The fungal frontier

A report of the 24th Fungal Genetics Conference, Asilomar, USA, 20-25 March 2007

Filamentation Involves Two Overlapping, but Distinct, Programs of Filamentation in the Pathogenic Fungus Candida albicans

The ability of the human pathogenic fungus Candida albicans to switch between yeast-like and filamentous forms of growth has long been linked to pathogenesis. Numerous environmental conditions, including growth at high temperatures, nutrient limitation, and exposure to serum, can trigger this morphological switch and are frequently used in in vitro models to identify genes with roles in filamentation. Previous work has suggested that differences exist between the various in vitro models both in the genetic requirements for filamentation and transcriptional responses to distinct filamentation-inducing media, but these differences had not been analyzed in detail. We compared 10 in vitro models for filamentation and found broad genetic and transcriptomic differences between model systems. The comparative analysis enabled the discovery of novel media-independent genetic requirements for filamentation as well as a core filamentation transcriptional profile. Our data also suggest that the physical environment drives distinct programs of filamentation in C. albicans, which has significant implications for filamentation in vivo

An Extensive Circuitry for Cell Wall Regulation in Candida albicans

Protein kinases play key roles in signaling and response to changes in the external environment. The ability of Candida albicans to quickly sense and respond to changes in its environment is key to its survival in the human host. Our guiding hypothesis was that creating and screening a set of protein kinase mutant strains would reveal signaling pathways that mediate stress response in C. albicans. A library of protein kinase mutant strains was created and screened for sensitivity to a variety of stresses. For the majority of stresses tested, stress response was largely conserved between C. albicans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. However, we identified eight protein kinases whose roles in cell wall regulation (CWR) were not expected from functions of their orthologs in the model fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe. Analysis of the conserved roles of these protein kinases indicates that establishment of cell polarity is critical for CWR. In addition, we found that septins, crucial to budding, are both important for surviving and are mislocalized by cell wall stress. Our study shows an expanded role for protein kinase signaling in C. albicans cell wall integrity. Our studies suggest that in some cases, this expansion represents a greater importance for certain pathways in cell wall biogenesis. In other cases, it appears that signaling pathways have been rewired for a cell wall integrity response

Calcineurin Is Required for Candida albicans To Survive Calcium Stress in Serum

The calcium-activated protein phosphatase calcineurin plays a critical role in the virulence of Candida albicans. Previous studies demonstrated that calcineurin is not required for the yeast-hypha dimorphic transition, host cell adherence, or host cell injury, which are all established virulence attributes of this organism. Calcineurin is, however, essential for survival in serum and disseminated infection. Here we identify the component of serum that is toxic to calcineurin mutant cells. Proteins, peptides, lipids, and other hydrophobic components were all excluded as essential toxic elements. Upon testing of small molecules present in serum, we discovered that calcineurin protects cells from stress caused by the endogenous levels of calcium ions present in serum. These studies illustrate how calcineurin functions in a calcium homeostatic pathway that enables a common human commensal to survive passage through the hostile environment of the bloodstream to establish deep-seated infections and cause disease

Analysis of gene expression in filamentous cells of Candida albicans grown on agar plates

Candida albicans (C. albicans) is a commensal organism of the human gastrointestinal and genitourinary tracts. C. albicans is also a major human pathogen, causing disease ranging from cutaneous infections to lethal systemic disease. The ability of this fungus to switch between yeast and filamentous forms of growth has long been linked to its pathogenesis. Filamentation can be induced by a variety of distinct environmental cues and can occur in either liquid or solid media. While some evidence suggests that there are differences between filamentation in solid and liquid media, gene expression analysis of filamentation in C. albicans has focused strictly on cells grown in liquid media. We have developed a method for analyzing gene expression of filamentous cells grown on solid induction media at early stages of filamentation, establishing cell plating densities, ideal collection times, and collection techniques. We have also demonstrated the utility of the approach not only in qRT-PCR assays, but high-throughput RNAseq assays as well. These assays will allow for comparison studies of C. albicans filamentation initiation in solid and liquid media

Candida albicans adds more weight to iron regulation.

The pathogen Candida albicans can occupy both the bloodstream and gastrointesintal (GI) tract, niches that differ in iron availability. Chen et al. report that a distinct transcription factor, Sef1, alters the conserved fungal iron regulatory paradigm. Sef1 is pivotal for bloodstream infection, but contributes to GI tract colonization as well.</p

Ergosterol Biosynthesis Inhibitors Become Fungicidal when Combined with Calcineurin Inhibitors against Candida albicans, Candida glabrata, and Candida krusei

Azoles target the ergosterol biosynthetic enzyme lanosterol 14α-demethylase and are a widely applied class of antifungal agents because of their broad therapeutic window, wide spectrum of activity, and low toxicity. Unfortunately, azoles are generally fungistatic and resistance to fluconazole is emerging in several fungal pathogens. We recently established that the protein phosphatase calcineurin allows survival of Candida albicans during the membrane stress exerted by azoles. The calcineurin inhibitors cyclosporine A (CsA) and tacrolimus (FK506) are dramatically synergistic with azoles, resulting in potent fungicidal activity, and mutant strains lacking calcineurin are markedly hypersensitive to azoles. Here we establish that drugs targeting other enzymes in the ergosterol biosynthetic pathway (terbinafine and fenpropimorph) also exhibit dramatic synergistic antifungal activity against wild-type C. albicans when used in conjunction with CsA and FK506. Similarly, C. albicans mutant strains lacking calcineurin B are markedly hypersensitive to terbinafine and fenpropimorph. The FK506 binding protein FKBP12 is required for FK506 synergism with ergosterol biosynthesis inhibitors, and a calcineurin mutation that confers FK506 resistance abolishes drug synergism. Additionally, we provide evidence of drug synergy between the nonimmunosuppressive FK506 analog L-685,818 and fenpropimorph or terbinafine against wild-type C. albicans. These drug combinations also exert synergistic effects against two other Candida species, C. glabrata and C. krusei, which are known for intrinsic or rapidly acquired resistance to azoles. These studies demonstrate that the activity of non-azole antifungal agents that target ergosterol biosynthesis can be enhanced by inhibition of the calcineurin signaling pathway, extending their spectrum of action and providing an alternative approach by which to overcome antifungal drug resistance

Cryptococcus neoformans Isolates from Transplant Recipients Are Not Selected for Resistance to Calcineurin Inhibitors by Current Immunosuppressive Regimens

The immunosuppressants tacrolimus (FK506) and cyclosporine A inhibit calcineurin and have potent antifungal activity. In this study, 24% of Cryptococcus neoformans isolates from solid-organ transplant patients exhibited altered sensitivity to these drugs, which may have an impact on the infectious course but does not appear to be the consequence of immunosuppressive therapy

Candida albicans Displays Differential Phenotypic and Genotypic Filamentation Profiles

In a large portion of the population, Candida albicans is a harmless member of the gut microbiome; however, this opportunistic fungal pathogen is the leading cause of candidiasis infections. The pathogenesis of C. albicans’ relies on alternating yeast and hyphal states via the process of filamentation, and characteristics of filamentation are major qualifications when clinically assessing the severity of infection. In a research setting, media type and state impact the filamentation of C. albicans, leading to observed phenotypes which do not necessarily reflect in vivo traits. To examine the effect various media have on filamentation a time course analysis of C. albicans strain SC5314 was performed. Cells from an overnight sample were plated on one of the following media: YPD, Lee’s, Spider, FBS, and RPMI, and grown at intervals 30, 60, 90, and 120 minutes at 30℃ or 37℃. Plates were imaged before cells were collected for RNA extraction. Evaluations of transcript levels at later stages of filamentation have been poor predictors of genes involved in filamentation; therefore, it was decided that RNA would be extracted from cells collected at 30 minutes post induction, a time point that precedes the development of filaments in inducing conditions based on imaging. RNAseq data has been analyzed to reveal the activity of genes at the initiation of filamentation. In a large portion of the population, Candida albicans is a harmless member of the gut microbiome; however, this opportunistic fungal pathogen is the leading cause of candidiasis infections. The pathogenesis of C. albicans’ relies on alternating yeast and hyphal states via the process of filamentation, and characteristics of filamentation are major qualifications when clinically assessing the severity of infection. In a research setting, media type and state impact the filamentation of C. albicans, leading to observed phenotypes which do not necessarily reflect in vivo traits. To examine the effect various media have on filamentation a time course analysis of C. albicans strain SC5314 was performed. Cells from an overnight sample were plated on one of the following media: YPD, Lee’s, Spider, FBS, and RPMI, and grown at intervals 30, 60, 90, and 120 minutes at 30℃ or 37℃. Plates were imaged before cells were collected for RNA extraction. Evaluations of transcript levels at later stages of filamentation have been poor predictors of genes involved in filamentation; therefore, it was decided that RNA would be extracted from cells collected at 30 minutes post induction, a time point that precedes the development of filaments in inducing conditions based on imaging. RNAseq data has been analyzed to reveal the activity of genes at the initiation of filamentation

An extensive circuitry for cell wall regulation in Candida albicans.

Protein kinases play key roles in signaling and response to changes in the external environment. The ability of Candida albicans to quickly sense and respond to changes in its environment is key to its survival in the human host. Our guiding hypothesis was that creating and screening a set of protein kinase mutant strains would reveal signaling pathways that mediate stress response in C. albicans. A library of protein kinase mutant strains was created and screened for sensitivity to a variety of stresses. For the majority of stresses tested, stress response was largely conserved between C. albicans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. However, we identified eight protein kinases whose roles in cell wall regulation (CWR) were not expected from functions of their orthologs in the model fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe. Analysis of the conserved roles of these protein kinases indicates that establishment of cell polarity is critical for CWR. In addition, we found that septins, crucial to budding, are both important for surviving and are mislocalized by cell wall stress. Our study shows an expanded role for protein kinase signaling in C. albicans cell wall integrity. Our studies suggest that in some cases, this expansion represents a greater importance for certain pathways in cell wall biogenesis. In other cases, it appears that signaling pathways have been rewired for a cell wall integrity response.</p