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.

FIG. 1.

URA3 mRNA abundance when URA3 is positioned at its native locus (CAF2-1) (A), the GCN4 locus (GTC43) (B), or the RPS10 locus after reintegration using the CIp10 vector (GTC45) (C). cDNA was synthesized from mRNA isolated from late-exponential-phase cells. URA3 mRNA abundance was measured relative to an internal control gene, EFB1, using multiplex RT-PCR.

FIG. 2.

Survival curves for female DBA/2 mice challenged iv. with C. albicans CAF2-1 (solid circles) and the following mutants derived from this strain: NGY152 (solid squares), GTC41 (GCN4/gcn4Δ; open circles), GTC43 (gcn4Δ; open squares), GTC45 (gcn4Δ/CIp10; solid triangles) and GTC49 (gcn4Δ/CIp10-GCN4; open triangles). Mice were in groups of 6 animals, except for CAF2-1, with 10 animals challenged.

FIG. 3.

Scatter plots of OMP decarboxylase specific activity in wild-type and mutant C. albicans strains versus three parameters of virulence in a mouse i.v. C. albicans challenge model: mean survival time (a), mean kidney burden (b), and mean brain burden (c). Each point represents the result with a single strain tested for BALB/c mice (open circles) or DBA/2 mice (solid circles) in groups of 6 animals, except for CAF2-1, NGY152, SC5314, BH1-1-1, and VC3.9, where group sizes of 12 to 30 animals were pooled from two to five replicate experiments. The strains tested were all derived originally from SC5314, whose OMP decarboxylase activity was taken as 100%. Details of the strains are shown in Table 1. Additional results for five natural isolates of C. albicans that differed in inherent virulence are individually identified on the graphs as solid squares; their virulence was tested in BALB/c mice. Data for brain burdens were not available for ATCC 44858.