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Published on: March 4, 2017
Altered expression of selectable marker URA3 in gene-disrupted Candida albicans strains complicates interpretation of
1Microbiology Department, University of Tennessee, Knoxville, Tennessee 37919, USA.
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.
Insights
The ura-blaster technique can reduce Candida albicans virulence factor gene expression. However, reduced orotidine 5'-monophosphate decarboxylase activity suggests the URA3 gene itself impacts virulence, complicating gene disruption studies.
Area of Science:
- Microbiology
- Molecular Biology
- Mycology
Background:
- The ura-blaster technique is used to disrupt genes in *Candida albicans* to identify virulence factors.
- The *URA3* gene in *C. albicans* has been previously identified as a virulence factor.
Purpose of the Study:
- To measure the orotidine 5 '-monophosphate (OMP) decarboxylase enzyme activities in *C. albicans* strains with ura-blaster-mediated genetic disruptions.
- To assess the impact of the ura-blaster construct on *URA3* gene expression and its implications for virulence studies.
Main Methods:
- Employing the ura-blaster technique for genetic disruption in *C. albicans*.
- Assaying orotidine 5 '-monophosphate (OMP) decarboxylase activity in genetically modified strains.
- Comparing enzyme activity levels between wild-type and disrupted strains.
Main Results:
- All *C. albicans* strains with ura-blaster-mediated genetic lesions exhibited reduced OMP decarboxylase activity compared to the wild type.
- Variations in activity levels among different gene disruptions suggest positional effects on gene expression.
- The observed reduction in virulence in ura-blaster constructed strains may not solely be due to the targeted gene disruption.
Conclusions:
- The ura-blaster technique's use of the *URA3* gene as a selectable marker can confound studies on *Candida albicans* virulence factors.
- Reduced OMP decarboxylase activity indicates that *URA3* itself influences virulence, complicating the interpretation of gene disruption experiments.
- Replacing *URA3* with an alternative selectable marker is recommended for accurate virulence studies and antifungal drug development.
