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.
INTRODUCTIONAspergillus fumigatus is the most common opportunistic fungal pathogen of humans; it causes fatal invasive aspergillosis in immunocompromised patients (Latgé, 1999(Latgé, , 2001Krappmann, 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 Latgé et al., 2005;Upadhyay & Shaw, 2006). Some cellsurface 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 Bruneau et al., 2001;Chabane et al., 2006;Romano et al., 2006;De Groot ...
