Analysis of the Candida albicans Als2p and Als4p adhesins suggests the potential for compensatory function within the Als family

Abstract

The ALS (agglutinin-like sequence) gene family encodes eight large cell-surface glycoproteins. The work presented here focuses on Als2p and Als4p, and is part of a larger effort to deduce the function of each Als protein. Both ALS4 alleles were deleted from the Candida albicans genome and the phenotype of the mutant strain (als4D/als4D; named 2034) studied. Loss of Als4p slowed germ tube formation of cells grown in RPMI 1640 medium and resulted in decreased adhesion of C. albicans to vascular endothelial cells. Loss of Als4p did not affect adhesion to buccal epithelial cells, biofilm formation in a catheter model, or adhesion to or destruction of oral reconstituted human epithelium (RHE). Although deletion of one ALS2 allele was achieved readily, a strain lacking the second allele was not identified despite screening thousands of transformants. The remaining ALS2 allele was placed under control of the C. albicans MAL2 promoter to create an als2D/PMAL2-ALS2 strain (named 2342). Real-time RT-PCR analysis of strain 2342 grown in glucose-containing medium (non-inducing conditions) showed that although ALS2 transcript levels were greatly reduced compared to wild-type cells, some ALS2 transcript remained. The decreased ALS2 expression levels were sufficient to slow germ tube formation in RPMI 1640 and Lee medium, reduce adhesion to vascular endothelial cells and to RHE, decrease RHE destruction, and impair biofilm formation. Growth of strain 2342 in maltose-containing medium (inducing conditions) restored the wild-type phenotype in all assays. Real-time RT-PCR analysis demonstrated that in maltose-containing medium, strain 2342 overexpressed ALS2 compared to wild-type cells; however no overexpression phenotype was apparent. Microarray analysis revealed little transcriptional response to ALS4 deletion, but showed twofold up-regulation of orf19.4765 in the glucose-medium-grown als2D/PMAL2-ALS2 strain. orf19.4765 encodes a protein with features of a glycosylated cell wall protein with similarity to Saccharomyces cerevisiae Ccw12p, although initial analysis suggested functional differences between the two proteins. Real-time RT-PCR measurement of ALS2 and ALS4 transcript copy number showed a 2?8-fold increase in ALS2 expression in the als4D/als4D strain and a 3?2-fold increase in ALS4 expression in the als2D/PMAL2-ALS2 strain, suggesting the potential for compensatory function between these related proteins.

Hyphal induction in the human fungal pathogen Candida albicans reveals a characteristic wall protein profile

“…On the other hand, Als4 and Pir1 levels were sharply decreased upon increasing the temperature from 30 to 37 u C and did not change significantly when inducers were added, indicating association with low temperature (Table 3). These results agree qualitatively with transcriptional data, which show that the expression of ALS4 (Zhao et al, 2005) and PIR1 decreases strongly upon switching the temperature from 30 to 37 u C. Pir1 is essential in C. albicans and is involved in wall regeneration and wall organization (Martínez et al, 2004) and probably also in b-1,3-glucan cross-linking (Ecker et al, 2006), while Als4 is important for the infection of buccal epithelial cells (Green et al, 2004). Interestingly, these proteins have also been shown to be contact-dependent, suggesting that they are involved in infections of the skin, where the body temperature is generally lower (Sorgo et al, 2011).…”

Candida albicansCell Wall Proteins

“…While this may reflect strain differences, it may also reflect the influence of environmental conditions, e.g., medium, on biofilm formation, as suggested by the differences in in vitro and in vivo biofilm formation described above. The loss of Als2p but not of Als4p reduced biofilm mass about 15% (416). This suggests that although there is some compensatory expression of ALS4 when ALS2 is deleted and vice versa (416), the compensation of Als4p is insufficient or does not extend to this function.…”

“…A side-by-side comparison of these strains should answer at least some of these questions. The deletion of ALS2 could not be achieved, and the gene was placed under the MAL2 promoter (416). The deletion of ALS4 resulted in an increased expression of ALS2 and vice versa with the uninduced conditional ALS2 strain, as determined by real-time RT-PCR.…”

Distinct domains of the Candida albicans adhesin Eap1p mediate cell–cell and cell–substrate interactions

“…C. albicans glycosylphosphatidylinositol-dependent cell wall proteins (GPI-CWP) are characterized by the presence of an Nterminal signal peptide and a C-terminal sequence signalling GPI anchor attachment (Dranginis et al, 2007). Many of these proteins are involved in mediating the adhesion of C. albicans cells to host materials and/or inert surfaces (Fu et al, 2002;Gaur & Klotz, 1997;Phan et al, 2007;Staab et al, 1999;Zhao et al, 2005Zhao et al, , 2007. Previous studies suggest that the N-terminal domains of the adhesins that are members of the GPI-CWP family mediate binding to substrates and the GPI anchor is required for incorporating these proteins into the cell wall (Frieman et al, 2002;Loza et al, 2004).…”

“…In C. albicans, EAP1 is expressed at similar levels in both yeast and filamentous forms (Li et al, 2007); however, the relative expression of EAP1 compared to other adhesins and the localization of Eap1p remain unknown. Many insights into the functions of C. albicans adhesins have been derived in C. albicans (Fu et al, 2002;Li et al, 2007;Phan et al, 2007;Staab et al, 1999;Zhao et al, 2005Zhao et al, , 2007. Heterologous expression of C. albicans adhesins in S. cerevisiae has also proven valuable in exploring C. albicans adhesive function (Loza et al, 2004;Rauceo et al, 2006).…”