From Genes to Networks: The Regulatory Circuitry Controlling Candida albicans Morphogenesis.

Candida albicans is a commensal yeast of most healthy individuals, but also one of the most prevalent human fungal pathogens. During adaptation to the mammalian host, C. albicans encounters different niches where it is exposed to several types of stress, including oxidative, nitrosative (e.g., immune system), osmotic (e.g., kidney and oral cavity) stresses and pH variation (e.g., gastrointestinal (GI) tract and vagina). C. albicans has developed the capacity to respond to the environmental changes by modifying its morphology, which comprises the yeast-to-hypha transition, white-opaque switching, and chlamydospore formation. The yeast-to-hypha transition has been very well characterized and was shown to be modulated by several external stimuli that mimic the host environment. For instance, temperature above 37 â„ƒ, serum, alkaline pH, and CO2 concentration are all reported to enhance filamentation. The transition is characterized by the activation of an intricate regulatory network of signaling pathways, involving many transcription factors. The regulatory pathways that control either the stress response or morphogenesis are required for full virulence and promote survival of C. albicans in the host. Many of these transcriptional circuitries have been characterized, highlighting the complexity and the interconnections between the different pathways. Here, we present the major signaling pathways and the main transcription factors involved in the yeast-to-hypha transition. Furthermore, we describe the role of heat shock transcription factors in the morphogenetic transition, providing an edifying example of the complex cross talk between pathways involved in morphogenesis and stress response.

The two-component response regulator Skn7 belongs to a network of transcription factors regulating morphogenesis in Candida albicans and independently limits morphogenesis-induced ROS accumulation.

Skn7 is a conserved fungal heat shock factor-type transcriptional regulator. It participates in maintaining cell wall integrity and regulates the osmotic/oxidative stress response (OSR) in S. cerevisiae, where it is part of a two-component signal transduction system. Here, we comprehensively address the function of Skn7 in the human fungal pathogen Candida albicans. We provide evidence reinforcing functional divergence, with loss of the cell wall/osmotic stress-protective roles and acquisition of the ability to regulate morphogenesis on solid medium. Mapping of the Skn7 transcriptional circuitry, through combination of genome-wide expression and location technologies, pointed to a dual regulatory role encompassing OSR and filamentous growth. Genetic interaction analyses revealed close functional interactions between Skn7 and master regulators of morphogenesis, including Efg1, Cph1 and Ume6. Intracellular biochemical assays revealed that Skn7 is crucial for limiting the accumulation of reactive oxygen species (ROS) in filament-inducing conditions on solid medium. Interestingly, functional domain mapping using site-directed mutagenesis allowed decoupling of Skn7 function in morphogenesis from protection against intracellular ROS. Our work identifies Skn7 as an integral part of the transcriptional circuitry controlling C. albicans filamentous growth and illuminates how C. albicans relies on an evolutionarily-conserved regulator to protect itself from intracellular ROS during morphological development.

Fungicidal Potency and Mechanisms of θ-Defensins against Multidrug-Resistant Candida Species.

Systemic candidiasis is a growing health care concern that is becoming even more challenging due to the growing frequency of infections caused by multidrug-resistant (MDR) Candida species. Thus, there is an urgent need for new therapeutic approaches to candidiasis, including strategies bioinspired by insights into natural host defense against fungal pathogens. The antifungal properties of θ-defensins, macrocyclic peptides expressed in tissues of Old World monkeys, were investigated against a panel of drug-sensitive and drug-resistant clinical isolates of Candida albicans and non-albicans Candida species. Rhesus θ-defensin 1 (RTD-1), the prototype θ-defensin, was rapidly and potently fungicidal against drug-sensitive and MDR C. albicans strains. Fungal killing occurred by cell permeabilization that was temporally correlated with ATP release and intracellular accumulation of reactive oxygen species (ROS). Killing by RTD-1 was compared with that by histatin 5 (Hst 5), an extensively characterized anticandidal peptide expressed in human saliva. RTD-1 killed C. albicans much more rapidly and at a >200-fold lower concentration than that of Hst 5. Unlike Hst 5, the anticandidal activity of RTD-1 was independent of mitochondrial ATP production. Moreover, RTD-1 was completely resistant to Candida proteases for 2 h under conditions that rapidly and completely degraded Hst 5. MICs and minimum fungicidal concentrations (MFCs) of 14 natural θ-defensins isoforms against drug-resistant C. albicans isolates identified peptides that are more active than amphotericin B and/or caspofungin against fluconazole-resistant organisms, including MDR Candida auris. These results point to the potential of macrocyclic θ-defensins as structural templates for the design of antifungal therapeutics.

Functional Portrait of Irf1 (Orf19.217), a Regulator of Morphogenesis and Iron Homeostasis in Candida albicans.

The alternate growth of Candida albicans between a unicellular yeast form and a multicellular hyphal form is crucial for its ability to cause disease. Interestingly, both morphological forms support distinct functions during proliferation in the human host. We previously identified ORF19.217 (C2_08890W_A), encoding a zinc-finger transcription factor of the C2H2 family, in a systematic screen of genes whose overexpression contributes to C. albicans' morphological changes. Conditional overexpression of ORF19.217 with the strong tetracycline-inducible promoter (P TET ) resulted in a hyperfilamentous phenotype. We examined growth of the orf19.217 knockout-mutant in different hypha-inducing conditions and found that the mutant still formed hyphae under standard hypha-inducing conditions. To further investigate the function of Orf19.217 in C. albicans, we combined genome-wide expression (RNA-Seq) and location (ChIP-Seq) analyses. We found that Orf19.217 is involved in regulatory processes comprising hyphal morphogenesis and iron acquisition. Comparative analysis with existing C. albicans hyphal transcriptomes indicates that Orf19.217-mediated filamentation is distinct from a true hyphal program. Further, the orf19.217 knockout-mutant did not show increased sensitivity to iron deprivation, but ORF19.217 overexpression was able to rescue the growth of a hap5-mutant, defective in a subunit of the CCAAT-complex, which is essential for iron acquisition. This suggested that Orf19.217 is involved in regulation of iron acquisition genes during iron deprivation and acts in a parallel pathway to the established CCAAT-complex. Interestingly, the orf19.217-mutant turned out to be defective in its ability to form filaments under iron-deficiency. Taken together our findings propose that the transcription factor Orf19.217 stimulates expression of the hyphal regulators EFG1 and BRG1 to promote filamentous growth under iron deprivation conditions, allowing the fungus to escape these iron-depleted conditions. The transcription factor therefore appears to be particularly important for adaptation of C. albicans to diverse environmental conditions in the human host. In regard to the newly identified functions, we have given the regulator the name Irf1, Iron-dependent Regulator of Filamentation.

Host Defense Peptides as Templates for Antifungal Drug Development.

Current treatment for invasive fungal diseases is limited to three classes of antifungal drugs: azoles, polyenes, and echinocandins. The most recently introduced antifungal class, the echinocandins, was first approved nearly 30 years ago. The limited antifungal drug portfolio is rapidly losing its clinical utility due to the inexorable rise in the incidence of invasive fungal infections and the emergence of multidrug resistant (MDR) fungal pathogens. New antifungal therapeutic agents and novel approaches are desperately needed. Here, we detail attempts to exploit the antifungal and immunoregulatory properties of host defense peptides (HDPs) in the design and evaluation of new antifungal therapeutics and discuss historical limitations and recent advances in this quest.

A conserved regulator controls asexual sporulation in the fungal pathogen Candida albicans.

Transcription factor Rme1 is conserved among ascomycetes and regulates meiosis and pseudohyphal growth in Saccharomyces cerevisiae. The genome of the meiosis-defective pathogen Candida albicans encodes an Rme1 homolog that is part of a transcriptional circuitry controlling hyphal growth. Here, we use chromatin immunoprecipitation and genome-wide expression analyses to study a possible role of Rme1 in C. albicans morphogenesis. We find that Rme1 binds upstream and activates the expression of genes that are upregulated during chlamydosporulation, an asexual process leading to formation of large, spherical, thick-walled cells during nutrient starvation. RME1 deletion abolishes chlamydosporulation in three Candida species, whereas its overexpression bypasses the requirement for chlamydosporulation cues and regulators. RME1 expression levels correlate with chlamydosporulation efficiency across clinical isolates. Interestingly, RME1 displays a biphasic pattern of expression, with a first phase independent of Rme1 function and dependent on chlamydospore-inducing cues, and a second phase dependent on Rme1 function and independent of chlamydospore-inducing cues. Our results indicate that Rme1 plays a central role in chlamydospore development in Candida species.

Rhesus Theta Defensin 1 Promotes Long Term Survival in Systemic Candidiasis by Host Directed Mechanisms.

Invasive candidiasis is an increasingly frequent cause of serious and often fatal infections in hospitalized and immunosuppressed patients. Mortality rates associated with these infections have risen sharply due to the emergence of multidrug resistant (MDR) strains of C. albicans and other Candida spp., highlighting the urgent need of new antifungal therapies. Rhesus theta (θ) defensin-1 (RTD-1), a natural macrocyclic antimicrobial peptide, was recently shown to be rapidly fungicidal against clinical isolates of MDR C. albicans in vitro. Here we found that RTD-1 was rapidly fungicidal against blastospores of fluconazole/caspofungin resistant C. albicans strains, and was active against established C. albicans biofilms in vitro. In vivo, systemic administration of RTD-1, initiated at the time of infection or 24 h post-infection, promoted long term survival in candidemic mice whether infected with drug-sensitive or MDR strains of C. albicans. RTD-1 induced an early (4 h post treatment) increase in neutrophils in naive and infected mice. In vivo efficacy was associated with fungal clearance, restoration of dysregulated inflammatory cytokines including TNF-α, IL-1ß, IL-6, IL-10, and IL-17, and homeostatic reduction in numbers of circulating neutrophils and monocytes. Because these effects occurred using peptide doses that produced maximal plasma concentrations (Cmax) of less than 1% of RTD-1 levels required for in vitro antifungal activity in 50% mouse serum, while inducing a transient neutrophilia, we suggest that RTD-1 mediates its antifungal effects in vivo by host directed mechanisms rather than direct fungicidal activity. Results of this study suggest that θ-defensins represent a new class of host-directed compounds for treatment of disseminated candidiasis.