Overexpression approaches to advance understanding of Candida albicans

Abstract

Gene perturbation is the standard strategy to elucidate gene function and it can be achieved by both knockout and mis-regulation approaches. The dramatic impact of aneuploidies in human and other model organism illustrates the importance of gene dosage for proper gene function (Sheltzer & Amon, 2011). In those instances, gene copy number variation without alteration to the basic genetic material is sufficient to cause a phenotype (Prelich, 2012;Tang & Amon, 2013). Naturally, molecular approaches aiming at either abolishing (knockout), reducing (knockdown), or increasing (overexpression) gene expression are commonly used by geneticists to decipher the role of a specific gene. The gene knockout strategy, used to understand the function of a gene through loss-of-function, faces several limitations: (a) many genes are expressed in a nichedependent manner, and thus the phenotypic effect linked to the absence of such genes can only be achieved in the specific conditions under which the gene is expressed; (b) the presence of functionally redundant genes will hamper the efficiency of such an approach when working with gene families; (c) the technical challenge of implementing knockout approaches in diploid organisms; and (d) only non-essential genes can be studied by knockout approaches (unless using conditional knockout approaches). Alternatively, gene overexpression has been exploited by geneticists to understand biological pathways as a parallel approach to the loss-of-function approach. By altering cellular localization, complex stoichiometry or proper expression regulation, overexpression of a gene could result in a phenotype that would provide information about the gene function.

Functional Portrait of Irf1 (Orf19.217), a Regulator of Morphogenesis and Iron Homeostasis in Candida albicans

“…Our transcriptome data do not indicate a direct influence of Irf1 on riboflavin biogenesis ( Figure S2 ), although we could detect direct binding of Irf1 in the regulatory sequence of the RIB3 gene, which encodes an enzyme of the riboflavin synthesis ( Echt et al., 2004 ). It should also be noted that Irf1 is a good example of the utility of the overexpression strategy in elucidating important processes in C. albicans ( Rai et al., 2022 ), as the mutant study did not reveal any insight into the function of the protein.…”

Emerging technologies in microbiology

“…The manipulation of gene expression has shed light on aspects of fungal and parasite biology that has been difficult to uncover with traditional loss of function/gene knockout approaches. The use of repressible or inducible promoters to alter gene expression in Candida albicans has facilitated studies on a range of fungal processes such as morphogenesis and biofilm formation (Rai et al, 2022). Similarly, in the apicomplexan parasites, Plasmodium and Toxoplasma, conditional gene expression has been applied to knockdown genes unable to be targeted by complete knockout.…”

Development and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans

“…Indeed, genetic overexpression in C. albicans occurs readily via gene copy number amplification and aneuploidy, and these copy number amplification events are frequently observed in genetically diverse clinical isolates (Coste et al 2006;Forche et al 2018;Todd et al 2019). The upregulation of gene expression plays a critical role in diverse aspects of fungal biology (Rai et al 2021), including the evolution of antifungal drug resistance (Selmecki et al 2008;Sanglard et al 2009;Schubert et al 2011;Flowers et al 2012;Lohberger et al 2014;Todd and Selmecki 2020), host colonization (Prieto et al 2017;Znaidi et al 2018), and virulence (Fu et al 2008;Lohberger et al 2014). Additionally, in other microbial systems, including bacteria, protozoa, and the model yeast Saccharomyces cerevisiae, genetic overexpression libraries have proven to be critical tools for identifying targets of drugs with uncharacterized mechanisms of action, as mutants overexpressing the drug target are typically more resistant to the drug in question (Li et al 2004;Luesch et al 2005;Smith et al 2010;Begolo et al 2014).…”

“…Overexpression screens can also help dissect molecular pathways (Sopko et al 2006;Sahni et al 2010;Polvi et al 2019). In C. albicans, several techniques exist for genetic overexpression (Rai et al 2021), including promoter replacement strategies (Delgado et al 2003;Park and Morschhäuser 2005;Eckert and Mühlschlegel 2009;Sahni et al 2010), the generation of ORFeome collection strains (Chauvel et al 2012;Legrand et al 2018), and CRISPR-based methods (Román et al 2019a). While these these tools have made important contributions to understanding overexpression in C. albicans, there remains a need for simple and rapid methods to efficiently target fungal genes of interest for overexpression, and to do so in diverse strain backgrounds.…”

“…2019). The upregulation of gene expression plays a critical role in diverse aspects of fungal biology (Rai et al . 2021), including the evolution of antifungal drug resistance (Selmecki et al .…”