leads to autophagy and possible necroptosis in Aspergillus fumigatus.

Previously, it has been shown that GPI proteins are required for cell wall synthesis and organization in Aspergillus fumigatus, a human opportunistic pathogen causing life-threatening invasive aspergillosis (IA) in immunocompromised patients. Blocking GPI anchor synthesis leads to severe phenotypes such as cell wall defects, increased cell death, and attenuated virulence. However, the mechanism by which these phenotypes are induced is unclear. To gain insight into global effects of GPI anchoring in A. fumigatus, in this study a conditional expression mutant was constructed and a genome wide transcriptome analysis was carried out. Our results suggested that suppression of GPI anchor synthesis mainly led to activation of phosphatidylinositol (PtdIns) signaling and ER stress. Biochemical and morphological evidence showed that autophagy was induced in response to suppression of the GPI anchor synthesis, and also an increased necroptosis was observed. Based on our results, we propose that activation of PtdIns3K and increased cytosolic Ca(2+), which was induced by both ER stress and PtdIns signaling, acted as the main effectors to induce autophagy and possible necroptosis.

[Cloning, expression and characterization of beta-glucosidase from Aspergillus fumigatus].

Exploring new beta-glucosidase genes is of great importance to industrialize beta-glucosidase. The genomes of Aspergillus fumigatus contain a bgl gene, which encodes a 65 kDa putative beta-glucosidase. The bgl gene was cloned into an expression plasmid and transformed to Escherichia coli BL21 (DE3). The bgl was expressed upon induction of Isopropyl beta-D-1-thiogalactopyranoside (IPTG). The recombinant protein was purified by GST-tag affinity chromatography. The purified recombinant Bgl was characterized using Esculin as substrate. The optimum temperature and pH were 45 degrees C and 5.0-6.0, respectively. The K(m) for Esculin was 17.7 mmol/L. The enzyme was stable in the range of pH 4-7. After incubation at 70 degrees C for 2 h, the recombinant Bgl remained 60% of its activity. Metal ions and chemical reagents had different influences on the activity of beta-glucosidase. Ca2+ (1 mmol/L) could increase enzyme activity slightly. On the contrary, the enzyme activity was greatly inhibited by 5 mmol/L Sodium dodecyl sulfate (SDS). Based on our results, the A. fumigatus Bgl was thermostable beta-glucosidase.

Repression of N-glycosylation triggers the unfolded protein response (UPR) and overexpression of cell wall protein and chitin in Aspergillus fumigatus.

Aspergillus fumigatus is the most common airborne fungal pathogen, causing fatal invasive aspergillosis in immunocompromised patients. The crude mortality is 60-90 % and remains around 29-42 % even with treatment. The main reason for patient death is the low efficiency of the drug therapies. As protein N-glycosylation is involved in cell wall biogenesis in A. fumigatus, a deeper understanding of its role in cell wall biogenesis will help to develop new drug targets. The Afstt3 gene encodes the essential catalytic subunit of oligosaccharyltransferase, an enzyme complex responsible for the transfer of the N-glycan to nascent polypeptides. To evaluate the role of N-glycosylation in cell wall biosynthesis, we constructed the conditional mutant strain CPR-stt3 by replacing the endogenous promoter of Afstt3 with the nitrogen-dependent niiA promoter. Repression of the Afstt3 gene in the CPR-stt3 strain led to a severe retardation of growth and a slight defect in cell wall integrity (CWI). One of the most interesting findings was that upregulation of the cell wall-related genes was not accompanied by an activation of the MpkA kinase, which has been shown to be a central element in the CWI signalling pathway in both Saccharomyces cerevisiae and A. fumigatus. Considering that the unfolded protein response (UPR) was found to be activated, which might upregulate the expression of cell wall protein and chitin, our data suggest that the UPR, instead of the MpkA-dependent CWI signalling pathway, is the major compensatory mechanism induced by repression but not abolition of N-glycosylation in A. fumigatus. Our finding is a key to understanding the complex compensatory mechanisms of cell wall biosynthesis and may provide a new strategy for drug development.

Reduced expression of the O-mannosyltransferase 2 (AfPmt2) leads to deficient cell wall and abnormal polarity in Aspergillus fumigatus.

Protein O-mannosyltransferases (PMTs) initiate O-mannosylation of secretory proteins, which are of fundamental importance in eukaryotes. The human fungal pathogen Aspergillus fumigatus possesses three genes encoding for PMTs, namely, Afpmt1, Afpmt2 and Afpmt4. We have previously shown that lack of AfPmt1 leads to a temperature-sensitive phenotype featured with severe defects in hyphal growth, conidiation, cell wall integrity and morphology at elevated temperatures. In this study, a conditional mutant P2 was constructed by replacing the native promoter of the Afpmt2 with the Aspergillus nidulans alcA promoter. Reduced expression of the Afpmt2 gene led to a lagged germination, retarded hyphal growth, reduced conidiation and defect in cell wall integrity; however, no temperature-sensitive growth was observed. Further analysis revealed that reduced expression of the Afpmt2 caused a failure of the actin re-arrangement. Our results suggest that Afpmt2 gene was required for growth and played a role distinct from that of the Afpmt1 in A. fumigatus.

Proteome analysis of Aspergillus fumigatus total membrane proteins identifies proteins associated with the glycoconjugates and cell wall biosynthesis using 2D LC-MS/MS.

We attempted to identify membrane proteins associated with the glycoconjugates and cell wall biosynthesis in the total membrane preparations of Aspergillus fumigatus. The total membrane preparations were first run on 1D gels, and then the stained gels were cut and submitted to in-gel digestion followed by 2D LC-MS/MS and database search. A total of 530 proteins were identified with at least two peptides detected with MS/MS spectra. Seventeen integral membrane proteins were involved in N-, O-glycosylation or GPI anchor biosynthesis. Nine membrane proteins were involved in cell wall biosynthesis. Eight proteins were identified as enzymes involved in sphingolipid synthesis. In addition, the proteins involved in cell wall and ergosterol biosynthesis can potentially be used as antifungal drug targets. Our method, for the first time, clearly provided a global view of the membrane proteins associated with glycoconjugates and cell wall biosynthesis in the total membrane proteome of A. fumigatus.

Characterization of the Aspergillus fumigatus phosphomannose isomerase Pmi1 and its impact on cell wall synthesis and morphogenesis.

Phosphomannose isomerase (PMI) is an enzyme catalysing the interconversion of mannose 6-phosphate (Man-6-P) and fructose 6-phosphate (Fru-6-P). The reaction catalysed by PMI is the first committed step in the synthesis of mannose-containing sugar chains and provides a link between glucose metabolism and mannosylation. In this study, the pmi1 gene was identified to encode PMI in the human fungal pathogen Aspergillus fumigatus. Characterization of A. fumigatus Pmi1 expressed in Escherichia coli revealed that this PMI mainly catalysed the conversion of Fru-6-P to Man-6-P and that its binding affinity for Man-6-P was similar to that of yeast PMIs, but different to those of PMIs from bacteria or animals. Loss of pmi1 was lethal unless Man was provided in the growth medium. However, a Deltapmi1 mutant cell showed a significantly reduced growth rate at a high concentration of Man. Biochemical analysis revealed that both inadequate and replete Man led to an accumulation of intracellular Man-6-P and a reduction in the amount of alpha-glucan in the cell wall. Uncoupling of the link between energy production and glycosylation by deletion of the pmi1 gene led to phenotypes such as defects in cell wall integrity, abnormal morphology and reduced conidiation. Our results reveal that PMI activity is essential for viability and plays a central regulatory role in both cell wall synthesis and energy production in A. fumigatus.

Comparative proteomic analysis of an Aspergillus fumigatus mutant deficient in glucosidase I (AfCwh41).

Alpha-glucosidase I regulates trimming of the terminal alpha-1,2-glucose residue in the N-glycan processing pathway, which plays an important role in quality control systems in mammalian cells. Previously, we identified the gene encoding alpha-glucosidase I in the opportunistic human fungal pathogen Aspergillus fumigatus, namely Afcwh41. Deletion of the Afcwh41 gene results in a severe reduction of conidia formation, a temperature-sensitive deficiency of cell wall integrity, and abnormalities of polar growth and septation. An upregulation of the genes encoding Rho-type GTPases was also observed, which suggests activation of the cell wall integrity pathway in the mutant. Using 2D gel analysis, we revealed that the proteins involved in protein assembly, ubiquitin-mediated degradation and actin organization are altered in the DeltaAfcwh41 mutant. Evidence was obtained for a defect in the polarized localization of the actin cytoskeleton in the mutant. Our results suggest that blocking of the glucose trimming in A. fumigatus might induce accumulation of misfolded proteins in the endoplasmic reticulum; these misfolded proteins are probably required for cell wall synthesis and thus activate the cell wall integrity pathway, which then causes the abnormal polarity associated with the DeltaAfcwh41 mutant.

Class IIC alpha-mannosidase AfAms1 is required for morphogenesis and cellular function in Aspergillus fumigatus.

The mammalian ER/cytosolic alpha-mannosidase (Man2C1p), yeast vacuolar alpha-mannosidase (Ams1p) and the Aspergillus nidulans alpha-mannosidase are members of Class IIC subgroup, which is involved in oligosaccharide catabolism and N-glycan processing. Unlike their mammalian counterparts, the yeast Ams1p and A. nidulans Class IIC alpha-mannosidase are not essential for morphogenesis and cellular function. In this study, the Afams1, a gene encoding a member of Class IIC alpha-mannosidases, was identified in the opportunistic pathogen Aspergillus fumigatus. Deletion of the Afams1 led to a severe defect in conidial formation, especially at a higher temperature. In addition, abnormalities of polarity and septation were associated with the DeltaAfams1 mutant. Our results showed that the Afams1 gene, in contrast to its homolog in yeast or A. nidulans, was required for morphogenesis and cellular function in A. fumigatus.

Afcwh41 is required for cell wall synthesis, conidiation, and polarity in Aspergillus fumigatus.

alpha-Glucosidase I regulates trimming of the terminal alpha-1,2-glucose residue in the N-glycan-processing pathway, which plays an important role in the quality control system in mammalian cells. However, the consequence of glucose trimming of the N-glycan in filamentous fungi is unclear. We identified the gene encoding alpha-glucosidase I in the human opportunistic fungal pathogen Aspergillus fumigatus, namely Afcwh41. Deletion of the Afcwh41 gene resulted in a defective N-glycan processing of the proteins secreted by A. fumigatus. Although the Afcwh41 was not essential for hyphal growth and virulence, a severe reduction in conidia formation and a temperature-sensitive deficiency of cell wall integrity (CWI) were observed. Also, abnormalities of polar growth and septation were observed during conidial germination and hyphal elongation of the mutant. Our results suggest that Afcwh41 was involved in CWI, polarity, septation, and conidiation in A. fumigatus, probably by affecting the proper function of the proteins that are required for cell wall synthesis.

GDP-mannose pyrophosphorylase is essential for cell wall integrity, morphogenesis and viability of Aspergillus fumigatus.

GDP-mannose pyrophosphorylase (GMPP) catalyses the synthesis of GDP-mannose, which is the precursor for the mannose residues in glycoconjugates, using mannose 1-phosphate and GTP as substrates. Repression of GMPP in yeast leads to phenotypes including cell lysis, defective cell wall, and failure of polarized growth and cell separation. Although several GMPPs have been isolated and characterized in filamentous fungi, the physiological consequences of their actions are not clear. In this study, Afsrb1, which is a homologue of yeast SRB1/PSA1/VIG9, was identified in the Aspergillus fumigatus genome. The Afsrb1 gene was expressed in Escherichia coli, and recombinant AfSrb1 was functionally confirmed as a GMPP. By the replacement of the native Afsrb1 promoter with an inducible Aspergillus nidulans alcA promoter, the conditional inactivation mutant strain YJ-gmpp was constructed. The presence of 3 % glucose completely blocked transcription of P(alcA)-Afsrb1, and was lethal to strain YJ-gmpp. Repression of Afsrb1 expression in strain YJ-gmpp led to phenotypes including hyphal lysis, defective cell wall, impaired polarity maintenance, and branching site selection. Also, rapid germination and reduced conidiation were documented. However, in contrast to yeast, strain YJ-gmpp retained the ability to direct polarity establishment and septation. Our results showed that the Afsrb1 gene is essential for cell wall integrity, morphogenesis and viability of Aspergillus fumigatus.

Deletion of the msdS/AfmsdC gene induces abnormal polarity and septation in Aspergillus fumigatus.

alpha-Mannosidases play an important role in the processing of mannose-containing glycans in eukaryotes. A deficiency in alpha-mannosidase is lethal in humans and cattle. In contrast to mammals, Saccharomyces cerevisiae does not require the endoplasmic reticulum alpha-mannosidase gene for growth. However, little is known of the consequence of loss of function of class I alpha-mannosidases in filamentous fungi. In this study, the msdS/AfmsdC gene was identified to encode 1,2-alpha-mannosidase MsdS in Aspergillus fumigatus. Soluble MsdS expressed in Escherichia coli was characterized as a typical class I alpha-mannosidase. The msdS gene was deleted by replacement of the msdS gene with a pyrG gene. Although the mutant showed a defect in N-glycan processing, as well as a reduction of cell wall components and a reduced ability of conidiation, it appeared that the rate of hyphal growth was not affected. Morphology analysis revealed abnormal polarity and septation at the stages of germination, hyphal growth and conidiation. Although the mechanism by which the N-glycan processing affects polarity and septation is unclear, our results show that msdS is involved in polarity and septation in A. fumigatus.