A conserved fungal morphogenetic kinase regulates pathogenic growth in response to carbon source diversity.

Fungal pathogens must exhibit strong nutritional plasticity, effectively sensing and utilizing diverse nutrients to support virulence. How the signals generated by nutritional sensing are efficiently translated to the morphogenetic machinery for optimal growth and support of virulence remains incompletely understood. Here, we show that the conserved morphogenesis-related kinase, CotA, imparts isoform-specific control over Aspergillus fumigatus invasive growth in host-mimicking environments and during infection.

The sterol C-24 methyltransferase encoding gene, erg6, is essential for viability of Aspergillus species.

Triazoles, the most widely used class of antifungal drugs, inhibit the biosynthesis of ergosterol, a crucial component of the fungal plasma membrane. Inhibition of a separate ergosterol biosynthetic step, catalyzed by the sterol C-24 methyltransferase Erg6, reduces the virulence of pathogenic yeasts, but its effects on filamentous fungal pathogens like Aspergillus fumigatus remain unexplored. Here, we show that the lipid droplet-associated enzyme Erg6 is essential for the viability of A.

A secondary mechanism of action for triazole antifungals in Aspergillus fumigatus mediated by hmg1.

Triazole antifungals function as ergosterol biosynthesis inhibitors and are frontline therapy for invasive fungal infections, such as invasive aspergillosis. The primary mechanism of action of triazoles is through the specific inhibition of a cytochrome P450 14-α-sterol demethylase enzyme, Cyp51A/B, resulting in depletion of cellular ergosterol. Here, we uncover a clinically relevant secondary mechanism of action for triazoles within the ergosterol biosynthesis pathway.

MOB-mediated regulation of septation initiation network (SIN) signaling is required for echinocandin-induced hyperseptation in .

is a major invasive mold pathogen and the most frequent etiologic agent of invasive aspergillosis. The currently available treatments for invasive aspergillosis are limited in both number and efficacy. Our recent work has uncovered that the β-glucan synthase inhibitors, the echinocandins, are fungicidal against strains of with defects in septation initiation network (SIN) kinase activity.

The gene contributes to cell wall homeostasis and fungal virulence.

In this study, two distinct infection models of , using murine macrophages (RAW264.7) and human lung epithelial cells (A549), were employed to identify the genes important for fungal adaptation during infection. Transcriptomic analyses of co-incubated uncovered 140 fungal genes up-regulated in common between both models that, when compared with a previously published transcriptomic study, allowed the identification of 13 genes consistently up-regulated in all three infection conditions.

The sterol C-24 methyltransferase encoding gene, , is essential for viability of species.

Ergosterol is a critical component of fungal plasma membranes. Although many currently available antifungal compounds target the ergosterol biosynthesis pathway for antifungal effect, current knowledge regarding ergosterol synthesis remains incomplete for filamentous fungal pathogens like . Here, we show for the first time that the lipid droplet-associated sterol C-24 methyltransferase, Erg6, is essential for viability.

and Mutations Are Drivers of -Independent Pan-Triazole Resistance in an Aspergillus fumigatus Clinical Isolate.

Aspergillus fumigatus is a ubiquitous environmental mold that can cause severe disease in immunocompromised patients and chronic disease in individuals with underlying lung conditions. Triazoles are the most widely used class of antifungal drugs to treat A. fumigatus infections, but their use in the clinic is threatened by the emergence of triazole-resistant isolates worldwide, reinforcing the need for a better understanding of resistance mechanisms.

Loss of Septation Initiation Network (SIN) kinases blocks tissue invasion and unlocks echinocandin cidal activity against Aspergillus fumigatus.

Although considered effective treatment for many yeast fungi, the therapeutic efficacy of the echinocandin class of antifungals for invasive aspergillosis (IA) is limited. Recent studies suggest intense kinase- and phosphatase-mediated echinocandin adaptation in A. fumigatus.

Analysis of the genes contribution to azole resistance in section with the CRISPR-Cas9 technique.

Cyp51 contribution to azole resistance has been broadly studied and characterized in , whereas it remains poorly investigated in other clinically relevant species of the genus, such as those of section In this work, we aimed to analyze the impact of genes ( and ) on the voriconazole (VRC) response and resistance of and We generated CRISPR-Cas9 and knock-out mutants from strains with different genetic backgrounds and diverse patterns of azole susceptibility. Single gene deletions of genes resulted in 2 to 16-fold decrease of the VRC Minimum Inhibitory Concentration (MIC) values, which were below the VRC Epidemiological Cutoff Value (ECV) established by the Clinical and Laboratory Standards Institute (CLSI) irrespective of their parental strains susceptibilities. Gene expression studies in the tested species confirmed that participates more actively than in the transcriptional response of azole stress.

Genome-Wide Association for Itraconazole Sensitivity in Non-resistant Clinical Isolates of .

is a potentially lethal opportunistic pathogen that infects over ~200,000 people and causes ~100,000 deaths per year globally. Treating infections is particularly challenging because of the recent emergence of azole-resistance. The majority of studies focusing on the molecular mechanisms underlying azole resistance have examined azole-resistant isolates.

Overexpression of the Small GTPase, RsrA, Promotes Polarity Establishment during Germination.

Cell polarization comprises highly controlled processes and occurs in most eukaryotic organisms. In yeast, the processes of budding, mating and filamentation require coordinated mechanisms leading to polarized growth. Filamentous fungi, such as , are an extreme example of cell polarization, essential for both vegetative and pathogenic growth.

Mechanisms of triazole resistance in Aspergillus fumigatus.

The ubiquitous fungal pathogen Aspergillus fumigatus is the primary cause of opportunistic mould infections in humans. Aspergilli disseminate via asexual conidia passively travelling through air currents to germinate within a broad range of environs, wherever suitable nutrients are found. Though the average human inhales hundreds of conidia daily, A.

Characterization of the Efflux Capability and Substrate Specificity of Aspergillus fumigatus PDR5-like ABC Transporters Expressed in Saccharomyces cerevisiae.

This research analyzed six genes encoding putative efflux proteins for their roles as transporters. Th genes , and were cloned into plasmids and overexpressed in a strain in which the highly active endogenous ABC transporter gene was deleted. The activity of each transporter was measured by efflux of rhodamine 6G and accumulation of alanine β-naphthylamide.

Differential requirements of protein geranylgeranylation for the virulence of human pathogenic fungi.

Protein prenylation is a crucial post-translational modification largely mediated by two heterodimeric enzyme complexes, farnesyltransferase and geranylgeranyltransferase type-I (GGTase-I), each composed of a shared α-subunit and a unique β-subunit. GGTase-I enzymes are validated drug targets that contribute to virulence in and to the yeast-to-hyphal transition in . Therefore, we sought to investigate the importance of the α-subunit, RamB, and the β-subunit, Cdc43, of the GGTase-I complex to hyphal growth and virulence.

Mutations in , Challenging the Paradigm of Clinical Triazole Resistance in Aspergillus fumigatus.

is the predominant pathogen of invasive aspergillosis, a disease state credited with over 200,000 life-threatening infections each year. The triazole class of antifungals are clinically essential to the treatment of invasive aspergillosis, both as frontline and as salvage therapy. Unfortunately, resistance to the triazoles among isolates is now increasingly reported worldwide, and a large proportion of this resistance remains unexplained.

SH3-class Ras guanine nucleotide exchange factors are essential for Aspergillus fumigatus invasive growth.

Proper hyphal morphogenesis is essential for the establishment and progression of invasive disease caused by filamentous fungi. In the human pathogen Aspergillus fumigatus, signalling cascades driven by Ras and Ras-like proteins orchestrate a wide variety of cellular processes required for hyphal growth. For activation, these proteins require interactions with Ras-subfamily-specific guanine nucleotide exchange factors (RasGEFs).

Emerging threat of triazole-resistant Aspergillus fumigatus.

Invasive aspergillosis is a leading cause of morbidity and mortality among immunocompromised populations and is predicted to cause more than 200 000 life-threatening infections each year. Aspergillus fumigatus is the most prevalent pathogen isolated from patients with invasive aspergillosis, accounting for more than 60% of all cases. Currently, the only antifungal agents available with consistent activity against A.

Whole-genome sequencing reveals highly specific gene targeting by in vitro assembled Cas9-ribonucleoprotein complexes in .

Background: CRISPR/Cas9-based genome editing is quickly becoming a powerful tool within the field of fungal genetics. Adaptation of CRISPR/Cas9 systems are allowing for rapid and highly efficient gene targeting within fungi. We recently reported the adaptation of a simple CRISPR/Cas9 system for gene deletion that is effective across multiple genetic backgrounds of .

Commonly Used Oncology Drugs Decrease Antifungal Effectiveness against Candida and Aspergillus Species.

The incidence of invasive fungal infections has risen significantly in recent decades as medical interventions have become increasingly aggressive. These infections are extremely difficult to treat due to the extremely limited repertoire of systemic antifungals, the development of drug resistance, and the extent to which the patient's immune function is compromised. Even when the appropriate antifungal therapies are administered in a timely fashion, treatment failure is common, even in the absence of microbial resistance.

C-terminus Proteolysis and Palmitoylation Cooperate for Optimal Plasma Membrane Localization of RasA in .

RasA is a major regulator of fungal morphogenesis and virulence in . The proper localization of RasA to the plasma membrane is essential for the formation of invasive hyphae during infection. In yeast, the localization of Ras2p to the plasma membrane is orchestrated by several post-translational modifications (PTM) at the C-terminal CAAX box that are thought to occur in sequential order.

A Simple and Universal System for Gene Manipulation in : -Assembled Cas9-Guide RNA Ribonucleoproteins Coupled with Microhomology Repair Templates.

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 is a novel genome-editing system that has been successfully established in . However, the current state of the technology relies heavily on DNA-based expression cassettes for delivering Cas9 and the guide RNA (gRNA) to the cell. Therefore, the power of the technology is limited to strains that are engineered to express Cas9 and gRNA.

The Aspergillus fumigatus farnesyltransferase β-subunit, RamA, mediates growth, virulence, and antifungal susceptibility.

Post-translational prenylation mechanisms, including farnesylation and geranylgeranylation, mediate both subcellular localization and protein-protein interaction in eukaryotes. The prenyltransferase complex is an αβ heterodimer in which the essential α-subunit is common to both the farnesyltransferase and the geranylgeranyltransferase type-I enzymes. The β-subunit is unique to each enzyme.

A Fungus-Specific Protein Domain Is Essential for RasA-Mediated Morphogenetic Signaling in .

Ras proteins function as conserved regulators of eukaryotic growth and differentiation and are essential signaling proteins orchestrating virulence in pathogenic fungi. Here, we report the identification of a novel N-terminal domain of the RasA protein in the filamentous fungus . Whereas this domain is absent in Ras homologs of higher eukaryotes, the N-terminal extension is conserved among fungi and is characterized by a short string of two to eight amino acids terminating in an invariant arginine.