Suzanne M. Noble

Strains and Strategies for Large-Scale Gene Deletion Studies of the Diploid Human Fungal Pathogen Candida albicans

ABSTRACT Candida albicans is the most common human fungal pathogen and causes significant morbidity and mortality worldwide. Nevertheless, the basic principles of C. albicans pathogenesis remain poorly understood. Of central importance to the study of this organism is the ability to generate homozygous knockout mutants and to analyze them in a mammalian model of pathogenesis. C. albicans is diploid, and current strategies for gene deletion typically involve repeated use of the URA3 selectable marker. These procedures are often time-consuming and inefficient. Moreover, URA3 expression levels—which are susceptible to chromosome position effects—can themselves affect virulence, thereby complicating analysis of strains constructed with URA3 as a selectable marker. Here, we describe a set of newly developed reference strains (leu2Δ/leu2Δ, his1Δ/his1Δ; arg4Δ/arg4Δ, his1Δ/his1Δ; and arg4Δ/arg4Δ, leu2Δ/leu2Δ, his1Δ/his1Δ) that exhibit wild-type or nearly wild-type virulence in a mouse model. We also describe new disruption marker cassettes and a fusion PCR protocol that permit rapid and highly efficient generation of homozygous knockout mutations in the new C. albicans strains. We demonstrate these procedures for two well-studied genes, TUP1 and EFG1, as well as a novel gene, RBD1. These tools should permit large-scale genetic analysis of this important human pathogen.

Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity

Candida albicans is the most common cause of serious fungal disease in humans. Creation of isogenic null mutants of this diploid organism, which requires sequential gene targeting, allows dissection of virulence mechanisms. Published analyses of such mutants show a near-perfect correlation between C. albicans pathogenicity and the ability to undergo a yeast-to-hypha morphological switch in vitro. However, most studies have used mutants constructed with a marker that is itself a virulence determinant and therefore complicates their interpretation. Using alternative markers, we created ∼3,000 homozygous deletion strains affecting 674 genes, or roughly 11% of the C. albicans genome. Screening for infectivity in a mouse model and for morphological switching and cell proliferation in vitro, we identified 115 infectivity-attenuated mutants, of which nearly half demonstrated normal morphological switching and proliferation. Analysis of such mutants revealed that virulence requires the glycolipid glucosylceramide. To our knowledge, this is the first C. albicans small molecule that has been found to be required specifically for virulence.

Systematic Genetic Analysis of Virulence in the Human Fungal Pathogen Cryptococcus neoformans

The fungus Cryptococcus neoformans is a leading cause of mortality and morbidity among HIV-infected individuals. We utilized the completed genome sequence and optimized methods for homologous DNA replacement using high-velocity particle bombardment to engineer 1201 gene knockout mutants. We screened this resource in vivo for proliferation in murine lung tissue and in vitro for three well-recognized virulence attributes-polysaccharide capsule formation, melanization, and growth at body temperature. We identified dozens of previously uncharacterized genes that affect these known attributes as well as 40 infectivity mutants without obvious defects in these traits. The latter mutants affect predicted regulatory factors, secreted proteins, and immune-related factors, and represent powerful tools for elucidating novel virulence mechanisms. In particular, we describe a GATA family transcription factor that inhibits phagocytosis by murine macrophages independently of the capsule, indicating a previously unknown mechanism of innate immune modulation.

A phenotypic profile of the Candida albicans regulatory network.

Candida albicans is a normal resident of the gastrointestinal tract and also the most prevalent fungal pathogen of humans. It last shared a common ancestor with the model yeast Saccharomyces cerevisiae over 300 million years ago. We describe a collection of 143 genetically matched strains of C. albicans, each of which has been deleted for a specific transcriptional regulator. This collection represents a large fraction of the non-essential transcription circuitry. A phenotypic profile for each mutant was developed using a screen of 55 growth conditions. The results identify the biological roles of many individual transcriptional regulators; for many, this work represents the first description of their functions. For example, a quarter of the strains showed altered colony formation, a phenotype reflecting transitions among yeast, pseudohyphal, and hyphal cell forms. These transitions, which have been closely linked to pathogenesis, have been extensively studied, yet our work nearly doubles the number of transcriptional regulators known to influence them. As a second example, nearly a quarter of the knockout strains affected sensitivity to commonly used antifungal drugs; although a few transcriptional regulators have previously been implicated in susceptibility to these drugs, our work indicates many additional mechanisms of sensitivity and resistance. Finally, our results inform how transcriptional networks evolve. Comparison with the existing S. cerevisiae data (supplemented by additional S. cerevisiae experiments reported here) allows the first systematic analysis of phenotypic conservation by orthologous transcriptional regulators over a large evolutionary distance. We find that, despite the many specific wiring changes documented between these species, the general phenotypes of orthologous transcriptional regulator knockouts are largely conserved. These observations support the idea that many wiring changes affect the detailed architecture of the circuit, but not its overall output.

Passage through the mammalian gut triggers a phenotypic switch that promotes Candida albicans commensalism

Among ∼5,000,000 fungal species, C. albicans is exceptional in its lifelong association with humans, either within the gastrointestinal microbiome or as an invasive pathogen. Opportunistic infections are generally ascribed to defective host immunity but may require specific microbial programs. Here we report that exposure of C. albicans to the mammalian gut triggers a developmental switch, driven by the Wor1 transcription factor, to a commensal cell type. Wor1 expression was previously observed only in rare genetic backgrounds, where it controls a white-opaque switch in mating. We show that passage of wild-type cells through the mouse gastrointestinal tract triggers WOR1 expression and a novel phenotypic switch. The resulting GUT (gastrointestinally induced transition) cells differ morphologically and functionally from previously defined cell types, including opaque cells, and express a transcriptome that is optimized for the digestive tract. The white-GUT switch illuminates how a microorganism can use distinct genetic programs to transition between commensalism and invasive pathogenesis.

Essential Yeast Protein with Unexpected Similarity to Subunits of Mammalian Cleavage and Polyadenylation Specificity Factor (CPSF)

The 3′ ends of most eukaryotic messenger RNAs are generated by internal cleavage and polyadenylation. In mammals, there is a strict dependence of both reactions on the sequence AAUAAA, which occurs upstream of polyadenylation [poly(A)] sites and which is recognized by CPSF. In contrast, cis-acting signals for yeast 3′-end generation are highly divergent from those of mammals, suggesting that trans-acting factors other than poly(A) polymerase would not be conserved. The essential yeast protein Brr5/Ysh1 shows sequence similarity to subunits of mammalian CPSF and is required for 3′-end processing in vivo and in vitro. These results demonstrate a structural and functional conservation of the yeast and mammalian 3′-end processing machineries despite a lack of conservation of the cis sequences.

Candida albicans cell-type switching and functional plasticity in the mammalian host

Candida albicans is a ubiquitous commensal of the mammalian microbiome and the most prevalent fungal pathogen of humans. A cell-type transition between yeast and hyphal morphologies in C. albicans was thought to underlie much of the variation in virulence observed in different host tissues. However, novel yeast-like cell morphotypes, including opaque(a/α), grey and gastrointestinally induced transition (GUT) cell types, were recently reported that exhibit marked differences in vitro and in animal models of commensalism and disease. In this Review, we explore the characteristics of the classic cell types — yeast, hyphae, pseudohyphae and chlamydospores — as well as the newly identified yeast-like morphotypes. We highlight emerging knowledge about the associations of these different morphotypes with different host niches and virulence potential, as well as the environmental cues and signalling pathways that are involved in the morphological transitions.

An RNA Transport System in Candida albicans Regulates Hyphal Morphology and Invasive Growth

Localization of specific mRNAs is an important mechanism through which cells achieve polarity and direct asymmetric growth. Based on a framework established in Saccharomyces cerevisiae, we describe a She3-dependent RNA transport system in Candida albicans, a fungal pathogen of humans that grows as both budding (yeast) and filamentous (hyphal and pseudohyphal) forms. We identify a set of 40 mRNAs that are selectively transported to the buds of yeast-form cells and to the tips of hyphae, and we show that many of the genes encoded by these mRNAs contribute to hyphal development, as does the transport system itself. Although the basic system of mRNA transport is conserved between S. cerevisiae and C. albicans, we find that the cargo mRNAs have diverged considerably, implying that specific mRNAs can easily move in and out of transport control over evolutionary timescales. The differences in mRNA cargos likely reflect the distinct selective pressures acting on the two species.

Candida albicans specializations for iron homeostasis: from commensalism to virulence.

Candida albicans is a fungal commensal-pathogen that persistently associates with its mammalian hosts. Between the commensal and pathogenic lifestyles, this microorganism inhabits host niches that differ markedly in the levels of bioavailable iron. A number of recent studies have exposed C. albicans specializations for acquiring iron from specific host molecules in regions where iron is scarce, while also defending against iron-related toxicity in regions where iron occurs in surfeit. Together, these results point to a central role for iron homeostasis in the evolution of this important human pathogen.

Genetics and molecular biology in Candida albicans.

Candida albicans is an opportunistic fungal pathogen of humans. Although a normal part of our gastrointestinal flora, C. albicans has the ability to colonize nearly every human tissue and organ, causing serious, invasive infections. In this chapter we describe current methodologies used in molecular genetic studies of this organism. These techniques include rapid sequential gene disruption, DNA transformation, RNA isolation, epitope tagging, and chromatin immunoprecipitation. The ease of these techniques, combined with the high-quality C. albicans genome sequences now available, have greatly facilitated research into this important pathogen. Candida albicans is a normal resident of the human gastrointestinal tract; it is also the most common fungal pathogen of humans, causing both mucosal and systemic infections, particularly in immune compromised patients. C. albicans and Saccharomyces cerevisiae last shared a common ancestor more than 900 million years ago; in terms of conserved coding sequences, the two species are approximately as divergent as fish and humans. Although C. albicans and S. cerevisiae share certain core features, they also exhibit many significant differences. This is not surprising as C. albicans has the ability to survive in nearly every niche of a mammalian host, a property not shared by S. cerevisiae. Research into C. albicans is important in its own right, particularly with regards to its ability to cause disease in humans; in addition, comparison with S. cerevisiae can reveal important insights into evolutionary processes. Many of the methodologies developed for use in S. cerevisiae have been adapted for C. albicans, and we describe some of the most common. Although alternative procedures are described in the literature, we have found those described below to be the most convenient. Because the C. albicans parasexual cycle is cumbersome to use in the laboratory, genetics in this organism has been based almost entirely on directed mutations. Because the organism is diploid, creating a deletion mutant requires two rounds of gene disruption. We describe a rapid method for creating sequential disruptions, one which can be scaled up to create large collections of C. albicans deletion mutants. We also describe a series of additional techniques including DNA transformation, mRNA isolation, epitope tagging, and chromatin immunoprecipitation (ChIP). The ease of these techniques, combined with the high-quality C. albicans genome sequences now available, has greatly increased the quality and pace of research into this important pathogen.