Expression of a chromosomally integrated, single-copy GFP gene in Candida albicans, and its use as a reporter of gene regulation

Abstract

Genetically engineered versions of the GFP gene, which encodes the green fluorescent protein of Aequorea victoria, were placed under the control of the constitutively active Candida albicans ACT1 promoter and integrated in single copy into the genome of this pathogenic yeast. Integrative transformants in which one of the two ACT1 alleles had been replaced by a GFP gene exhibited a homogeneous, constitutive fluorescent phenotype. Cells expressing GFP with the wild-type chromophore exhibited very weak fluorescence compared to those GFP proteins with the S65T or S65A, V68L, S72A (GFPmut2) chromophore mutations. Substitution of the CTG codon, which specifies serine instead of leucine in C. albicans, by TTG was absolutely necessary for GFP expression. Although GFP mRNA levels in cells containing a GFP gene with the CTG codon were comparable to those of transformants containing GFP with the TTG substitution, only the latter produced GFP protein, as detected by Western blotting, suggesting that the frequent failure to express heterologous genes in C. albicans is principally due to the noncanonical codon usage. Transformants expressing the modified GFP gene from the promoter of the SAP2 gene, which encodes one of the secreted acid proteinases of C. albicans, showed fluorescence only under conditions which promote proteinase expression, thereby demonstrating the utility of stable, chromosomally integrated GFP reporter genes for the study of gene activation in C. albicans.

Sensing of mammalian IL-17A regulates fungal adaptation and virulence

“…Study approval was provided by the local ethics committee (Umbria Regional Hospital Ethics Committee, CEAS Umbria), and informed written consent was obtained from all participants in accordance with the Declaration of Helsinki. The CAG31A C. albicans mutant strain expressing the GFP gene was obtained as described 42 . When observed by fluorescence microscopy, both yeasts and hyphae of the CAG31A strain were intensively fluorescent 43 .…”

Activation of the Multiple Drug Resistance Gene MDR1 in Fluconazole-Resistant, Clinical Candida albicans Strains Is Caused by Mutations in a trans -Regulatory Factor

“…Plasmid pGFP41 has been described previously (18). It contains a GFP gene, genetically modified for expression in C. albicans under control of the SAP2 promoter, and the URA3 selection marker in the vector pBluescript.…”

“…To avoid recombination between the MDR1 promoter from strain CAI4 and MDR1 upstream sequences in the host strains, integration was targeted to an ectopic site in the genome. The CDR4 locus was chosen for integration, as this region had been characterized previously by our group and it had been shown that inactivation of one of the CDR4 alleles did not result in a detectable phenotype (18). In addition, CDR4 expression levels did not differ between the fluconazolesusceptible and resistant isolates (9).…”

Depletion of a Polo-like Kinase inCandida albicansActivates Cyclase-dependent Hyphal-like Growth

“…Primers CB51F and CB51R were designed to bind immediately up and downstream of the STOP codon, respectively, and to reverse-amplify the plasmid sequence, into which a XhoI/SpeI PCR fragment (produced with primers CB44F and CB44R) of GFP (Morschhauser et al, 1998) was cloned, creating plasmid pCB105. pCB105 was digested with BstEII for directed integration at the endogenous TUB1 locus, creating full-length 3Ј-tagged TUB1-GFP, under control of its own promotor, and a 5Ј-truncated copy.…”