The Candida albicans gene HWP1 encodes a surface protein that is required for normal hyphal development in vitro. We used mutants lacking one or both alleles of HWP1 to investigate the role of this gene in virulence. Mice infected intravenously with the homozygous hwp1 null mutant, CAL3, survived a median of >14 days, whereas mice infected with a control strain containing two functional alleles of HWP1 survived only 3.5 days. After 1 day of infection, all strains produced similar levels of infection in the kidneys, spleen, and blood. However, after 2 and 3 days, there was a significant decrease in the number of organisms in the kidneys of the mice infected with CAL3. This finding suggests that the hwp1 homozygous null mutant is normal in its ability to initiate infection but deficient in its capacity to maintain infection. CAL3 also germinated minimally in the kidneys. The ability of the heterozygous null mutant to germinate and cause mortality in mice was intermediate to CAL3, suggesting a gene dosage effect. To investigate potential mechanisms for the diminished virulence of CAL3, we examined its interactions with endothelial cells and neutrophils in vitro. CAL3 caused less endothelial cell injury than the heterozygous hwp1 mutant. We conclude that the HWP1 gene product is important for both in vivo hyphal development and pathogenicity of C. albicans. Also, the ability to form filaments may be critical for candidal virulence by enabling the fungus to induce cellular injury and maintain a deep-seated infection.

The morphological plasticity of Candida albicans is an important determinant of pathogenicity, and nonfilamentous mutants are avirulent. HWP1, a hypha-specific gene, was identified in a genetic screen for developmentally regulated genes and encodes a cell surface protein of unknown function. Heterozygous and homozygous deletions of HWP1 resulted in a medium-conditional defect in hyphal development. HWP1 expression was blocked in a Δefg1 mutant, reduced in an Δrbf1 mutant, and derepressed in a Δtup1 mutant. Therefore,HWP1 functions downstream of the developmental regulatorsEFG1, TUP1, and RBF1. Mutation ofCPH1 had no effect on HWP1 expression, suggesting that the positive regulators of hyphal development,CPH1 and EFG1, are components of separate pathways with different target genes. The expression of a second developmentally regulated gene, ECE1, was similarly regulated by EFG1. Since ECE1 is not required for hyphal development, the regulatory role of EFG1apparently extends beyond the control of cell shape determinants. However, expression of ECE1 was not influenced byTUP1, suggesting that there may be some specificity in the regulation of morphogenic elements during hyphal development.

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.