Roles of TUP1 in switching, phase maintenance, and phase-specific gene expression in Candida albicans

Abstract

Candida albicans strain WO-1 switches spontaneously and reversibly between a "white" and "opaque" phenotype that affects colony morphology, cellular phenotype, and expression of a number of phase-specific genes and virulence traits. To assess the role of the transcription regulator Tup1p in this phenotypic transition, both TUP1 alleles were deleted in the mutant delta tup1. Delta tup1 formed "fuzzy large" colonies made up of cells growing exclusively in the filamentous form. Delta tup1 cells did not undergo the white-opaque transition, but it did switch spontaneously, at high frequency (approximately 10(-3)), and unidirectionally through the following sequence of colony (and cellular) phenotypes: "fuzzy large" (primarily hyphae) --> "fuzzy small" (primarily pseudohyphae) --> "smooth" (primarily budding yeast) --> "revertant fuzzy" (primarily pseudohyphae). Northern analysis of white-phase, opaque-phase, and hypha-associated genes demonstrated that Tup1p also plays a role in the regulation of select phase-specific genes and that each variant in the delta tup1 switching lineage differs in the level of expression of one or more phase-specific and/or hypha-associated genes. Using a rescued delta tup1 strain, in which TUP1 was placed under the regulation of the inducible MET3 promoter, white- and opaque-phase cells were individually subjected to a regime in which TUP1 was first downregulated and then upregulated. The results of this experiment demonstrated that (i) downregulation of TUP1 led to exclusive filamentous growth in both originally white- and opaque-phase cells; (ii) the white-phase-specific gene WH11 continued to be expressed in TUP1 downregulated cultures originating from white-phase cells, while WH11 expression remained repressed in TUP1-downregulated cultures originating from opaque-phase cells, suggesting that cells maintained phase identity in the absence of TUP1 expression; and (iii) subsequent upregulation of TUP1 resulted in mass conversion of originally white-phase cells to the opaque phase and maintenance of originally opaque-phase cells in the opaque phase and in the resumption in both cases of switching, suggesting that TUP1 reexpression turns on the switching system in the opaque phase.

FIG. 1.

Protocol for creating TUP1 deletion mutants. The plasmid pGEMΔTUP was derived from pGEMTUP by replacing 886 bp of the TUP1 open reading frame with the hisG-URA3-hisG cassette. The plasmid pGEMΔTUP was cleaved with ApaI and SacI to release the TUP1-hisG cassette, which was used to transform the C. albicans ura3 auxotrophic strain TS3.3. Heterozygous transformants were grown on 5-FOA to obtain a pop-out derivative, which was used to obtain the homozygous deletion mutant by repeating the method.

FIG. 2.

Southern blot analysis of the null mutant Δtup1-1. Approximately 3 μg each of total genomic DNA from the parental strain TU17, the heterozygous deletion mutant Thet1, the pop-out derivative of Thet1, Thet1-p1, the original deletion mutant Δtup1-1 exhibiting the “fuzzy large” phenotype, and the Δtup1-1 variant exhibiting the “fuzzy small” phenotype were individually digested with SalI and subjected to Southern blotting. Blots were hybridized with the TUP1 probe. The size in kilobases of the fragments is shown to the left of the patterns. The small differences in migration of the 5.8- and 2.7-kb bands were due to differences in the amount of DNA loaded.

FIG. 3.

Cellular phenotypes. (A and B) White- and opaque-phase cells, respectively, of control strain TU17. (C and D) White- and opaque-phase cells, respectively, of the TUP1 heterozygous deletion mutant Thet1. (E) Hyphal phenotype of Δtup1-1 “fuzzy large” colonies. (F) Pseudohyphal phenotype of Δtup1-1 “fuzzy small” colonies. (G) Budding phenotype of Δtup1-1 “smooth” colonies. (H) Pseudohyphal phenotype of Δtup1-1 “revertant fuzzy” colonies. Scale bar, 5 μm.

FIG. 4.

Summary of switching in TU17 (TUP1/TUP1), Thet1 (TUP1/tup1), and Δtup1-1 (tup1/tup1). (A) The white-opaque transition in TU17 and Thet1. Colonies were photographed with direct lighting after 7 days of incubation at 25°C. (B) Unidirectional switching in Δtup1-1. The frequencies along each arrow represent the number of variants (phenotype pointed to) in a population of phenotype from which each arrow emanates. Fuzzy large, fuzzy small, and smooth colonies were photographed with indirect lighting after 11 days incubation at 25°C. Revertant fuzzy colonies were photographed with indirect lighting after 14 days incubation at 25°C.

FIG. 5.

Northern analysis of the expression of phase-regulated genes (WH11, EFG1, OP4, SAP1, SAP3, and MCM1) and TUP1 in white (Wh)- and opaque (Op)-phase cells of TU17 and Thet1, white-phase hyphae in TU17 (TU) and Thet1 (Thet), and the “fuzzy large” and “fuzzy small” phenotypes of Δtup1-1 grown at 25 and 37°C. Northern blots were probed with the noted genes. The ethidium bromide-stained 18S rRNA bands are presented at the bottom of the hybridization patterns to demonstrate comparable loading.

FIG. 6.

Northern analysis of the expression of hypha-associated genes (ECE1, RBT5, and HWP1), phase-specific genes (WH11, EFG1, and OP4), and TUP1 in white (Wh)- and opaque (Op)-phase cells of TU17, and Δtup1-1 fuzzy large (Fuz. Lg.), fuzzy small (Fuz. Sm.), smooth (Sm.), and revertant fuzzy (Rev. Fuz.) cells. RBT5 and HWP1 have been demonstrated to be regulated by TUP1 (7, 39). Northern blots were probed with the noted genes. The ethidium bromide-stained 18S rRNA bands are presented at the bottom of the hybridization patterns to demonstrate comparable loading.

FIG. 7.

Northern analysis of the expression of the white phase-specific gene WH11 in the white (Wh) and opaque (Op) phases of parental strain TU17, in the white (Wh) and opaque (Op) phases of the rescued strain Δtup1-1res1 grown in the absence of methionine, which upregulates TUP1 (a, b), in hyphae of the large fuzzy phenotype of original white- and original opaque-phase cells transferred to medium containing methionine, which downregulates TUP1 (a′, b′), and after the hyphae from original white- and opaque-phase cultures were transferred to medium lacking methionine, which again upregulates TUP1 (a″, b″).