Gene Essentiality Analyzed by In Vivo Transposon Mutagenesis and Machine Learning in a Stable Haploid Isolate of Candida albicans

Abstract

Comprehensive understanding of an organism requires that we understand the contributions of most, if not all, of its genes. Classical genetic approaches to this issue have involved systematic deletion of each gene in the genome, with comprehensive sets of mutants available only for very-well-studied model organisms. We took a different approach, harnessing the power of in vivo transposition coupled with deep sequencing to identify >500,000 different mutations, one per cell, in the prevalent human fungal pathogen Candida albicans and to map their positions across the genome. The transposition approach is efficient and less labor-intensive than classic approaches. Here, we describe the production and analysis (aided by machine learning) of a large collection of mutants and the comprehensive identification of 1,610 C. albicans genes that are essential for growth under standard laboratory conditions. Among these C. albicans essential genes, we identify those that are also essential in two distantly related model yeasts as well as those that are conserved in all four major human fungal pathogens and that are not conserved in the human genome. This list of genes with functions important for the survival of the pathogen provides a good starting point for the development of new antifungal drugs, which are greatly needed because of the emergence of fungal pathogens with elevated resistance and/or tolerance of the currently limited set of available antifungal drugs.

Exploiting homologous recombination increases SATAY efficiency for loss- and gain-of-function screening

“…2013; Sanchez et al, 2019;Segal et al, 2018;Uhse et al, 2018;van Opijnen et al, 2009;Zhu et al, 2018). We have recently developed a protocol dubbed SATAY (for SAturated Transposon Analysis in Yeast), which utilizes a galactoseinducible Ac transposase to mobilize a minimal transposon (miniDs) (Michel et al, 2017).…”

Pseudouridine synthase 7 impacts Candida albicans rRNA processing and morphological plasticity

“…effects. KTI11 an essential gene known to play roles in tRNA wobble base modification was the most up-regulated gene in pus7/pus7 (Segal et al, 2018). Significant increase in abundance of a key translational regulator such as Kti11 could have dramatic effects on proteins required for rRNA processing (Huang et al, 2005).…”

“…ScPus7 is promiscuous, and although we do not observe modification by CaPus7 at all analogous sites, it is possible that CaPus7 modifies a broad range of substrates at lower frequency or that modification of a small set of substrates has complex downstream effects. KTI11 an essential gene known to play roles in tRNA wobble base modification was the most up‐regulated gene in pus7/pus7 (Segal et al, ). Significant increase in abundance of a key translational regulator such as Kti11 could have dramatic effects on proteins required for rRNA processing (Huang et al, ).…”

The CRISPR toolbox in medical mycology: State of the art and perspectives

“…Genetic manipulation of fungal pathogens has been a milestone in deepening our understanding of virulence and host-pathogen interactions. The generation of single-gene mutant libraries (e.g., in C. neoformans [62], C. albicans [63], and C. parapsilosis [64], as well as other approaches like promoter replacement [65], transposon mutagenesis to study gene essentiality [66], and heterozygous libraries generated for haploinsufficiency-based genetic interaction studies in C. albicans [67] allowed the dissection of the molecular players of fungal biology and pathogenicity.…”