High-Throughput Screening Identifies Genes Required for Induction of Macrophage Pyroptosis.

The innate immune system is the first line of defense against invasive fungal infections. As a consequence, many successful fungal pathogens have evolved elegant strategies to interact with host immune cells. For example, undergoes a morphogenetic switch coupled to cell wall remodeling upon phagocytosis by macrophages and then induces macrophage pyroptosis, an inflammatory cell death program.

A Genome-Wide Screen of Deletion Mutants in the Filamentous Saccharomyces cerevisiae Background Identifies Ergosterol as a Direct Trigger of Macrophage Pyroptosis.

Phagocytic cells such as macrophages play an important role in the host defense mechanisms mounted in response to the common human fungal pathogen , triggers macrophage NLRP3--mediated pyroptosis, an inflammatory programmed cell death pathway. Here, we provide evidence that -dependent pyroptosis occurs in the kidney of infected mice during the early stages of infection. We have also used a genome-wide screen of nonessential Σ1278b genes to identify genes required for yeast-triggered macrophage pyroptosis.

Systematic Complex Haploinsufficiency-Based Genetic Analysis of 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 , 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.

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).

The Celecoxib Derivative AR-12 Has Broad-Spectrum Antifungal Activity In Vitro and Improves the Activity of Fluconazole in a Murine Model of Cryptococcosis.

Only one new class of antifungal drugs has been introduced into clinical practice in the last 30 years, and thus the identification of small molecules with novel mechanisms of action is an important goal of current anti-infective research. Here, we describe the characterization of the spectrum of in vitro activity and in vivo activity of AR-12, a celecoxib derivative which has been tested in a phase I clinical trial as an anticancer agent. AR-12 inhibits fungal acetyl coenzyme A (acetyl-CoA) synthetase in vitro and is fungicidal at concentrations similar to those achieved in human plasma.

Antitumor/Antifungal Celecoxib Derivative AR-12 is a Non-Nucleoside Inhibitor of the ANL-Family Adenylating Enzyme Acetyl CoA Synthetase.

AR-12/OSU-03012 is an antitumor celecoxib-derivative that has progressed to Phase I clinical trial as an anticancer agent and has activity against a number of infectious agents including fungi, bacteria and viruses. However, the mechanism of these activities has remained unclear. Based on a chemical-genetic profiling approach in yeast, we have found that AR-12 is an ATP-competitive, time-dependent inhibitor of yeast acetyl coenzyme A synthetase.

Estrogen receptor antagonists are anti-cryptococcal agents that directly bind EF hand proteins and synergize with fluconazole in vivo.

Unlabelled: Cryptococcosis is an infectious disease of global significance for which new therapies are needed. Repurposing previously developed drugs for new indications can expedite the translation of new therapies from bench to beside. Here, we characterized the anti-cryptococcal activity and antifungal mechanism of estrogen receptor antagonists related to the breast cancer drugs tamoxifen and toremifene.

Candida albicans triggers NLRP3-mediated pyroptosis in macrophages.

Pyroptosis is an inflammasome-mediated programmed cell death pathway triggered in macrophages by a variety of stimuli, including intracellular bacterial pathogens. Activation of pyroptosis leads to the secretion of interleukin-1β (IL-1β) and pore-mediated cell lysis. Although not considered an intracellular pathogen, Candida albicans is able to kill and, thereby, escape from macrophages.

Candida albicans morphogenesis is not required for macrophage interleukin 1β production.

Unlabelled: The interaction of Candida albicans with macrophages induces the production of interleukin 1β (IL-1β) through inflammasome activation in a process that is required for host survival. C. albicans hypha formation has been linked to IL-1β production, but the question of whether hyphae are sufficient to trigger IL-1β production has not been examined directly.

A repurposing approach identifies off-patent drugs with fungicidal cryptococcal activity, a common structural chemotype, and pharmacological properties relevant to the treatment of cryptococcosis.

New, more accessible therapies for cryptococcosis represent an unmet clinical need of global importance. We took a repurposing approach to identify previously developed drugs with fungicidal activity toward Cryptococcus neoformans, using a high-throughput screening assay designed to detect drugs that directly kill fungi. From a set of 1,120 off-patent medications and bioactive molecules, we identified 31 drugs/molecules with fungicidal activity, including 15 drugs for which direct antifungal activity had not previously been reported.

Cryptococcus neoformans phosphoinositide-dependent kinase 1 (PDK1) ortholog is required for stress tolerance and survival in murine phagocytes.

Cryptococcus neoformans PKH2-01 and PKH2-02 are orthologous to mammalian PDK1 kinase genes. Although orthologs of these kinases have been extensively studied in S. cerevisiae, little is known about their function in pathogenic fungi.

The Spx regulator modulates stress responses and virulence in Enterococcus faecalis.

The ability to cope with endogenous or host-generated reactive oxygen species is considered a key virulence attribute of the opportunistic pathogen Enterococcus faecalis, a leading cause of hospital-acquired infections. In this study, we used in silico and mutational analyses to identify and characterize the role of the Spx global regulator in oxidative stress tolerance and virulence in E. faecalis.