Genetic analysis of the Candida albicans biofilm transcription factor network using simple and complex haploinsufficiency

Abstract

Biofilm formation by Candida albicans is a key aspect of its pathobiology and is regulated by an integrated network of transcription factors (Bcr1, Brg1, Efg1, Ndt80, Rob1, and Tec1). To understand the details of how the transcription factors function together to regulate biofilm formation, we used a systematic genetic interaction approach based on generating all possible double heterozygous mutants of the network genes and quantitatively analyzing the genetic interactions between them. Overall, the network is highly susceptible to genetic perturbation with the six network heterozygous mutants all showing alterations in biofilm formation (haploinsufficiency). In addition, many double heterozygous mutants are as severely affected as homozygous deletions. As a result, the network shows properties of a highly interdependent ‘small-world’ network that is highly efficient but not robust. In addition, these genetic interaction data indicate that TEC1 represents a network component whose expression is highly sensitive to small perturbations in the function of other networks TFs. We have also found that expression of ROB1 is dependent on both auto-regulation and cooperative interactions with other network TFs. Finally, the heterozygous NDT80 deletion mutant is hyperfilamentous under both biofilm and hyphae-inducing conditions in a TEC1-dependent manner. Taken together, genetic interaction analysis of this network has provided new insights into the functions of individual TFs as well as into the role of the overall network topology in its function.

Genetic interaction analysis comes to the diploid human pathogen Candida albicans

“…Our initial studies in this area indicate that specific TFs work with others and that these relationships are dynamic [10]. As demonstrated by our CHI experiments [10,21] and the double mutant array approach of Shapiro and colleagues [9], modestly sized, focused arrays of strains containing systematic deletions of pairs of genes with previously characterized functions can provide new insights into genetic network function without recourse to large scale sets of mutant arrays. Thus, genetic interaction analysis does not need to be genome wide to be informative.…”

“…If a double homozygous mutant is constructed with such a TF, then only positive genetic interactions will be measurable. We and others have shown that double heterozygous mutants can be used to identify genetic interactions by virtue of a phenomenon called complex haploinsufficiency (CHI) [10,21].…”

“…CHI is observed when a strain with heterozygous mutations in 2 different loci ( Fig 2C) shows a phenotype relative to the single mutants that is consistent with a genetic interaction ( Fig 1C). We used a CHI to identify genetic interactions among a set of 6 TFs that are required for biofilm formation ( Fig 2D); because deletion of any of the TFs essentially abolished biofilm formation, double homozygous mutants would not provide useful information [20,21]. From this set of data, we identified a new function for TEC1.…”

“…Thus, TEC1 plays an important co-regulatory function during adherence that would not be evident from single gene analysis. At the mature biofilm stage, interestingly, TEC1 interacts with only 3 of the other TFs [21], indicating that the functional relationships between this network of TFs are dynamic and dependent on the stage of biofilm formation. CHI, therefore, can be a useful complement to the study of homozygous deletions, particularly when homozygous deletions are either lethal or completely abolish the phenotype of interest.…”

The Impact of Gene Dosage and Heterozygosity on the Diploid Pathobiont Candida albicans

“…Haploinsufficiency has been extensively used by Krysan and colleagues to quantitate genetic interactions in C. albicans [84][85][86][87][88]. This includes the examination of strains both for simple haploinsufficiency (observable phenotypes in strains heterozygous for a single gene) and for complex haploinsufficiency (phenotypes in strains heterozygous for two different genes).…”