Genetic interaction analysis of transcription factors and in the regulation of respiration and fluconazole susceptibility.

is the second most common cause of invasive candidiasis and is widely known to have reduced susceptibility to fluconazole relative to many other spp. Upc2A is a transcription factor that regulates ergosterol biosynthesis gene expression under conditions of sterol stress such as azole drug treatment or hypoxia. Through an microevolution experiment, we found that loss-of-function mutants of the ATF/CREB transcription factor suppresses the fluconazole hyper-susceptibility of the ∆ mutant.

Deletion of the Candida albicans TLO gene family results in alterations in membrane sterol composition and fluconazole tolerance.

Development of resistance and tolerance to antifungal drugs in Candida albicans can compromise treatment of infections caused by this pathogenic yeast species. The uniquely expanded C. albicans TLO gene family is comprised of 14 paralogous genes which encode Med2, a subunit of the multiprotein Mediator complex which is involved in the global control of transcription.

Alternative ergosterol biosynthetic pathways confer antifungal drug resistance in the human pathogens within the species complex.

Unlabelled: Mucormycoses are emerging fungal infections caused by a variety of heterogeneous species within the Mucorales order. Among the species complex, is the most frequently isolated pathogen in mucormycosis patients and despite its clinical significance, there is an absence of established genome manipulation techniques to conduct molecular pathogenesis studies. In this study, we generated a spontaneous uracil auxotrophic strain and developed a genetic transformation procedure to analyze molecular mechanisms conferring antifungal drug resistance.

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.

Alternative ergosterol biosynthetic pathways confer antifungal drug resistance in the human pathogens within the Mucor species complex.

Mucormycoses are emerging fungal infections caused by a variety of heterogeneous species within the Mucorales order. Among the Mucor species complex, Mucor circinelloides is the most frequently isolated pathogen in mucormycosis patients and despite its clinical significance, there is an absence of established genome manipulation techniques to conduct molecular pathogenesis studies. In this study, we generated a spontaneous uracil auxotrophic strain and developed a genetic transformation procedure to analyze molecular mechanisms conferring antifungal drug resistance.

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.

Molecular Characterization and Sterol Profiles Identify Nonsynonymous Mutations in as a Major Mechanism Conferring Reduced Susceptibility to Amphotericin B in Candida kefyr.

The molecular basis of reduced susceptibility to amphotericin B (rs-AMB) among any yeasts is poorly defined. Genetic alterations in genes involved in ergosterol biosynthesis and total cell sterols were investigated among clinical Candida kefyr isolates. C.

Subpalpebral lavage placement for remote topical administration of ocular medications in 12 dogs: A retrospective review and assessment of owner perception.

Objective: The aim of this study was to describe the placement of subpalpebral lavage (SPL) systems in 12 dogs (15 eyes) intolerant of topical ocular medications to assess the suitability, complications encountered and owner perception of use.

Animals Studied: Retrospective review of dogs that underwent SPL placement for treatment of ocular disease at the Ophthalmology Department, University of Bristol Small Animal Hospital between 2017 and 2021.

Procedure(s): Data recorded included signalment, history, diagnosis, treatment, reason for SPL placement, uni- or bilateral placement, duration of placement, complications, and outcome.

A prospective, masked, randomized, controlled superiority study comparing the incidence of corneal injury following general anesthesia in dogs with two methods of corneal protection.

Objective: To compare the incidence of corneal injury during general anesthesia (GA) and the immediate post-operative period in eyes protected with topical ocular lubricant alone with eyes protected with topical lubricant followed by complete eyelid closure using tape.

Animals Studied: One hundred client-owned dogs (200 eyes) undergoing GA for MRI scan.

Methods: Patients had ocular lubricant applied to both eyes upon induction of anesthesia.

Titration of C-5 Sterol Desaturase Activity Reveals Its Relationship to Candida albicans Virulence and Antifungal Susceptibility Is Dependent upon Host Immune Status.

The azole antifungals inhibit sterol 14α-demethylase (S14DM), which depletes cellular ergosterol and promotes synthesis of the dysfunctional lipid 14α-methylergosta-8,24(28)-dien-3β,6α-diol, ultimately arresting growth. Mutations that inactivate sterol Δ-desaturase (Erg3p), the enzyme that produces the sterol-diol upon S14DM inhibition, enhances Candida albicans growth in the presence of the azoles. However, null mutants are sensitive to some physiological stresses and can be less virulent than the wild type.

Cytochrome P450 168A1 from Pseudomonas aeruginosa is involved in the hydroxylation of biologically relevant fatty acids.

The cytochrome P450 CYP168A1 from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli followed by purification and characterization of function. CYP168A1 is a fatty acid hydroxylase that hydroxylates saturated fatty acids, including myristic (0.30 min-1), palmitic (1.

Loss-of-Function Mutations Suppress the Fluconazole Susceptibility of Δ Mutation in Candida glabrata, Implicating Additional Positive Regulators of Ergosterol Biosynthesis.

Two of the major classes of antifungal drugs in clinical use target ergosterol biosynthesis. Despite its importance, our understanding of the transcriptional regulation of ergosterol biosynthesis genes in pathogenic fungi is essentially limited to the role of hypoxia and sterol-stress-induced transcription factors such as Upc2 and Upc2A as well as homologs of sterol response element binding (SREB) factors. To identify additional regulators of ergosterol biosynthesis in Candida glabrata, an important human fungal pathogen with reduced susceptibility to ergosterol biosynthesis inhibitors relative to other spp.

In vivo emergence of high-level resistance during treatment reveals the first identified mechanism of amphotericin B resistance in Candida auris.

Objective: Candida auris has emerged as a health-care-associated and multidrug-resistant fungal pathogen of great clinical concern. As many as 50% of C. auris clinical isolates are reported to be resistant to amphotericin B, but no mechanisms contributing to this resistance have been identified.

Small-Molecule Inhibitors Targeting Sterol 14α-Demethylase (CYP51): Synthesis, Molecular Modelling and Evaluation Against Candida albicans.

Fungal infections are a global issue affecting over 150 million people worldwide annually, with 750 000 of these caused by invasive Candida infections. Azole drugs are the frontline treatment against fungal infections; however, resistance to current azole antifungals in C. albicans poses a threat to public health.

Controlled in vitro delivery of voriconazole and diclofenac to the cornea using contact lenses for the treatment of Acanthamoeba keratitis.

Acanthamoeba keratitis is caused by a protozoal infection of the cornea, with 80% of cases involving the improper use of contact lenses. The infection causes intense pain and is potentially blinding. However, early diagnosis improves treatment efficacy and the chances of healing.

The negative cofactor 2 complex is a key regulator of drug resistance in Aspergillus fumigatus.

The frequency of antifungal resistance, particularly to the azole class of ergosterol biosynthetic inhibitors, is a growing global health problem. Survival rates for those infected with resistant isolates are exceptionally low. Beyond modification of the drug target, our understanding of the molecular basis of azole resistance in the fungal pathogen Aspergillus fumigatus is limited.

Comparative Genomics for the Elucidation of Multidrug Resistance in Candida lusitaniae.

Multidrug resistance (MDR) has emerged in hospitals due to the use of several agents administered in combination or sequentially to the same individual. We reported earlier MDR in during therapy with amphotericin B (AmB), azoles, and candins. Here, we used comparative genomic approaches between the initial susceptible isolate and 4 other isolates with different MDR profiles.

Isavuconazole and voriconazole inhibition of sterol 14α-demethylases (CYP51) from Aspergillus fumigatus and Homo sapiens.

Here we report the first evaluation of isavuconazole inhibition of Aspergillus fumigatus CYP51 and thus sterol biosynthesis in the fungus. Voriconazole and isavuconazole both bound tightly to recombinant A. fumigatus CYP51 isoenzymes A and B (AfCYP51A and AfCYP51B) isolated in Escherichia coli membranes.

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.

The Evolution of Azole Resistance in Sterol 14α-Demethylase (CYP51) through Incremental Amino Acid Substitutions.

Recombinant CYP51 (CaCYP51) proteins containing 23 single and 5 double amino acid substitutions found in clinical strains and the wild-type enzyme were expressed in and purified by Ni-nitrilotriacetic acid agarose chromatography. Catalytic tolerance to azole antifungals was assessed by determination of the concentration causing 50% enzyme inhibition (IC) using CYP51 reconstitution assays. The greatest increase in the IC compared to that of the wild-type enzyme was observed with the five double substitutions Y132F+K143R (15.