Figure 1.
Adherence of wild-type and mutant strains.
Adherence to silicone was measured in a Fluxion flow cell as described in Methods, and is expressed relative to the wild-type reference strain. Panel A. Transcription factor insertion mutants. The mutants presented had statistically significant decreases (p value ≤0.05) in adherence when compared to reference strain DAY286. The zap1Δ/Δ mutant is included for reference. Measurements indicate mean and standard deviation for 1–3 isolates, as indicated in Table S1 worksheet 1A. Panel B. Analysis of Bcr1 and its target genes. The wild-type strain DAY185 was used as a standard for comparison to mutants bcr1Δ/Δ (CJN702), bcr1Δ/Δ+pBCR1 (CJN698), hwp1Δ/Δ (CAH7-1A1E2), als3Δ/Δ (CAYF178U), als1Δ/Δ (CAYC2YF1U), bcr1Δ/Δ+ALS1-OE (CJN1144), and als1Δ/Δ+pALS1 (CAYC1). Asterisks indicate statistically significant decreases in adherence compared to the wild-type strain.
Table 1.
Summary of adherence mutant properties.
Figure 2.
Gene expression profiles of adherence mutants.
Panel A. Hierarchical clustering of gene expression data. NanoString expression data (Table S2) were analyzed as described in Methods. Briefly, averages of three independent determinations for each mutant strain were divided by averages of six independent determinations of the reference wild-type strain DAY185 to obtain the fold change values for each of 293 genes. All mutant strains were insertion homozygotes except for ace2, arg81, crz2, zap1, and zfu2, which were deletion homozygotes. Transcription factor mutants with adherence defects are indicated with underlined gene names; the remaining mutants were controls included for comparison. Color scale limits were set at (−2.0, 0.0, 2.0), so that the brightest yellow represents 4 fold up-regulation compared to wild-type, and the brightest blue represents 4 fold down-regulation. We define the clusters by representative genes. HYVIR: over 50% of the genes in this cluster are known to play roles in hyphal growth or virulence. RAM: top targets of Ace2 (Regulation of Ace2 and polarized morphogenesis), which are also regulated by Cbk1, Snf5, Cas5, Bcr1, and Met4. ZAPT: known Zap1 targets. CSTAR: Cell surface targets of adherence regulators. Additional small clusters of co-regulated genes did not have unifying functional or structural features. Panel B. Summary of regulatory relationships among the 30 adherence regulators, Zap1, and the four clusters of target genes defined in panel 2A. Black circles: target gene clusters. Yellow circles: transcription factors. Yellow circles with black border: adherence regulators whose defects in adherence can be rescued by ZAP1 overexpression. Blue lines: negative regulation for at least 2/3 of the target genes in the cluster. Orange lines: positive regulation for at least 2/3 of the target genes in the cluster.
Figure 3.
Functional relationship between Snf5 and Ace2.
Strains indicated at the top of each column include SNF5/SNF5 (DAY185), snf5Δ/Δ (DHY02), snf5Δ/Δ+pSNF5 (DHY8), and snf5Δ/Δ+ACE2-OE (DHY20). Panel A. Adherence and biofilm formation assays. Each strain was assayed for adherence, biofilm formation in vitro (48 hr biomass measurements and 24 hr confocal imaging assays), and 24 hr biofilm formation in vivo (catheter lumen surfaces imaged via scanning electron microscopy at 50× or 1000× magnification as indicated). Panel B. Pleiotropic phenotypic assays. Yeast cells were visualized to assess aggregation after 8 hr growth (mid-exponential phase) in YPD at 30°C. Hypha formation visualized after 4 hr of growth in Spider medium at 37°C. Cell wall inhibitor sensitivity was measured by spot dilution assays: overnight cultures were serially diluted five-fold from left to right and assayed for growth on YPD, YPD+200 µg/ml Congo Red and YPD+62.5 µg/ml caspofungin after 48 hours at 30°C.
Figure 4.
Expression of ZAP1 and novel Zap1 dependent genes.
Strains were grown in Spider medium for 8 hr at 37°C and QRTPCR assays were used to determine RNA levels for of ZAP1, ORF19.4652, PGA39 and QDR1. RNA levels were normalized to control TDH3 RNA and then expressed as relative units compared to each RNA in the wild-type strain. Strains included wild type (DAY185), zap1Δ/Δ (CJN1201), zcf28Δ/Δ (JF144), zcf28Δ/Δ+ZAP1-OE (JFY261), try2−/− (EHY97), try2Δ/Δ+ZAP1-OE (JFY337), try3−/− (EHY30), and try3−/−+ZAP1-OE (JFY251).
Figure 5.
Restoration of adherence by increased ZAP1 expression.
Adherence of mutant strains, without the ZAP1-OE allele (blue) or with the ZAP1-OE allele (red), is indicated relative to the wild-type reference strain DAY185.
Figure 6.
Portrait of C. albicans adherence regulators.
Our main findings are summarized with transcription factors (blue boxes) connected to cell surface genes (green boxes) and the target process of adherence. Bcr1 promotes adherence through stimulation of ALS1 expression. Snf5 promotes adherence through stimulation of ACE2 expression. Try2, Try3, Try4, Try5, Suc1, Fgr27, Zcf28, and Uga33 are required for adherence and required for the expression of CSTAR genes. CSTAR gene products include numerous predicted cell wall proteins; we hypothesize that many CSTAR gene products mediate adherence. Zap1 is also a positive regulator of CSTAR genes, but it is not required for adherence in our assays. Ada2, Met4, and Try6 are negative regulators of many CSTAR genes. Finally, many transcription factors are required for adherence (Arg81, Cas5, Czf1, Crz2, Dal81, Fcr3, Leu3, Not3, Taf14, War1, Znc1, Zfu2, Zcf8, Zcf31, Zcf34, Zcf39), and govern expression of one or several classes of genes (summarized in Figure 2B), but cannot be connected to specific functional targets.