Ectopic Expression of URA3 Can Influence the Virulence Phenotypes and Proteome of Candida albicans but Can Be Overcome by Targeted Reintegration of URA3 at the RPS10 Locus

Abstract

Uridine auxotrophy, based on disruption of both URA3 alleles in diploid Candida albicans strain SC5314, has been widely used to select gene deletion mutants created in this fungus by "Ura-blasting" and PCR-mediated disruption. We compared wild-type URA3 expression with levels in mutant strains where URA3 was positioned either within deleted genes or at the highly expressed RPS10 locus. URA3 expression levels differed significantly and correlated with the specific activity of Ura3p, orotidine 5-monophosphate decarboxylase. Reduced URA3 expression following integration at the GCN4 locus was associated with an attenuation of virulence. Furthermore, a comparison of the SC5314 (URA3) and CAI-4 (ura3) proteomes revealed that inactivation of URA3 caused significant changes in the levels of 14 other proteins. The protein levels of all except one were partially or fully restored by the reintegration of a single copy of URA3 at the RPS10 locus. Transcript levels of genes expressed ectopically at this locus in reconstituted heterozygous mutants also matched the levels found when the genes were expressed at their native loci. Therefore, phenotypic changes in C. albicans can be associated with the selectable marker rather than the target gene. Reintegration of URA3 at an appropriate expression locus such as RPS10 can offset most problems related to the phenotypic changes associated with gene knockout methodologies.Genes encoding enzymes in the uridine biosynthetic pathway are used as positive selectable markers in gene disruption strategies for a wide range of eukaryotic organisms. For the major human pathogenic fungus Candida albicans, this approach, based on the URA3 gene, was the only means available for gene disruption for more than 10 years. C. albicans is diploid and has no known natural sexual cycle that leads to a haploid form. Therefore, molecular genetic evidence associating phenotypic changes with individual gene functions requires the successive disruption of two copies of the gene of interest. "Ura-blasting," used for gene disruption in C. albicans (28), is based on a similar approach in Saccharomyces cerevisiae (1) and makes use of a C. albicans ura3 auxotroph. Transformants are selected on the basis of uridine prototrophy. The marker is then recycled by use of 5-fluoroorotic acid to select for Ura Ϫ segregants that have lost URA3 through a reciprocal crossover between identical flanking DNA repeats. The "Ura-blaster" can then be used to disrupt the second allele in these ura3 segregants. PCR-based gene disruption protocols also exploit the URA3 marker but often do not recycle it (90).One area of investigation that has made extensive use of specific gene disruption based on the URA3 selection strategy is the study of virulence in C. albicans. At least 50 genes have been implicated as putative virulence factors in this fungus based on results of mouse intravenous challenge experiments in which the homozygous C. albicans null mutants of interest were shown to be less lethal than wild-type controls (61). In most o...

Flanking direct repeats of hisG alter URA3 marker expression at the HWP1 locus of Candida albicans

“…The phenotypic deficiencies correlated with reduced OMPase activity, and the filamentation defects were bypassed by uridine supplementation (Cheng et al, 2003). Similar results were obtained by Brand et al (2004), who further showed that integration of URA3 at the RPS10 locus allowed for normal expression.…”

“…Similarly Brand et al (2004) demonstrated reduced URA3 expression in a number of mutants and restoration of normal expression with integration of the marker at the RPS10 locus. Again, the poorly expressed marker was within the context of flanked hisG repeats, which were not included at the RPS10 locus (Brand et al, 2004).…”

Superoxide dismutases and glutaredoxins have a distinct role in the response of Candida albicans to oxidative stress generated by the chemical compounds menadione and diamide

“…1A). The URA3 gene was reintegrated at the neutral RPS1 locus to avoid ectopic URA3 expression problems (Brand et al 2004), creating the mutant GCY 214. The GRX2 gene and URA3 were integrated at the RPS1 locus of the Δ sod1/ grx2 null mutant to generate the Δ sod1/grx2 + GRX2 reintegrant (GCY 215) (Fig.…”

“…Gene disruption was confirmed by polymerase chain reaction (PCR). To avoid potential problems associated with ectopic expression of URA3 (Brand et al 2004), the Ura-Δsod1/grx2 mutant was transformed with StuI-digested CIp10 plasmid (Murad et al 2000), thus ensuring URA3 expression at the neutral RPS1 locus (orf19.3002).…”

A conserved G protein (Drg1p) plays a role in regulation of invasive filamentation in Candida albicans

“…5A); therefore, the mutant strain exhibited delayed lethality. The location of the URA3 gene in XC414 (DRG1 + complemented strain) differed from its location in XC101 (WT), and may have influenced the virulence of the complemented strain, as observed previously (Brand et al, 2004;Lay et al, 1998).…”