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

Abstract

HWP1 encodes an adhesin of Candida albicans and has been implicated in filamentation and virulence. URA3, an often-used transformation selection marker, is apparently incorrectly expressed when integrated at the HWP1 locus, which results in an attenuated virulence phenotype. Expression of URA3 is compromised by ectopic integration at other loci as well. In contrast, prior studies from the authors' laboratory had demonstrated that the filamentation deficiency and attenuated virulence of hwp1D mutants were fully restored in rescued strains in which URA3 was integrated at the HWP1 locus. This discrepancy prompted a reinvestigation of these mutants. A series of congenic strains were constructed which demonstrated that the filamentation and virulence defects of a homozygous hwp1D mutant could be rescued without introduction of a functional HWP1 allele. Despite the absence of detectable differences in URA3 expression, analysis of suppressor mutations suggested that reduced URA3 expression gave rise to the mutant phenotypes. Several independent spontaneous suppressor mutations that restored filamentation to strains of genotype hwp1D : : hisG-URA3-hisG/hwp1D : : hisG had acquired a tandem duplication of the hisG-URA3-hisG marker cassette. The hwp1 null mutant and rescued strains differed by the presence or absence of flanking hisG sequence. Substitution of the hisG-URA3-hisG insert of the hwp1 null mutant with URA3 alone largely rescued the filamentation and virulence phenotypes. The presence of a single copy of hisG adjacent to URA3 had no effect. It is concluded that flanking direct repeats of hisG, present as part of a recyclable disruption cassette, negatively influenced URA3 expression and are responsible for the previously reported phenotypes of the hwp1 mutants.

Phenotypic Consequences of a Spontaneous Loss of Heterozygosity in a Common Laboratory Strain of Candida albicans

“…Proper expression of C. albicans URA3 (orf19.1716 ) is important for virulence and for dimorphic transition (Lay et al 1998;Brand et al 2004;Sharkey et al 2005). As such, phenotypic assays often compare Uri + versions of mutants, which have been constructed in a CAI4 background, to CAF2-1 (URA3/ura3).…”

“…Relevant to this work is a short region of Chr3R nearby CEN3 that homozygosed during the construction of CAI4 (ura3D/ura3D) from CAF2-1 (ura3D/URA3). Phenotypic analysis of mutants derived from CAI4 is complicated because URA3 expression levels affect filamentation and virulence properties (Bain et al 2001;Chen et al 2004;Noble and Johnson 2005;Sharkey et al 2005;Noble et al 2010) as well as replication stress (Poulter 1990). Yet we found that, (1) URA3 reintegration into its endogenous locus does not affect MMS sensitivity and (2) supplementing YPD with additional uridine did not modify MMS sensitivity (Figure 1).…”

Candida albicansCell Wall Proteins

“…The biofilm had few hyphae, while both planktonic yeast cells and hyphae were in the surrounding medium. Several years ago, a study showed that the deletion of HWP1 does not cause a filamentation defect (347). Biofilms formed when bcr1⌬/⌬ mutant cells were mixed in various ratios with cells of strains with wild-type HWP1 or with heterozygous or homozygous null mutations (277).…”

Environmental Sensing and Signal Transduction Pathways Regulating Morphopathogenic Determinants of Candida albicans

“…The most widely used marker is the URA3 gene, which encodes orotidine-5Ј-phosphate decarboxylase and confers uracil prototrophy. However, this marker must be used with caution; URA3 expression levels can affect virulence and are susceptible to chromosome position effects, thereby complicating the analysis of strains constructed with URA3 as a selectable marker (62,261,281,292). To avoid the use of the URA3 marker, various other auxotrophic markers, as well as dominant markers, have been developed and are currently used (206,224,241,263).…”