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State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China
Correspondence
Cheng Jin
jinc{at}sun.im.ac.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the nucleotide sequence of Afsrb1 is DQ017035.
A table of primers used in this study is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 2001; Krappmann, 2006) and is the leading cause of death in patients with leukaemia and AIDS, and those that have had bone-marrow-transplants. The crude mortality from invasive aspergillosis is above 90 %, and falls to around 50–70 % if treatment is given (Steinbach et al., 2003). The main reason for patient death is the low efficiency of the drug therapies available to treat invasive aspergillosis. Therefore, there is an urgent need for a deep understanding of A. fumigatus at the molecular level.
The cell wall helps the fungus to battle against adverse environments, and it has a variety of biological functions, such as maintaining morphogenesis, and regulating the selective permeability (Yoda et al., 2000; Agaphonov et al., 2001). The A. fumigatus cell wall consists mainly of a covalently connected polysaccharide skeleton (glucans and chitin) that is interlaced and coated with glycoproteins, which contain mannose and galactose derived primarily from the process of glycosylation (Fontaine et al., 2000; Latgé et al., 2005; Upadhyay & Shaw, 2006). Some cell-surface proteins are further modified at their C terminus by the addition of a glycosylphosphatidylinositol (GPI) anchor, and they are transported to the plasma membrane and cell wall. These GPI proteins are involved in morphogenesis and cell-wall organization (Mouyna et al., 2000, 2005; Bruneau et al., 2001; Chabane et al., 2006; Romano et al., 2006; De Groot et al., 2005; Li et al., 2007).
GDP-mannose, an activated form of mannose, is the precursor of mannose residues in galactomannan, glycoprotein and the GPI anchor. Activation of mannose requires three enzymes: phosphomannose isomerase, phosphomannomutase and GDP-mannose pyrophosphorylase (GMPP). To date, several GMPPs have been identified and characterized in different species (Griffin et al., 1997; Ning & Elbein, 1999; Ohta et al., 2000; Warit et al., 1998; Sacchetti et al., 2004). In Saccharomyces cerevisiae and Candida albicans, GMPP is essential (Hashimoto et al., 1997; Warit et al., 2000), while in Leishmania mexicana the GMPP is not required for viability (Garami & Ilg, 2001). Depletion of the GMPP in S. cerevisiae and C. albicans leads to pleiotropic phenotypes, including cell lysis, failure of cell separation, impaired budding and hyphal switching, clumping and flocculation, and defect of the cell wall (Warit et al., 2000). The aforementioned reports imply that GMPPs are specifically crucial for synthesis and organization of the cell wall, and thus essential for species that have a cell wall.
To evaluate the impact of GMPP on the cell wall of A. fumigatus, we identified a homologue of yeast SRB1/PSA1/VIG9 from a genomic database through bioinformatics analysis. In this report, the putative Afsrb1 gene was expressed, and confirmed to encode a GMPP. In addition, the phenotypes associated with depletion of the Afsrb1 gene were analysed.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). A. fumigatus strain CEA17 (Weidner et al., 1998), a gift from C. d'Enfert, Institut Pasteur, Paris, France, was propagated at 37 °C on YGA (0.5 % yeast extract, 2 % glucose, 1.5 % Bacto-agar), complete medium (CM), or minimal medium with 0.5 mM sodium glutamate as a nitrogen source (MM) (Cove, 1966). Uridine and uracil were added at a concentration of 5 mM when strain CEA17 was grown.
Strains were grown in liquid CM at 37 °C, with shaking at 250 r.p.m. At the specified culture time point, mycelia were harvested, washed with distilled water, frozen in liquid N2, and then ground by hand. The powder was stored at –70 °C for DNA, RNA and protein extraction. Conidia were prepared by growing A. fumigatus strains on solid CM with uridine and uracil for 48 h at 37 °C. The spores were collected, washed twice with 0.01 % Tween 20 in physiological saline, resuspended in 0.01 % Tween 20 in saline, and the concentration of spores was confirmed by haemocytometer counting and viable counting. Vectors and plasmids were propagated in Escherichia coli DH5
(Bethesda Research Laboratories, Bethesda, Maryland, USA).
Computer analysis.
Sequence alignments were analysed by Vector NTI, and a BLAST search was performed.
Isolation of the Afsrb1 gene from A. fumigatus.
A homologue of yeast SRB1/PSA1/VIG9 was identified by a tBLASTn search of the A. fumigatus genome database (http://www.tigr.org/tdb/e2k1/afu1/), and it was designated Afsrb1. cDNA of Afsrb1 was isolated by RT-PCR. A. fumigatus total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer's instruction, and cDNA was synthesized with primer 1 (see supplementary Table S1, available with the online version of this paper) using M-MuLV (NEB). Thirty cycles (94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min) of PCR reaction were carried out using primers 2 and 3 (Table S1). The PCR products were subcloned into pGEM-T vector (Promega) to yield pGEM-T-GMPP, and then sequenced. The position of the intron was determined by comparing the cDNA with the genomic sequence.
Expression of the Afsrb1 gene in E. coli.
The cDNA of the Afsrb1 gene was amplified from pGEM-T-GMPP with primers 4 and 5 (Table S1), and subcloned into pET-32a (Novagen). The recombinant expression vector containing the Afsrb1 gene fused to the gene encoding thioredoxin (Trx) was confirmed by PCR, restriction enzymes and sequencing. The resulting recombinant plasmid was designated pET-32GMP.
The recombinant strain E. coli BL21(DE3) (Novagen), harbouring pET32-GMP, was grown in 5 ml Luria–Bertani (LB) medium containing 0.1 mg ampicillin ml–1, at 37 °C overnight. A 1 ml volume of cell culture was inoculated into 100 ml LB containing 0.1 mg ampicillin ml–1, and incubated at 30 °C. When the OD600 value of the cell culture reached 0.6, the recombinant protein was induced by the addition of IPTG (final concn 0.4 mM; Sigma) at 30 °C for 4–6 h. The cells were harvested by centrifugation (12 000 r.p.m. for 15 min in an Avanti J-25 Centrifuge; Beckman), and resuspended in 100 ml 1x binding buffer (0.02 M sodium phosphate, 0.5 M NaCl, 40 mM imidazole, pH 7.4) containing 0.3 mg lysozyme ml–1. After incubation at 37 °C for 15 min, the cells were sonicated. The cell lysate was collected by centrifugation (12 000 r.p.m. for 15 min), filtered through a 0.45 µm membrane, and run on a HiTrap affinity column (Ni Sepharose 6 Fast Flow; GE Healthcare). After washing with 20 column vols binding buffer, the recombinant protein was eluted with a gradient of imidazole (40–500 mM), and dialysed against 50 mM Tris buffer, pH 7.5.
The purity of the recombinant protein was confirmed by SDS-PAGE. The protein concentration was determined by the Bradford assay (Bradford, 1976).
Enzyme assay.
GMPP activity was determined as described by Ohta et al. (2000). Briefly, the reaction mixture contained 50 mM Tris (pH 7.5), 8 mM MgCl2, 100 µM GTP, 100 µM mannose 1-phosphate (Sigma), 1 mM DTT, 0.1 unit inorganic pyrophosphatase ml–1 (Sigma), and 0.1 µg recombinant GMPP, in a total volume of 100 µl. The reaction was carried out at 30 °C for 30 min. A 40 µl volume of the reaction mixture was diluted with 120 µl water, mixed with 40 µl dye reagent (containing malachite green, sulfuric acid, ammonium molybdate and Tween 20), and incubated at 30 °C for 10 min. The amount of inorganic phosphate generated from the pyrophosphate was determined by measuring the absorbance at 630 nm. Trx was used as a negative control.
Construction of the conditional inactivation mutant.
The conditional inactivation mutant was constructed by replacement of the native promoter (Psrb1) of Afsrb1 with the alcA promoter (PalcA). In order to achieve this, an 869 bp fragment from –14 to +855 of the A. fumigatus Afsrb1 genomic sequence was amplified with primers 6 and 7 (see supplementary Table S1), and cloned into the BamHI site of pAL3 (a gift from J. R. D. Lucas, Universidad de Alcalá, Madrid, Spain) to generate pALGMP. The upstream fragment of Psrb1 was amplified from A. fumigatus genomic DNA using primers 18 and 15 (94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min) (Table S1), and the downstream fragment of the Psrb1 was amplified from pALGMP using primers 14 and 7 (94 °C for 1 min, 56 °C for 1 min, and 72 °C for 3 min) (Table S1). The two PCR products were used as templates for the second-round PCR reaction, in which a fragment containing (in sequence) the upstream sequence of Psrb1, the pyr-4 gene, PalcA, and the downstream sequence of Psrb1, was amplified with primers 7 and 18 (94 °C for 1 min, 56 °C for 1 min, and 72 °C for 4 min) (Table S1). The products from the second round of PCR were subcloned into pGEM-T to generate pGEM-P, and then sequenced.
The linearized pGEM-T was transformed into A. fumigatus strain CEA17 by PEG-mediated fusion of protoplasts (Langfelder et al., 2002), and screened for cells with uridine/uracil autotrophy. The transformants were chosen by PCR, and the transformation was confirmed by Southern blot analysis. For PCR analysis, three pairs of primers (8 and 9, 10 and 11, and 12 and 13) (Table S1) were employed with the following cycling conditions: 94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min. The first pair of primers was utilized as a positive control that could yield a 1268 bp Afsrb1 fragment. The other two pairs of primers corresponded to a 1217 bp fragment of pyr-4, and a 1210 bp fragment of PalcA–Afsrb1, respectively, in the transformant. For Southern blotting, genomic DNA was digested with BamHI, separated by electrophoresis, and transferred to a nylon membrane (Zeta-probe+; Bio-Rad). A fragment of 876 bp, amplified from Afsrb1 (+197 to +1072) with primers 16 and 17 (Table S1), was used as a probe. Labelling and visualization were performed using the DIG DNA labelling and detection kit (Roche Applied Science), according to the manufacturer's instructions.
Phenotypic analysis of the conditional inactivation mutant.
For the antifungal reagent sensitivity test, A. fumigatus strains were grown on solid repressive medium (RM) (MM containing 1 % glucose and 0.05 M threonine) containing 200 µg Calcofluor white ml–1, 20 µg hygromycin B ml–1, 150 µg Congo red ml–1, 40 µg G418 ml–1 or 0.01 % SDS. After incubation at 37 °C for 2–3 days, the plates were taken out and photographed.
For growth characteristics, a 10 µl slurry of spores (1x108 ml–1) was spotted onto solid RM. The radius of each colony was measured at time intervals, and plotted. The experiment was repeated three times.
Chemical analysis of the cell wall.
Conidia were inoculated into 100 ml liquid RM to a concentration of 1x106 ml–1, and the suspension was shaken (250 r.p.m.) at 37 °C for 36 h. The mycelia were harvested as described above, and lyophilized. Three aliquots of 10 mg dry mycelium were used as independent samples for analysis. Each sample was boiled for 5 min in 2 ml 50 mM Tris/HCl buffer containing 2 % SDS, 100 mM Na-EDTA, 40 mM β-mercaptoethanol and 1 mM PMSF (Elorza et al., 1985; Hearn & Sietsma, 1994; Schoffelmeer et al., 1999) to remove unbound cell-wall proteins and water-soluble sugar. After treatment with 3 % NaOH at 75 °C for 1 h, proteins were released, and quantified by using the Lowry protein assay (Lowry et al., 1951). Glucan and chitin were digested in 96 % formic acid at 100 °C for 4 h. Formic acid was evaporated by lyophilization, and the residues were dissolved in 10 ml distilled water. Glucan and chitin were estimated by determining the amount of glucose and N-acetylglucosamine released after digestion, respectively. Glucose was measured by using the phenol sulphuric acid method (Dubois et al., 1956). N-Acetylglucosamine was measured by using the method described by Lee et al. (2005). The experiment was repeated twice.
Microscopic analysis.
A 10 ml volume of liquid RM was inoculated with 107 freshly harvested conidia, poured into a Petri dish containing glass coverslips, and incubated at 37 °C for the time indicated in each experiment. At the specified times, coverslips with adhering germlings were removed, and spore germination was observed and counted by using a differential interference contrast (DIC) microscope.
Propidium iodide (PI; Molecular Probes) staining was carried out by following the manufacturer's instruction. A 100 µl volume containing 1x109 ml–1 conidia was inoculated into 100 ml liquid RM, and incubated at 37 °C with shaking (250 r.p.m.) for 25 h until the culture reached mid-exponential phase. The mycelia were collected, washed with PBS, and resuspended in 1 ml PBS. A 10 µl volume of 50 µg PI ml–1 and 1 µl 1 % NP-40 were added to 10 µl mycelium suspension, followed by incubation at room temperature for 5 min. After centrifugation, mycelia were washed three times with PBS, and viewed with a fluorescence microscope.
Staining of the nuclei, septa and cell walls was performed as follows. Conidia were incubated as described above. The coverslips with adhering germlings were removed, and fixed in the fixative solution (8 % formaldehyde, 50 mM phosphate buffer, pH 7.0, and 0.2 % Triton X-100) for 30 min. Coverslips were then washed with water, incubated for 5 min with 10 µg fluorescent brightener 28 ml–1 (Sigma) and 1 mg 4',6-diamidino-2-phenylindole ml–1 (Sigma) plus 0.1 % NP-40. After washing with water, germlings were photographed using a microscope.
Conidia or mycelia were fixed in 2.5 % glutaraldehyde in 0.1 M phosphate buffer, pH 7.0, at room temperature for 4 h or at 4 °C overnight. After fixation, cells were washed three times in 0.1 M phosphate, post-fixed in 1 % osmium tetroxide and 0.1 M phosphate for 2–4 h, placed in increasing concentrations of methanol (30, 50, 70, 85, 95 and 100 %), and post-fixed in 2 % uranyl acetate/30 % methanol. Cells were rinsed, dehydrated, and embedded in Epon 812 for the floating sheet method. Sections were examined with an H-600 electron microscope (Hitachi).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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From the expression of the Afsrb1 gene in pET-32a/BL21(DE3), a 58 kDa fusion protein was expressed, and purified to homogeneity (Fig. 1). Enzyme activity was determined via a colorimetric assay coupled with inorganic pyrophosphatase, as described in Methods. Compared with the 0.03±0.02 A630 (mean±SD) value of the Trx control, Trx-AfSrb1 had a corresponding value of 0.55±0.02. The recombinant enzyme showed maximal activity toward mannose 1-phosphate and GTP at 35 °C and pH 8.5, and required 7.5 mM Mg2+. It also exhibited 20 % activity towards glucose 1-phosphate, and 40 % activity towards UTP, as compared with mannose 1-phosphate and GTP, respectively. When ATP, CTP or ITP was used as the substrate, only 30 % activity was detected, as compared with GTP. These results demonstrate that the Afsrb1 gene encodes a GMPP in A. fumigatus.
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; Romero et al., 2003). A fragment in which the pyr-4 gene and PalcA were sandwiched with two flanking sequences of the Psrb1 promoter was amplified via recombinant PCR (Zarrin et al., 2005), and transformed into strain CEA17. One strain, namely YJ-gmpp, was obtained. PCR analysis showed that a 1217 bp fragment of pyr-4 and a 1210 bp fragment of PalcA–Afsrb1 could be amplified from the genomic DNA of strain YJ-gmpp, while no such fragments were amplified from the wild-type strain YJ-407 (Fig. 2b, c). Southern blotting analysis of the BamHI-digested genomic DNA of strain YJ-gmpp confirmed that wild-type 4082 bp BamHI fragment was converted into a 2597 bp BamHI fragment (Fig. 2d). These results demonstrated that native Psrb1 was replaced by the pyr-4 gene and PalcA in strain YJ-gmpp.
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). When an osmotic stabilizer (1 M sorbitol or 0.6 M KCl) was added to the MM medium containing 3 % glucose, the growth of strain YJ-gmpp was compensated to some extent (Fig. 3b). These observations indicate that the Afsrb1 gene is required for cell wall integrity, and that it is an essential gene in A. fumigatus.
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). Under this repressive condition, the expression of the Afsrb1 gene was reduced to a minimal level that could not be detected by Northern blot (Fig. 4b). Thus, we cultivated strain YJ-gmpp in RM at 37 °C for phenotypic analyses.
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, the growth rate of strain YJ-gmpp was approximately 30 % of that of the wild-type. In liquid RM, hyphae of strain YJ-gmpp were swollen and hyperbranched, whereas the wild-type exhibited long and dispersed filaments (Fig. 5b). It was apparent that depletion of Afsrb1 expression induced impaired polar maintenance in A. fumigatus.
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); this suggested a significant increase in cell lysis, and is an observation similar to that seen in yeast srb1 mutants (Warit et al., 2000). However, in contrast to the yeast mutants (Warit et al., 2000), YJ-gmpp formed septa normally, though its basal cells became shorter (Fig. 6d, e). Moreover, despite a small portion of the YJ-gmpp conidia being arrested at the stage of isotropic growth or early germination (Fig. 6d), most conidial cells retained the ability to establish polarized growth (Fig. 6e). Examination of the ultrastructure of the hyphal cell wall revealed that strain YJ-gmpp had a thinner cell wall under repressive conditions (Fig. 6f).
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). To test whether there is a similar phenotype associated with the conditional depletion of the Afsrb1, we inoculated strain YJ-gmpp and the wild-type onto RM containing 150 µg Congo Red ml–1 or 200 µg Calcofluor white ml–1. As expected, strain YJ-gmpp was hypersensitive to these two reagents (Fig. 7), suggesting a defect in the cell wall. In addition, depletion of Afsrb1 also led to hypersensitivity to 40 µg G418 ml–1, 20 µg hygromycin B ml–1, and 0.01 % SDS (Fig. 7).
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, the alkali-soluble -glucan and alkali-insoluble β-glucan contents remained unchanged in strain YJ-gmpp depleted of Afsrb1, while the alkali-soluble protein and alkali-insoluble chitin contents were 1.3- and 2.0-fold higher, respectively, than those of the wild-type. These results suggested that repression of Afsrb1 gene expression in A. fumigatus induced an increased content of chitin and protein in the cell wall.
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, the conidia of strain YJ-gmpp germinated earlier and more rapidly than those of the wild-type. The earliest emergence of the first germ tube occurred after incubation for 7 h; this is about 1 h earlier than the wild-type. Statistical analysis revealed that the emergence of the first germ tube in strain YJ-gmpp occurred at rates of 29, 42, 60 and 65 % after incubation at 37 °C for 7, 8, 9 and 10 h, respectively. Meanwhile, the corresponding rates for the wild-type strain were 0, 20, 52 and 65 % at 7, 8, 9 and 10 h, respectively. About 6–14 % of strain YJ-gmpp conidia formed the second germ tube after 8–9 h, while only 5 % of the wild-type cells formed a second germ tube after 10 h. These results demonstrated that depletion of the Afsrb1 gene led to an earlier and more rapid germination in A. fumigatus.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Bruneau et al., 2001; De Groot et al., 2005; Upadhyay & Shaw, 2006). In S. cerevisiae and C. albicans, GMPP is indispensable, and depletion leads to pleiotropic phenotypes, including lysis, failure of cell separation, impaired budding, clumping and flocculation, as well as defects in the cell wall, and impaired hyphal switching ability (Warit et al., 2000). In contrast to yeasts, little is known about the physiological consequences of the action of GMPP in filamentous fungi.
In this study, the Afsrb1 gene was identified to encode the GMPP in A. fumigatus. To evaluate its physiological consequences in A. fumigatus, a conditional inactivation mutant, strain YJ-gmpp, was constructed by replacing the Afsrb1 promoter with an inducible promoter, PalcA. Three promoters have been successfully used in A. fumigatus, including the A. nidulans alcA promoter, the E. coli tetracycline-regulated promoter, and the A. fumigatus NiiA nitrogen-regulated promoter (Romero et al., 2003; Hu et al., 2007). The alcA promoter is induced, through the positive transcriptional regulator alcR, by various substrates such as ethanol or threonine, and repressed by glucose in the presence of the negative regulator CreA (Panozzo et al., 1998). In strain YJ-gmpp, the Afsrb1 gene was expressed under the control of the promoter of alcA. The presence of 3 % glucose in the medium effectively blocked transcription of the PalcA–Afsrb1 expression cassette. Glucose-mediated repression of the Afsrb1 gene caused a lethality of strain YJ-gmpp, suggesting that, as documented in S. cerevisiae and C. albicans, the Afsrb1 gene is essential for viability of A. fumigatus.
Under repressive conditions, strain YJ-gmpp displayed some phenotypes similar to the yeast mutants, such as increased cell lysis and a defective cell wall (Warit et al., 2000). On the other hand, in contrast to the yeast mutants, strain YJ-gmpp retained the ability to direct polarity establishment and cell separation, despite the fact that its mycelia were shorter, swollen and hyperbranched. Clearly, these phenotypes are different from those observed in the yeast mutants.
Recently, we have shown that the GPI anchor is not required for the viability of A. fumigatus. Blocking of GPI-anchor synthesis causes increased death or cell lysis (Li et al., 2007). In the present study, a similar, but more severe, sensitivity to PI staining was associated with the repression of the Afsrb1 gene. This significant increase in death might be attributed to the depletion of both GPI anchoring and protein glycosylation.
In yeast, a defect in the cell wall requires the cells to induce the cell-wall integrity pathway to survive, and the compensatory mechanism featured with increased chitin content is triggered (Carotti et al., 2002). In the present study, we observed an increased content of chitin in the cell wall of strain YJ-gmpp depleted of Afsrb1. Although the mechanism by which the increased content of chitin is triggered remains unclear, it is likely that A. fumigatus compensates for its cell-wall defect by synthesizing more chitin. In addition, an increased content of protein in the cell wall was also observed, and we hypothesize that this is also a compensatory mechanism. There are two lines of evidence to indirectly support our hypothesis: (i) the hypersensitivity of strain YJ-gmpp to G418 and hygromycin B, which are known to inhibit protein synthesis, may suggest a compensated role of protein in the cell wall; (ii) the hypersensitivity of strain YJ-gmpp to SDS may imply that the increased numbers of proteins are loosely attached to the cell wall, and this may be due to lack of GDP-mannose under repressive conditions.
Germination is characterized by a series of ordered morphological events, including the switch from isotropic to polar growth, the emergence of second germ tube from the conidium, and septation. In A. fumigatus, depletion of Afsrb1 resulted in an earlier emergence of the first and second germ tubes. A similar phenotype has also been observed in A. fumigatus mutants with defective ECM33 or afpig-a (Romano et al., 2006; Li et al., 2007); both mutants are defective in the cell wall. Since ECM33 is a GPI protein, it is reasonable to conclude that the rapid germination induced by repression of the Afsrb1 might be due to a decrease or loss of some GPI proteins in the cell wall of A. fumigatus.
Some A. fumigatus mutants with reduced conidiation have been identified previously. One example is the A. fumigatus 
Afpmt1 mutant, which exhibits a severe reduction of conidiation, especially at high temperatures, and this suggests that O-mannosylation is required for the conidiation of A. fumigatus (Zhou et al., 2007). We also found a reduction in the amount of conidiation in strain YJ-gmpp depleted of Afsrb1. Thus, we postulate that depletion of O-mannosylation in A. fumigatus might be one of the causes for the reduced conidiation, since GDP-mannose is the precursor of O-glycan. Interestingly, unlike the case of the Afpmt1 mutant, strain YJ-gmpp was able to form normal conidia under repressive conditions, suggesting that A. fumigatus may possess a mechanism to ensure its survival in conditions of GDP-mannose starvation, by which fewer, but normal, conidia are formed.
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ACKNOWLEDGEMENTS |
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Edited by: N. L. Glass
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Received 6 April 2008;
revised 3 June 2008;
accepted 5 June 2008.
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