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 ofC. 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 aURA3-disrupted gene as a target for antifungal drug development could be devised, it is clearly desirable to replaceURA3 with a different selectable marker that does not influence virulence.

Correlation between phospholipase of Candida albicans and resistance to fluconazole

“…15 However, the same group later reported that the ÔUra-blasterÕ technique used to produce the knockout mutants might itself have attenuated the virulence of the microorganism. 24 Furthermore, Lyon and Resende demonstrated that phospholipase activity was stronger in Candida strains whose MICs for fluconazole were below 2 lg ml )1 . 25 Fekete-forgacs et al used two C. albicans strains (a fluconazole-sensitive clinical isolate and a fluconazole-resistant laboratorymutant derived from the former by growth at increasing fluconazole concentrations) to examine the changes in virulence traits accompanying the development of resistance to fluconazole.…”

The Candida albicans CaACE2 gene affects morphogenesis, adherence and virulence

“…We chose the MPA R system to delete the ORF because the resulting Caace2 null mutant is otherwise isogenic to the wild-type strain, except for the addition of one short recognition site for the FLP recombinase. Several studies have shown that gene disruption using URA3 is associated with phenotypes that reflect the expression of URA3 rather than the deletion (Lay et al, 1998;Bain et al, 2001;Sundstrom et al, 2002;Cheng et al, 2003;Staab and Sundstrom, 2003). However, to ensure that the phenotypes that we report are caused by deleting CaACE2 and are not artifacts, we also generated constructs in which the two CaACE2 alleles in C. albicans RM1000 were replaced with functional URA3 and HIS1 genes.…”

Virulence genes in the pathogenic yeast Candida albicans

“…Nevertheless, the case of housekeeping and/or essential genes in virulence is a delicate matter since it is very di¤cult to ascertain their e¡ect with respect to the host. In this sense, some auxotrophic genes could be included in this group of`housekeeping' genes and they have been considered by other authors to be virulence determinants [51,90]. Nucleotide metabolism may be important for virulence as has been shown in other fungal species, a result that suggests that nucleotide uptake may be rate-limiting in vivo, that is, inside the animal host.…”

“…In addition, it was later shown that ura3 and ade2 strains obtained entirely through molecular biology methods (strain CAI-4) were also avirulent when assayed in a rat model of oral candidiasis [62]. This result, in fact, has questioned the interpretation of several virulence studies, due to the dual nature of URA3 as an intrinsic pathogenic determinant and a genetic marker [90]. Since the behaviour of these strains with respect to other phenotypic traits not related to pathogenicity is apparently normal, a plausible explanation for this result is that the availability of these nutritional requirements during infection is low and the existence of both phosphoribosylaminoimidazole carboxylase and orotidine 5P-phosphate decarboxylase activity is required for a correct supply of nucleic acid precursors and full growth and virulence.…”