Drivers of diversification in fungal pathogen populations.

To manage and treat chronic fungal diseases effectively, we require an improved understanding of their complexity. There is an increasing appreciation that chronic infection populations are often heterogeneous due to diversification and drift, even within a single microbial species. Genetically diverse populations can contribute to persistence and resistance to treatment by maintaining cells with different phenotypes capable of thriving in these dynamic environments. In chronic infections, fungal pathogens undergo prolonged challenges that can drive trait selection to convergent adapted states through restricted access to critical nutrients, assault by immune effectors, competition with other species, and antifungal drugs. This review first highlights the various genetic and epigenetic mechanisms that promote diversity in pathogenic fungal populations and provide an additional barrier to assessing the actual heterogeneity of fungal infections. We then review existing studies of evolution and genetic heterogeneity in fungal populations from lung infections associated with the genetic disease cystic fibrosis. We conclude with a discussion of open research questions that, once answered, may aid in diagnosing and treating chronic fungal infections.

Mrs4 loss of function in fungi during adaptation to the cystic fibrosis lung.

The genetic disease cystic fibrosis (CF) frequently leads to chronic lung infections by bacteria and fungi. We identified three individuals with CF with persistent lung infections dominated by Clavispora (Candida) lusitaniae. Whole-genome sequencing analysis of multiple isolates from each infection found evidence for selection for mutants in the gene MRS4 in all three distinct lung-associated populations. In each population, we found one or two unfixed, non-synonymous mutations in MRS4 relative to the reference allele found in multiple environmental and clinical isolates including the type strain. Genetic and phenotypic analyses found that all evolved alleles led to loss of function (LOF) of Mrs4, a mitochondrial iron transporter. RNA-seq analyses found that Mrs4 variants with decreased activity led to increased expression of genes involved in iron acquisition mechanisms in both low iron and replete iron conditions. Furthermore, surface iron reductase activity and intracellular iron were much higher in strains with Mrs4 LOF variants. Parallel studies found that a subpopulation of a CF-associated Exophiala dermatitidis infection also had a non-synonymous LOF mutation in MRS4. Together, these data suggest that MRS4 mutations may be beneficial during chronic CF lung infections in diverse fungi, perhaps, for the purposes of adaptation to an iron-restricted environment with chronic infections. IMPORTANCE The identification of MRS4 mutations in Clavispora (Candida) lusitaniae and Exophiala dermatitidis in individuals with cystic fibrosis (CF) highlights a possible adaptive mechanism for fungi during chronic CF lung infections. The findings of this study suggest that loss of function of the mitochondrial iron transporter Mrs4 can lead to increased activity of iron acquisition mechanisms, which may be advantageous for fungi in iron-restricted environments during chronic infections. This study provides valuable information for researchers working toward a better understanding of the pathogenesis of chronic lung infections and more effective therapies to treat them.

In host evolution of Exophiala dermatitidis in cystic fibrosis lung micro-environment.

Individuals with cystic fibrosis (CF) are susceptible to chronic lung infections that lead to inflammation and irreversible lung damage. While most respiratory infections that occur in CF are caused by bacteria, some are dominated by fungi such as the slow-growing black yeast Exophiala dermatitidis. Here, we analyze isolates of E. dermatitidis cultured from two samples, collected from a single subject 2 years apart. One isolate genome was sequenced using long-read Nanopore technology as an in-population reference to use in comparative single nucleotide polymorphism and insertion-deletion variant analyses of 23 isolates. We then used population genomics and phylo-genomics to compare the isolates to each other as well as the reference genome strain E. dermatitidis NIH/UT8656. Within the CF lung population, three E. dermatitidis clades were detected, each with varying mutation rates. Overall, the isolates were highly similar suggesting that they were recently diverged. All isolates were MAT 1-1, which was consistent with their high relatedness and the absence of evidence for mating or recombination between isolates. Phylogenetic analysis grouped sets of isolates into clades that contained isolates from both early and late time points indicating there are multiple persistent lineages. Functional assessment of variants unique to each clade identified alleles in genes that encode transporters, cytochrome P450 oxidoreductases, iron acquisition, and DNA repair processes. Consistent with the genomic heterogeneity, isolates showed some stable phenotype heterogeneity in melanin production, subtle differences in antifungal minimum inhibitory concentrations, and growth on different substrates. The persistent population heterogeneity identified in lung-derived isolates is an important factor to consider in the study of chronic fungal infections, and the analysis of changes in fungal pathogens over time may provide important insights into the physiology of black yeasts and other slow-growing fungi in vivo.

Mrs4 loss of function in fungi during adaptation to the cystic fibrosis lung.

The genetic disease cystic fibrosis (CF) frequently leads to chronic lung infections by bacteria and fungi. We identified three individuals with CF with persistent lung infections dominated by Clavispora ( Candida ) lusitaniae . Whole genome sequencing analysis of multiple isolates from each infection found evidence for selection for mutants in the gene MRS4 in all three distinct lung-associated populations. In each population, we found one or two unfixed, non-synonymous mutations in MRS4 relative to the reference allele found in multiple environmental and clinical isolates including the type strain. Genetic and phenotypic analyses found that all evolved alleles led to loss of function of Mrs4, a mitochondrial iron transporter. RNA Seq analyses found that Mrs4 variants with decreased activity led to increased expression of genes involved in iron acquisition mechanisms in both low iron and replete iron conditions. Furthermore, surface iron reductase activity and intracellular iron was much higher in strains with Mrs4 loss of function variants. Parallel studies found that a subpopulation of a CF-associated Exophiala dermatiditis infection also had a non-synonymous loss of function mutation in MRS4. Together, these data suggest that MRS4 mutations may be beneficial during chronic CF lung infections in diverse fungi perhaps for the purposes of adaptation to an iron restricted environment with chronic infections.

Loss-of-Function ROX1 Mutations Suppress the Fluconazole Susceptibility of upc2AΔ Mutation in Candida glabrata, Implicating Additional Positive Regulators of Ergosterol Biosynthesis.

Two of the major classes of antifungal drugs in clinical use target ergosterol biosynthesis. Despite its importance, our understanding of the transcriptional regulation of ergosterol biosynthesis genes in pathogenic fungi is essentially limited to the role of hypoxia and sterol-stress-induced transcription factors such as Upc2 and Upc2A as well as homologs of sterol response element binding (SREB) factors. To identify additional regulators of ergosterol biosynthesis in Candida glabrata, an important human fungal pathogen with reduced susceptibility to ergosterol biosynthesis inhibitors relative to other Candida spp., we used a serial passaging strategy to isolate suppressors of the fluconazole hypersusceptibility of a upc2AΔ deletion mutant. This led to the identification of loss-of-function mutations in two genes: ROX1, the homolog of a hypoxia gene transcriptional suppressor in Saccharomyces cerevisiae, and CST6, a transcription factor that is involved in the regulation of carbon dioxide response in C. glabrata. Here, we describe a detailed analysis of the genetic interaction of ROX1 and UPC2A. In the presence of fluconazole, loss of Rox1 function restores ERG11 expression to the upc2AΔ mutant and inhibits the expression of ERG3 and ERG6, leading to increased levels of ergosterol and decreased levels of the toxic sterol 14α methyl-ergosta-8,24(28)-dien-3ß, 6α-diol, relative to the upc2AΔ mutant. Our observations establish that Rox1 is a negative regulator of ERG gene biosynthesis and indicate that a least one additional positive transcriptional regulator of ERG gene biosynthesis must be present in C. glabrata. IMPORTANCE Candida glabrata is one of the most important human fungal pathogens and has reduced susceptibility to azole-class inhibitors of ergosterol biosynthesis. Although ergosterol is the target of two of the three classes of antifungal drugs, relatively little is known about the regulation of this critical cellular pathway. Sterols are both essential components of the eukaryotic plasma membrane and potential toxins; therefore, sterol homeostasis is critical for cell function. Here, we identified two new negative regulators in C. glabrata of ergosterol (ERG) biosynthesis gene expression. Our results also indicate that in addition to Upc2A, the only known activator of ERG genes, additional positive regulators of this pathway must exist.

Structural Characterization of the Reaction and Substrate Specificity Mechanisms of Pathogenic Fungal Acetyl-CoA Synthetases.

Acetyl CoA synthetases (ACSs) are Acyl-CoA/NRPS/Luciferase (ANL) superfamily enzymes that couple acetate with CoA to generate acetyl CoA, a key component of central carbon metabolism in eukaryotes and prokaryotes. Normal mammalian cells are not dependent on ACSs, while tumor cells, fungi, and parasites rely on acetate as a precursor for acetyl CoA. Consequently, ACSs have emerged as a potential drug target. As part of a program to develop antifungal ACS inhibitors, we characterized fungal ACSs from five diverse human fungal pathogens using biochemical and structural studies. ACSs catalyze a two-step reaction involving adenylation of acetate followed by thioesterification with CoA. Our structural studies captured each step of these two half-reactions including the acetyl-adenylate intermediate of the first half-reaction in both the adenylation conformation and the thioesterification conformation and thus provide a detailed picture of the reaction mechanism. We also used a systematic series of increasingly larger alkyl adenosine esters as chemical probes to characterize the structural basis of the exquisite ACS specificity for acetate over larger carboxylic acid substrates. Consistent with previous biochemical and genetic data for other enzymes, structures of fungal ACSs with these probes bound show that a key tryptophan residue limits the size of the alkyl binding site and forces larger alkyl chains to adopt high energy conformers, disfavoring their efficient binding. Together, our analysis provides highly detailed structural models for both the reaction mechanism and substrate specificity that should be useful in designing selective inhibitors of eukaryotic ACSs as potential anticancer, antifungal, and antiparasitic drugs.

New Mitochondrial Targets in Fungal Pathogens.

In eukaryotic cells, mitochondria are responsible for the synthesis of ATP using power generated by the electron transport chain (ETC). While much of what is known about mitochondria has been gained from a study of a small number of model species, including the yeast Saccharomyces cerevisiae, the general mechanisms of mitochondrial respiration have been recognized as being highly conserved across eukaryotes. Now, Sun et al. (N. Sun, R. S. Parrish, R. A. Calderone, and W. A. Fonzi, mBio 10:e00300-19, 2019, https://doi.org/10.1128/mBio.00300-19) take the next steps in understanding mitochondrial function by identifying proteins that are unique to a smaller phylogenetic group of microbes. Using the combination of in silico, biochemical, and microbiological assays, Sun and colleagues identified seven genes that are unique to the CTG fungal clade, which contains multiple important human pathogens, including Candida albicans, and showed that they are required for full ETC function during respiratory metabolism. Because respiratory metabolism is critical for fungal pathogenesis, these clade-specific mitochondrial factors may represent novel therapeutic targets.

Systematic Complex Haploinsufficiency-Based Genetic Analysis of Candida albicans Transcription Factors: Tools and Applications to Virulence-Associated Phenotypes.

Genetic interaction analysis is a powerful approach to the study of complex biological processes that are dependent on multiple genes. Because of the largely diploid nature of the human fungal pathogen Candida albicans, genetic interaction analysis has been limited to a small number of large-scale screens and a handful for gene-by-gene studies. Complex haploinsufficiency, which occurs when a strain containing two heterozygous mutations at distinct loci shows a phenotype that is distinct from either of the corresponding single heterozygous mutants, is an expedient approach to genetic interactions analysis in diploid organisms. Here, we describe the construction of a barcoded-library of 133 heterozygous TF deletion mutants and deletion cassettes for designed to facilitate complex haploinsufficiency-based genetic interaction studies of the TF networks in C. albicans We have characterized the phenotypes of these heterozygous mutants under a broad range of in vitro conditions using both agar-plate and pooled signature tag-based assays. Consistent with previous studies, haploinsufficiency is relative uncommon. In contrast, a set of 12 TFs enriched in mutants with a role in adhesion were found to have altered competitive fitness at early time points in a murine model of disseminated candidiasis. Finally, we characterized the genetic interactions of a set of biofilm related TFs in the first two steps of biofilm formation, adherence and filamentation of adherent cells. The genetic interaction networks at each stage of biofilm formation are significantly different indicating that the network is not static but dynamic.

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

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.