Candida albicans is a commensal fungus of the human gastrointestinal tract and a prevalent opportunistic pathogen. To examine diversity within this species, extensive genomic and phenotypic analyses were performed on 21 clinical C. albicans isolates. Genomic variation was evident in the form of polymorphisms, copy number variations, chromosomal inversions, subtelomeric hypervariation, loss of heterozygosity (LOH), and whole or partial chromosome aneuploidies. All 21 strains were diploid, although karyotypic changes were present in eight of the 21 isolates, with multiple strains being trisomic for Chromosome 4 or Chromosome 7. Aneuploid strains exhibited a general fitness defect relative to euploid strains when grown under replete conditions. All strains were also heterozygous, yet multiple, distinct LOH tracts were present in each isolate. Higher overall levels of genome heterozygosity correlated with faster growth rates, consistent with increased overall fitness. Genes with the highest rates of amino acid substitutions included many cell wall proteins, implicating fast evolving changes in cell adhesion and host interactions. One clinical isolate, P94015, presented several striking properties including a novel cellular phenotype, an inability to filament, drug resistance, and decreased virulence. Several of these properties were shown to be due to a homozygous nonsense mutation in the EFG1 gene. Furthermore, loss of EFG1 function resulted in increased fitness of P94015 in a commensal model of infection. Our analysis therefore reveals intra-species genetic and phenotypic differences in C. albicans and delineates a natural mutation that alters the balance between commensalism and pathogenicity.

Highlights d Proteogenomic characterization reveals the functional impact of genomic alterations d Phosphoproteomics uncovers putative therapeutic targets downstream of KRAS d Multiomics links endothelial cell remodeling and glycolysis to immune exclusion d Proteomics and glycoproteomics reveal candidates for early detection or intervention

Highlights d Proteogenomics characterization of 218 pediatric brain tumor samples of 7 histologies d Proteomic clusters reveal actionable biological features spanning histological boundaries d Proteomics reveal downstream effects of DNA alterations not evident in transcriptomics d Kinase activity analyses provide insights into pathway activities and druggable targets

Highlights d TF loss frequently occurs during murine infections and drives host adaptation d Hemizygosity of the EFG1 TF licenses a phenotypic transition in C. albicans cells d Numerous clinical isolates are EFG1 hemizygous and thus can undergo this transition d This ''phenotypic switch'' corresponds to mutational loss of EFG1 function

Mutations introduce variation into the genome upon which selection can act. Defining the nature of these changes is critical for determining species evolution, as well as for understanding the genetic changes driving important cellular processes. The heterozygous diploid fungus Candida albicans is both a frequent commensal organism and a prevalent opportunistic pathogen. A prevailing theory is that C. albicans evolves primarily through the gradual buildup of mitotic mutations, and a pressing issue is whether sexual or parasexual processes also operate within natural populations. Here, we establish that the C. albicans genome evolves by a combination of localized mutation and both short-tract and long-tract loss-of-heterozygosity (LOH) events within the sequenced isolates. Mutations are more prevalent within noncoding and heterozygous regions and LOH increases towards chromosome ends. Furthermore, we provide evidence for genetic exchange between isolates, establishing that sexual or parasexual processes have contributed to the diversity of both nuclear and mitochondrial genomes.

19The opportunistic fungal pathogen Candida albicans lacks a conventional sexual 20 program and is thought to evolve, at least primarily, through the clonal acquisition of genetic 21 changes. Here, we performed an analysis of heterozygous diploid genomes from 21 clinical 22 AUTHOR SUMMARY 40Mutations introduce variation into the genome upon which selection can act. Defining 41 the nature of these changes is critical for determining species evolution, as well as for 42 understanding the genetic changes driving important cellular processes such as carcinogenesis. 43The fungus Candida albicans is a heterozygous diploid species that is both a frequent 44 commensal organism and a prevalent opportunistic pathogen. Prevailing theory is that C. 45 albicans evolves primarily through the gradual build-up of mutations, and a pressing question is 46 whether sexual or parasexual processes also operate within natural populations. Here, we 47 58A wide variety of genetic events contribute to the evolution of eukaryotic genomes. In 59 asexual cells, haploid genomes evolve via the accumulation of point mutations as well as 60 undergo recombination events that drive DNA expansions/contractions (indels). Heterozygous 61 diploid genomes also have the capacity to experience loss of heterozygosity (LOH) events, in 62 which genetic information is lost from one of the two chromosome homologs. In addition, both 63 haploid and diploid genomes may experience large-scale chromosomal changes such as gross 64 rearrangements, acquisition of supernumerary chromosomes or other forms of aneuploidy [1, 2]. 65Many eukaryotic species also generate genetic diversity via sexual reproduction. Here, 66 recombination between individuals provides an efficient mechanism for producing diverse 67 progeny. Sexual reproduction can therefore promote adaptation to new environments more 68 rapidly than asexual propagation [3, 4]. However, this comes at a fitness cost due to the 69 associated energetic requirements and the fact that only 50% of parental alleles are passed on 70 to single progeny [5][6][7]. Sex can also be detrimental by breaking up beneficial allelic 71 combinations [5,8]. Facultative sexuality, the ability to alternate between sexual and asexual 72 forms of reproduction, promotes a flexible lifestyle that can accelerate adaptation in response to 73 environmental pressures [4, 9]. 74Sexual reproduction has been extensively studied in the Saccharomyces clade, where 75 the model yeast Saccharomyces cerevisiae divides mitotically but can also undergo mating and 76 meiosis to generate recombinant progeny. The related Candida clade includes some of the 77 most important human fungal pathogens encountered in the clinic [10, 11], although the 78 Saccharomyces and Candida clades diverged from one another ~235 million years ago [12]. 79The most clinically-relevant Candida species is C. albicans that, like all Candida species, was 80 originally designated an obligate asexual organism. However, mating of diploid cells has been 81 observed in the laboratory an...

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The human commensal and opportunistic fungal pathogen Candida albicans displays extensive genetic and phenotypic variation across clinical isolates. Here, we performed RNA sequencing on 21 well-characterized isolates to examine how genetic variation contributes to gene expression differences and to link these differences to phenotypic traits. C. albicans adapts primarily through clonal evolution, and yet hierarchical clustering of gene expression profiles in this set of isolates did not reproduce their phylogenetic relationship. Strikingly, strain-specific gene expression was prevalent in some strain backgrounds. Association of gene expression with phenotypic data by differential analysis, linear correlation, and assembly of gene networks connected both previously characterized and novel genes with 23 C. albicans traits. Construction of de novo gene modules produced a gene atlas incorporating 67% of C. albicans genes and revealed correlations between expression modules and important phenotypes such as systemic virulence. Furthermore, targeted investigation of two modules that have novel roles in growth and filamentation supported our bioinformatic predictions. Together, these studies reveal widespread transcriptional variation across C. albicans isolates and identify genetic and epigenetic links to phenotypic variation based on coexpression network analysis. IMPORTANCE Infectious fungal species are often treated uniformly despite clear evidence of genotypic and phenotypic heterogeneity being widespread across strains. Identifying the genetic basis for this phenotypic diversity is extremely challenging because of the tens or hundreds of thousands of variants that may distinguish two strains. Here, we use transcriptional profiling to determine differences in gene expression that can be linked to phenotypic variation among a set of 21 Candida albicans isolates. Analysis of this transcriptional data set uncovered clear trends in gene expression characteristics for this species and new genes and pathways that were associated with variation in pathogenic processes. Direct investigation confirmed functional predictions for a number of new regulators associated with growth and filamentation, demonstrating the utility of these approaches in linking genes to important phenotypes.