Altered expression of selectable marker URA3 in gene-disrupted Candida albicans strains complicates interpretation of virulence studies

Abstract

The ura-blaster technique for the disruption of Candida albicans genes has been employed in a number of studies to identify possible genes encoding virulence factors of this fungal pathogen. In this study, the URA3-encoded orotidine 5'-monophosphate (OMP) decarboxylase enzyme activities of C. albicans strains with ura-blaster-mediated genetic disruptions were measured. All strains harboring genetic lesions via the ura-blaster construct showed reduced OMP decarboxylase activities compared to that of the wild type when assayed. The activity levels in different gene disruptions varied, suggesting a positional effect on the level of gene expression. Because the URA3 gene of C. albicans has previously been identified as a virulence factor for this microorganism, our results suggest that decreased virulence observed in strains constructed with the ura-blaster cassette cannot accurately be attributed, in all cases, to the targeted genetic disruption. Although revised methods for validating a URA3-disrupted gene as a target for antifungal drug development could be devised, it is clearly desirable to replace URA3 with a different selectable marker that does not influence virulence.

FIG. 1

Relationship between OMP decarboxylase specific activities in C. albicans strains and the known experimental virulence of the strains previously reported. Relative virulence is represented as the day of death for 50% of a group of immunocompetent mice injected with 10⁶ C. albicans cells via the lateral tail vein. Mice injected with avirulent strains remain healthy and do not achieve 50% death after 24 to 30 days of observation. OMP decarboxylase specific activities are reported as units of enzyme activity per milligram of protein within the assayed cell lysate of each strain.