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Presence of a mannoprotein, MnpAp, in the hyphal cell wall of Aspergillus nidulans

     Division of Biological Sciences, Chonbuk National University, Chonju, Chonbuk 561-756, Republic of Korea

Sung Soo Whang ¹

     Division of Science Education, Chonbuk National University, Chonju, Chonbuk 561-756, Republic of Korea

	ABSTRACT

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The presence of a mannoprotein, MnpAp, in the hyphal cell wall of Aspergillus nidulans was examined by immunogold electron microscopy using a mnpA-null mutant as a negative control. The hyphal cell wall of wild type consisted of two layers—an electron-dense smooth outer layer and an electron-translucent inner layer—while the hyphal cell wall of the mnpA-null mutant had an electron-dense irregular outer layer together with the electron-translucent inner layer. In wild type, MnpAp was present throughout the electron-translucent layer of the hyphal cell wall but was absent from the conidial cell wall. In the mnpA-null mutant, MnpAp was absent from the cell walls of both cell types. These results indicate that MnpAp is present in the hyphal cell wall and that it influences cell wall surface structure.

Key words: Aspergillus nidulans, cell wall, immunoelectron microscopy, mannoprotein, mnpA

	INTRODUCTION

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The cell wall of the filamentous fungus Aspergillus nidulans plays an important role in determining and maintaining various cellular structures, including vegetative and aerial hyphae, conidiphores, conidia, cleistothecia and asci. It also functions in protection, recognition of external stimuli, transport and storage of carbohydrates (Bull 1970, Aronson 1981, Claveri-Martin et al 1988). The fungus first develops tubular undifferentiated hyphae, which in turn give rise to various types of cells (Martinelli and Kinghorn 1994, Adams et al 1988). Hyphal growth is polar due to the synthesis of cell wall components at apical and/or subapical regions. Analyses of cell wall components of Aspergillus have determined that walls are composed of ß-1,3-glucan, ß-1,6-glucan, linear ß-1,3/1,4-glucan, chitin and mannoproteins (Bull 1970, Zonneveld 1971, Fontaine et al 2000) but they have a simple structure compared with other organisms (Fontaine et al 2000). This organism appears to be well suited for studing cell-wall morphogenesis and components using genetic and ultrastructural approaches.

Many genes have been identified as being involved in the synthesis of the cell walls of A. nidulans, including orlA, tsE, orlC, orlD (Borgia 1992, Borgia and Dodge 1992), chsA, chsB, chsC, chsD (Bowen et al 1992), fksA (Nam et al 1998), csmA (Fujiwara et al 1997) and bimG11 (Borgia 1992, Doonan 1994). Four of these, orlA (Borgia and Dordge 1992), chsA, chsC (Specht et al 1996) and fksA (Nam et al 1998), related to the synthesis of both carbohydrates and lipids, have been cloned and characterized. However, little is known regarding genes encoding structural proteins in or on the cell walls of A. nidulans (Smith and Payton 1994).

We previously have described mnpA, a single-copy A. nidulans gene that encodes a mannoprotein, MnpAp (Jeong et al 2003). The nucleotide sequences of genomic DNA and cDNA revealed that the gene has an open-reading frame of 261 amino acids without introns. The deduced amino acid sequence showed 60% identity to that of A. fumigatus galactomannoprotein MP1. MnpAp also has homologs in A. flavus, as well as in a more distantly related species Penicillium marneffei (Woo et al 2003). To date, however, mutants for this gene have not been described.

We report here the first characterization of strains mutant for mnpA. Comparisons of wild type and mutant strains confirmed that MnpAp is present in hyphal cell walls. Results further indicate that MnpAp has a role in maintaining cell wall structure.

	MATERIALS AND METHODS

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Strains and media – Aspergillus nidulans strains used in the study were VER7 (pJYargB) (pabaA1; yA2; argB::trpC; trpC801; argB⁺) (Han et al 2001) and DMPV1 (pabaA1; yA2; argB::trpC; trpC801; mnpA::argB) (Jeong et al 2003). Aspergillus nidulans DMPV1 is a presumed null mutant, which possesses a deletion in the mnpA gene and can synthesize only a 28 amino-acid N-terminal portion of MnpAp (Jeong et al 2003). Complex medium (CM), minimal medium (MM) and CM containing 0.6 M potassium chloride (CMK) were prepared as previously described (Pontecorvo et al 1953, Jeong et al 200).

Manipulation of A. nidulans – Conidia were harvested by gentle rotation of plates with 0.08% (v/v) Tween 80. Sexual development was induced according to the method described previously (Jeong et al 2000).

Immnunogold staining and transmission electron microscopy (TEM) – To produce MnpAp protein, the mnpA cDNA was amplified by PCR with a primer pair, 5'-ACC GGA TCC AAG CGC CAG TC-3' (MPUP3, 853872) and 5'-GCG TTG GTG ATT CCG GCA GA-3' (MPL3, 12641283), digested with BamHI, and ligated into pET28a plasmid digested with BamHI and EcoICRI. As a result, the MnpAp protein, lacking 20 amino acids from the N-terminus and 100 amino acids from the C-terminus, was fused in frame with a His-Tag at both ends. Numbers associated with primer sequences correspond with nucleotides in the mnpA sequence deposited in GenBank (accession number AF497720). The His-Tag fusion protein was expressed and purifed with the pET expression system as described by the manufacturer (Novagen, Darmstadt, Germany).

To produce polyclonal antibodies against the recombinant MnpAp, approximately 100 µg of MnpAp was mixed with an equal volume of adjuvant complete Freund's (Difco, Detroit, Michigan) and injected subcutaneously into two female BALB/c mice. Incomplete Freund's adjuvant (Sigma, St. Louis, Missouri) was used in a subsequent injection 2 wk after the first. Four days after the second injection, serum containing polyclonal anti-MnpAp antibodies was obtained from blood pre-incubated at room temperature for 30 min and at 0 C for 30 min.

For ultrastructural analyses, cells were fixed in 2.5% (v/v) glutaraldehyde and 5% (v/v) acrolein in 0.1 M sodium cacodylate buffer (pH 7.3) for 2 h at 0 C, dehydrated in a graded series of ethanol and embedded in LR White (Electron Microscopy Sciences, Washington, Pennsylvania) following the manufacturer's instructions. After polymerization of the resin, thin sections were cut to a thickness of 60 nm with a diamond knife and mounted onto 300 mesh nickel grids for immunostaining. For immunostaining, sections were blocked for 20 min in 0.5% (w/v) bovine serium albumin (BSA) (Sigma) and 0.1% gelatin in PBS. Mouse anti-MnpAp serum (diluted 1:500 in 20 mM Tris buffer, pH 8.2) with 0.1% BSA (TBSA) was added and incubated with cell sections for 90 min. After rinsing with TBSA, specimens were incubated with TBSA containing 1:25-diluted goat antimouse immunoglobulin G conjugated with 10 nm gold particles (Amersham, Pharmacia Biotech Ltd., Buckinghamshire, U.K.). After washing with TBSA, sections were counterstained with uranyl acetate and lead citrate, followed by examination with a JEOL 1200EX TEM operating at 80 kV.

	RESULTS

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Distribution of MnpAp in the cell wall of A. nidulans – The mnpA gene encoding a mannoprotein of A. nidulans recently was isolated and characterized (Jeong et al 2003). To look for MnpAp in the cell wall of A. nidulans, the cell wall ultrastructure of an mnpA-null mutant, DMPV1 was compared with that of wild-type strain VER7 (pJYargB). Fixed sections of hyphal cells were immunogold-stained with mouse anti-MnpA antibodies (Fig. 1). MnpAp conjugated with immunogold particles was found in the hyphal cell wall of wild type (Fig. 1A, C) but was absent from that of the mnpA mutant (Fig. 1B, D). Immunoelectron micrographs further revealed that MnpAp was evenly distributed throughout the electron-translucent layer of cell walls but was not localized in a specific layer (Fig. 1B).

View larger version (184K): [in this window] [in a new window]   FIG. 1. Immunoelectron microscopy of the hyphal cell of A. nidulans stained with mouse anti-MnpAp antibodies in cross sectional view. (A) Wild-type cell wall, showing immunogold particles specifically located at the cell wall. Arrow indicates MnpAp protein. Bar, 100 nm. (B) An mnpA-null mutant showing the absence of immunogold particles in the cell wall. Bar, 100 nm. (C) A wild-type cell at lower magnification of (A), Bar, 200 nm. (D) An mnpA-null mutant at lower magnification of (B), Bar, 200 nm

View larger version (75K): [in this window] [in a new window]   FIG. 2. Transmission electron micrographs of A. nidulans in cross-sectioned hyphal cells. (A) A wild type, showing an electron-dense outer layer of the cell wall. Bar, 200 nm. (B) An mnpA-null mutant, showing an irregular and disrupted electron-dense outer layer and a broad electron translucent inner layer of the cell wall. Bar, 200 nm

	DISCUSSION

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It was hypothesized that MnpAP is located in the cell wall because its deduced amino acid sequence shares common sequence motifs with many cell wall proteins previously reported, including a signal sequence, O-glycosylation sites and a glycosylated phophatidylinositol (GPI)-anchoring motif (Klis 1994, Caro et al 1997, Kapteyn et al 2000, Jeong et al 2003). Immunogold staining with anti-MnpAp antibodies confirmed that the MnpAp protein is present in the hyphal cell wall.

Because the cell wall in yeast has a highly dynamic structure (Klis 1994, Kapteyn et al 1999), it has been hypothesised that the cell wall of A. nidulans also might have a flexible architecture. In this study, the wild-type cell wall was found to possess a bilayered structure consisting of an electron-dense smooth outer layer and an electron-translucent inner layer, in agreement with an earlier report (Sewall et al 1990). Although the overall shape of the cell remained unchanged, hyphal cell walls in the mnpA-null mutant comprised an electron-dense irregular and disrupted outer layer and a broad electron-translucent inner layer. MnpAp was absent from the hyphal cell wall of the mnpA-null mutant but was located throughout the hyphal cell wall of wild type. The results suggest that the MnpAp influences the surface structure. Furthermore, MnpAp was absent from the conidial cell wall of wild type (data not shown), which is consistent with the previous result that the mnpA transcript is absent from conidia but present in hyphae (Jeong et al 2003).

Koch and Rademacher (1980) have mentioned that the distinction between the external protein layer and the internal glucan layer is not clear-cut. Electron microscopic evidence indicates that mannoproteins are interwoven with the underlying glucan layer or, as in Schizosaccharomyces pombe (Walther et al 1988), may even penetrate the entire wall. The distribution pattern of MnpAp in the cell wall of A. nidulans was similar to that of Pir-related protein in Candida albicans, because Pir-related protein incorporated into its cell wall through an alkali-sensitive linkage to ß-1,3-glucan (Mormeneo et al 1994, Kapteyn et al 2000).

	ACKNOWLEDGMENTS

 
The comments of reviewer, Dr. Donald Natvig, Mycologia associate editor, made this article better than it was. We thank Prof. R.S. Hill for reading of manuscript. This work was supported by Korea Research Foundation Grant (KRF-2000-015-DP0373) to S.S.W. through the Basic Science Research Institute at Chonbuk National University in 2000.

	FOOTNOTES

 
¹ Corresponding author. E-mail: whang{at}chonbuk.ac.kr

Accepted for publication June 3, 2003.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Adams TH, Wieser JK, Yu J-H., 1998 Asexual sporulation in Aspergillus nidulans. Microbiol Mol Biol Rev 62:35-54[Abstract/Free Full Text]

Aronson JM., 1981 Cell wall chemistry, ultrastructure and metabolism. In: Cole GT, ed. Biology of conidial fungi. Vol. 2. London: Academic Press. p 459–507

Borgia PT., 1992 The role of the orlA, tsE and bimG genes of Aspergillus nidulans in chitin synthesis. J Bacteriol 174:384-389[Abstract/Free Full Text]

———, Dordge CL., 1992 Characterization of Apergillus nidulans mutants deficient in cell-wall chitin or glucan. J Bacteriol 174:377-383[Abstract/Free Full Text]

Bowen AR, Chen-Wu JL, Momany M, Young R, Szaniszlo PJ, Robbins PW., 1992 Classification of fungal chitin synthesis. Proc Natl Acad Sci USA 89:519-523[Abstract/Free Full Text]

Bull AT., 1970 Chemical composition of wild-type and mutant Aspergillus nidulans cell walls. The nature of polysaccharide and melanin constituents. J Genet Microbiol 63:75-94

Caro LHP, Tettelin H, Vossen JH, Ram AF, van den Ende H, Klis FM., 1997 In silicio indentification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae. Yeast 13:1477-1489[Medline]

Claverie-Martin F, Diaz-Torres MR, Geoghegan MJ., 1988 Chemical composition and ultrastructure of wild-type and white mutant Aspergillus nidulans conidial walls. Curr Microbiol 16:281-287

Doonan JH., 1994 Control of cell growth. In: Aspergillus, 50 years on. The Netherland: Elsevier. p 455–478

Fontaine T, Simenel C, Dubreucq G, Adam O, Delepierre M, Lemoine J, Vorgias CE, Diaquin M, Latge JP., 2000 Molecular organization of the alkali-insoluble fraction of Aspergillus fumigatus cell wall. J Biol Chem 275:27594-27607[Abstract/Free Full Text]

Fujiwara M, Horiuchi H, Ohta A, Takagi M., 1997 Biochem Biophys Res Commun 236:75-78[Medline]

Han K-H, Han K-Y, Yu J-H, Chae K-S, Jahng K-Y, Han D-M., 2001 The nsdD gene encodes a putative GATA type transcription factor necessary for sexual development of Aspergillus nidulans. Mol Microbiol 41:299-309[Medline]

Jeong H-Y, Han D-M, Jahng K-Y, Chae K-S., 2000 The rpl16a gene for ribosomal protein L16A identified from expressed sequence tags is differentially expressed during sexual development of Aspergillus nidulans. Fung Genet Biol 31:69-78

———, Kim H, Han D-M, Jahng K-Y, Chae K-S., 2003 Expression of the mnpA gene that encodes the mannoprotein of Aspergillus nidulans is dependent on fadA and flbA as well as veA. Fung Genet Biol 38:228-236

Kapteyn JC, van den Ende H, Klis FM., 1999 The contribution of cell wall proteins to the organization of the yeast cell wall. Biochim Biophys Acta 1426:373-383[Medline]

———, Hoyer LL, Hecht JE, Müller WH, Andel A, Verkleij AJ, Makarow M, van den Ende H, Klis FM., 2000 The cell wall architecture of Candida albicans wild-type cells and cell wall defective mutants. Mol Microbiol 35:601-611[Medline]

Klis FM., 1994 Review: cell wall assembly in yeast. Yeast 10:851-869[Medline]

Koch Y, Rademacher KH., 1980 Chemical and enzymatic changes in the cell walls of Candida albicans and Saccharomyces cerevisiae by scanning electron microscopy. Can J Microbiol 26:965-970[Medline]

Lee D-W, Kim S, Kim S-J, Han D-M, Jahang K-Y, Chae K-S., 2001 The lsdA gene is necessary for sexual development inhibition by a salt in Aspergillus nidulans. Curr Genet 39:237-243[Medline]

Martinelli SD, Kinghorn JR., 1994 Aspergillus: 50 years on. In: The Netherlands: Elsevier Science B. V

Mormeneo S, Marcilla A, Iranzo M, Sentandreu R., 1994 Structural mannoproteins released by ß-elimination from Candida albicans cell walls. FEMS Microbiol Lett 123:131-136[Medline]

Nam JS, Lee DH, Lee KH, Park HM, Bae KS., 1998 Cloning and phylogenetic analysis of chitin synthase genes from the insect pathogenic fungus, Matarhizium anisopliae var. anisopliae. FEMS Microbiol Lett 159:77-84[Medline]

Pontecorve G, Roper JA, Hemmons LM, MacDonald KD, Bufton AWJ., 1953 The genetics of Aspergillus nidulans. Advan Genet 5:141-238

Sewall TC, Mims CM, Timberlake WE., 1990 Conidium differentiation in Aspergillus nidulans wild type and wet-white (wetA) mutant strains. Dev Biol 138:499-508[Medline]

Smith DJ, Payton MA., 1994 Hyphal tip expression in Aspergillus nidulans requires in manA gene, which encodes phosphomannose isomerase. Mol Cell Biol 14:6030-6038[Abstract/Free Full Text]

Specht CA, Liu Y, Robbins PW, Bulawa CE, Iartchouk N, et al 1996 The chsD and chsE genes of Aspergillus nidulans and their roles in chitin synthesis. Fung Genet Biol Newsl 20:153-167

Walther P, Schweingruber AM, Müller M, Schweingruber ME., 1988 Morphological organization of glycoprotein-containing cell surface structures in yeast. J Ultrastruc Mol Struc Res 101:123-136

Woo PCY, Chong KTK, Leung ASP, Wong SSY, Lau SKP, Yuen KY., 2003 AFLMP1 encodes an antigenic cell wall protein in Aspergillus flavus. J Clin Microbiol 41:845-850[Abstract/Free Full Text]

Zonneveld BJM., 1971 Biochemical analysis of the cell wall of Aspergillus nidulans. Biochim Biophys Acta 249:506-514[Medline]

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