The Aspergillus nidulans alcA promoter drives tightly regulated conditional gene expression in Aspergillus fumigatus permitting validation of essential genes in this human pathogen

Romero, Beatriz; Turner, Geoffrey; Olivas, Israel; Laborda, F.; Lucas, J. Ramón De

doi:10.1016/s1087-1845(03)00090-2

Cited by 59 publications

(84 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…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, P alcA . 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;. 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).…”

Section: Discussionmentioning

confidence: 99%

“…A conditional inactivation mutant was constructed by expressing the Afsrb1 gene under the control of P alcA , which is a strictly regulated promoter that can be triggered by ethanol or threonine, and is repressed completely by 3 % glucose (Waring et al, 1989;Romero et al, 2003). A fragment in which the pyr-4 gene and P alcA were sandwiched with two flanking sequences of the P srb1 promoter was amplified via recombinant PCR (Zarrin et al, 2005), and transformed into strain CEA17.…”

Section: Construction Of the Conditional Inactivation Mutantmentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2008

View full text Add to dashboard Cite

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 ...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Construction Of the Conditional Inactivation Mutantmentioning

confidence: 99%

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

et al. 2008

View full text Add to dashboard Cite

show abstract

“…Activity with 1,2-dichloro-4-nitrobenzene (DCNB) was performed at 345 nm in the same way as for CDNB, with some exceptions; 100 mM phosphate buVer (pH 7.5), 100 mM DCNB in 100% ethanol, and 50 mM GSH in phosphate Table 1 Nucleotide sequence of oligonucleotide primers used to amplify Afugst genes A-C from A. fumigatus genomic DNA and cDNA, respectively Nucleotide sequence of control calmodulin primers (Romero et al, 2003) are also given. Oligonucleotide primers were designed based on sequence data obtained from the A. fumigatus genome sequencing eVort (http://www.tigr.org).…”

Section: Gst Activity Assaysmentioning

confidence: 99%

“…Subsequent PCR of GST cDNA was performed as described above. Control PCRs were performed with primers LCALM and RCALM (Table 1) which amplify 348 and 617 bp regions from A. fumigatus cDNA and genomic DNA, respectively (Romero et al, 2003). Densitiometric quantiWcation of PCR products was performed using Genetools software (Syngene).…”

Section: Induction Of Gst Expression In a Fumigatus And Analysis By mentioning

confidence: 99%

Identification, cloning, and functional expression of three glutathione transferase genes from Aspergillus fumigatus

Burns¹,

Geraghty²,

Neville³

et al. 2005

Fungal Genetics and Biology

View full text Add to dashboard Cite

Analysis of the genome of the human pathogen, Aspergillus fumigatus, revealed the presence of several putative glutathione transferase (GST) open reading frames. Three A. fumigatus GST genes, termed gstA, B, and C, were cloned and recombinant proteins expressed in Escherichia coli. Functional analysis of recombinant gstA-C conWrms that the enzymes exhibit GST activity and glutathione peroxidase activity. RT-PCR conWrmed low basal expression of gstA and gstC which was markedly up-regulated (at least 4£-10£) in the presence of either H 2 O 2 or 1-chloro-2,4-dinitrobenzene (CDNB). GstB expression was only observed in the presence of CDNB. These results demonstrate for the Wrst time the existence of three functional GSTs in A. fumigatus and strongly suggest a role for these enzymes in the response of the organism to both oxidative stress and xenobiotic presence. 

show abstract

“…Os picos que aparecem no espectro obtido em modo positivo, com m/z 396, 374 e 356, correspondem, respectivamente, à molécula de equisetina cationizada com sódio, à molécula protonada, e à molécula protonada após sofrer uma perda neutra de água. (SHOJI et al, 2005;ROMERO et al, 2003;NIKOLAEV et al, 2002;TADA et al, 1991 Mais decisivas que mutações, que possuem relativamente baixa taxa de ocorrência, falhas nas construções genéticas poderiam levar a uma enzima definitivamente não-funcional. Planejar as construções, e determinar os pontos de fusão, é uma parte bastante desafiadora dentro do processo de hibridização.…”

Section: Construções No Vetor Deg2unclassified

Cultura mista, manipulação química e genética de micro-organismos: estratégias para a diversificação do metabolismo secundário

Chagas¹

View full text Add to dashboard Cite

The Aspergillus nidulans alcA promoter drives tightly regulated conditional gene expression in Aspergillus fumigatus permitting validation of essential genes in this human pathogen

Cited by 59 publications

References 46 publications

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

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

Identification, cloning, and functional expression of three glutathione transferase genes from Aspergillus fumigatus

Cultura mista, manipulação química e genética de micro-organismos: estratégias para a diversificação do metabolismo secundário

Contact Info

Product

Resources

About