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 PalcA–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.
BradfordM. M.1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
CarottiC.,
FerrarioL.,
RonceroC.,
ValdiviesoM. H.,
DuranA.,
PopoloL.2002; Maintenance of cell integrity in the gas1 mutant of Saccharomyces cerevisiae requires the Chs3p-targeting and activation pathway and involves an unusual Chs3p localization. Yeast 19:1113–1124
DuboisM.,
GillesK. A.,
HamiltonJ. K.,
RebersP. A.,
SmithF.1956; Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356
ElorzaM. V.,
MurguiA.,
SentandreuR.1985; Dimorphism in Candida albicans : contribution of mannoproteins to the architecture of yeast and mycelial cell walls. J Gen Microbiol 131:2209–2216
GaramiA.,
IlgT.2001; Disruption of mannose activation in Leishmania mexicana : GDP-mannose pyrophosphorylase is required for virulence, but not for viability. EMBO J 20:3657–3666
GriffinA. M.,
PoelwijkE. S.,
MorrisV. J.,
GassonM. J.1997; Cloning of the aceF gene encoding the phosphomannose isomerase and GDP-mannose pyrophosphorylase activities involved in acetan biosynthesis in Acetobacter xylinum . FEMS Microbiol Lett 154:389–396
HashimotoH.,
SakakibaraA.,
YamasakiM.,
YodaK.1997; Saccharomyces cerevisiae VIG9 encodes GDP-mannose pyrophosphorylase, which is essential for protein glycosylation. J Biol Chem 272:16308–16314
LangfelderK.,
GattungS.,
BrakhageA. A.2002; A novel method used to delete a new Aspergillus fumigatus ABC transporter-encoding gene. Curr Genet 41:268–274
LatgéJ.-P.,
MouynaI.,
TekaiaF.,
BeauvaisA.,
DebeaupuisJ. P.,
NiermanW.2005; Specific molecular features in the organization and biosynthesis of the cell wall of Aspergillus fumigatus . Med Mycol 43:S15–S22
LeeJ. I.,
YuY. M.,
RhoY. M.,
ParkB. C.,
ChoiJ. H.,
ParkH. M.,
MaengP. J.2005; Differential expression of the chsE gene encoding a chitin synthase of Aspergillus nidulans in response to developmental status and growth conditions. FEMS Microbiol Lett 249:121–129
MouynaI.,
FontaineT.,
VaiM.,
MonodM.,
FonziW. A.,
DiaquinM.,
PopoloL.,
HartlandR. P.,
LatgéJ.-P.2000; Glycosylphosphatidylinositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall. J Biol Chem 275:14882–14889
OhtaA.,
ChibanaH.,
ArisawaM.,
SudohM.2000; The VIG9 gene products from the human pathogenic fungi Candida albicans and Candida glabrata encode GDP-mannose pyrophosphorylase. Biochim Biophys Acta1475265–272
PanozzoC.,
CornillotE.,
FelenbokB.1998; The CreA repressor is the sole DNA-binding protein responsible for carbon catabolite repression of the alcA gene in Aspergillus nidulans via its binding to a couple of specific sites. J Biol Chem 273:6367–6372
RomeroB.,
TurnerG.,
OlivasI.,
LabordaF.,
De LucasJ. R.2003; The Aspergillus nidulans alcA promoter drives tightly regulated conditional gene expression in Aspergillus fumigatus permitting validation of essential genes in this human pathogen. Fungal Genet Biol 40:103–114
SteinbachW. J.,
StevensD. A.,
DenningD. W.2003; Combination and sequential antifungal therapy for invasive aspergillosis: review of published in vitro and in vivo interactions and 6281 clinical cases from 1966 to 2001. Clin Infect Dis 37:S188–S224
UpadhyayS.,
ShawB. D.2006; A phosphoglucose isomerase mutant in Aspergillus nidulans is defective in hyphal polarity and conidiation. Fungal Genet Biol 43:739–751
WaringR. B.,
MayG. S.,
MorrisN. R.1989; Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin-coding genes. Gene 79:119–130
WaritS.,
WalmsleyR. M.,
StatevaL. I.1998; Cloning and sequencing of the Candida albicans homologue of SRB1/PSA1/VIG9 , the essential gene encoding GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae . Microbiology 144:2417–2426
WaritS.,
ZhangN.,
ShortA.,
WalmsleyR. M.,
OliverS. G.,
StatevaL. I.2000; Glycosylation deficiency phenotypes resulting from depletion of GDP-mannose pyrophosphorylase in two yeast species. Mol Microbiol 36:1156–1166
WeidnerG.,
d'EnfertC.,
KochA.,
MolP. C.,
BrakhageA. A.1998; Development of a homologous transformation system for the human pathogenic fungus Aspergillus fumigatus based on the pyrG gene encoding orotidine 5′-monophosphate decarboxylase. Curr Genet 33:378–385
XiaG.,
JinC.,
ZhouJ.,
YangS.,
ZhangS.,
JinC.2001; A novel chitinase having a unique mode of action from Aspergillus fumigatus YJ-407. Eur J Biochem 268:4079–4085
YodaK.,
KawadaT.,
KaibaraC.,
FujieA.,
AbeM.,
HashimotoH.,
ShimizuJ.,
TomishigeN.,
NodaY.,
YamasakiM.2000; Defect in cell wall integrity of the yeast Saccharomyces cerevisiae caused by a mutation of the GDP-mannose pyrophosphorylase gene VIG9. Biosci Biotechnol Biochem 64:1937–1941
ZhouH.,
HuH.,
ZhangL.,
LiR.,
OuyangH.,
MingJ.,
JinC.2007; O -Mannosyltransferase 1 in Aspergillus fumigatus (AfPmt1p) is crucial for cell wall integrity and conidium morphology, especially at an elevated temperature. Eukaryot Cell 6:2260–2268