Olorofim Effectively Eradicates Dermatophytes In Vitro and In Vivo.

Superficial fungal infections are prevalent worldwide, with dermatophytes as the most common cause. Various antifungal agents including azoles and allylamines are commonly used to treat dermatophytosis. However, their overuse has yielded drug-resistant strains, calling for the development of novel antimycotic compounds. Olorofim is a newly developed antifungal compound that targets pyrimidine biosynthesis in molds. The purpose of this study was to determine the in vitro and in vivo antifungal effects of olorofim against common dermatophytes. The in vitro activity of olorofim against dermatophytes was assessed by microtiter broth dilution method. Bioinformatic analysis of olorofim binding to dihydroorotate dehydrogenase (DHODH) of dermatophytes was also performed, using Aspergillus fumigatus DHODH as a template. The in vivo efficacy of the drug was investigated, using a guinea pig model, experimentally infected with Microsporum gypseum. Microtiter assays confirmed the high in vitro sensitivity of dermatophytes to olorofim (MIC = 0.015-0.06 mg/liter). Amino acid sequence analysis indicated that DHODH is highly conserved among dermatophytes. The critical residues, in dermatophytes, involved in olorofim binding were similar to their counterparts in A. fumigatus DHODH, which explains their susceptibility to olorofim. Typical skin lesions of dermatophyte infection were observed in the guinea pig model at 7 days postinoculation. Following 1 week of daily topical administration of olorofim, similar to the clotrimazole group, the skin lesions were resolved and normal hair growth patterns appeared. In light of the in vitro and in vivo activity of olorofim against dermatophytes, this novel agent may be considered as a treatment of choice against dermatophytosis.

Production of Mycophenolic Acid by a Newly Isolated Indigenous Penicillium glabrum.

Soil-occupant fungi produce a variety of mycotoxins as secondary metabolites, one of which is mycophenolic acid (MPA), an antibiotic and immunosuppressive agent. MPA is mainly produced by several species of Penicillium, especially Penicillium brevicompactum. Here, we present the first report of MPA production by a local strain belonging to Penicillium glabrum species. We screened ascomycete cultures isolated from moldy food and fruits, as well as soils, collected from different parts of Iran. MPA production of one hundred and forty Penicillium isolates was analyzed using HPLC. Three MPA producer isolates were identified, among which the most producer was subjected to further characterization, based on morphological and microscopic analysis, as well as molecular approach (ITS, rDNA and beta-tubulin gene sequences). The results revealed that the best MPA producer belongs to P. glabrum IBRC-M 30518, and can produce 1079 mg/L MPA in Czapek-Dox medium.

Absence of AfuXpot, the yeast Los1 homologue, limits Aspergillus fumigatus growth under amino acid deprived condition.

In Saccharomyces cerevisiae, los1 encodes a nuclear tRNA exporter. Despite the non-essentiality, the deletion of los1 has been shown to extend replicative life span in yeast. Here, we characterized AfuXpot, the los1 homologue in human pathogen Aspergillus fumigatus and found that it is continuously expressed during fungal growth. Microscopic examination of an AfuXpot-GFP-expressing transformant confirmed the nuclear localization of the fusion protein. The targeted gene deletion affirmed the non-essential role of AfuXpot in hyphal growth and sporulation. However, the growth of the deletion mutant was affected by amino acid, but not glucose, deprivation. The susceptibility of the deletant strain to protein and DNA/RNA synthesis inhibitors was also altered. Using bioinformatics tools, some transcription factor binding sites were predicted in AfuXpot promoter. Expression analyses of potential AfuXpot-interacting genes showed a marked down-regulation of sfp1 and mtr10 homologues in ΔAfuXpot strain. Our data demonstrates some conserved aspects of AfuXpot as a tRNA exporter in A. fumigatus.

New insights into resistance of Helicobacter pylori against third- and fourth-generation fluoroquinolones: A molecular docking study of prevalent GyrA mutations.

BACKGROUND: Fluoroquinolones hinder bacterial DNA replication by inhibiting DNA gyrase. However, mutations, in the QRDR segment of its A subunit (GyrA), cause antibiotic resistance. Here, the interactions of levofloxacin (LVX), gemifloxacin (GXN), and moxifloxacin (MXN) with Helicobacter pylori GyrA, in LVX-resistant vs -sensitive strains, were studied. METHODS: Levoflixacin-sensitive (n = 4) and -resistant (n = 9) H pylori strains, randomly selected from another antibiotic susceptibility study, underwent PCR amplification of gyrA gene, spanning the QRDR segment. The amplified gene fragments were sequenced and aligned. The homology model of H pylori GyrA was built based on that of Escherichia coli, and energy minimization was done. The interaction patterns of LVX, GXN, and MXN with GyrA were analyzed via molecular docking studies. RESULTS: Sequence alignment of the 13 studied strains, created 5 categories of strains: (A) wild type-like (H pylori ATCC26695), (B) N87K-only, (C) D91N-only, (D) N87K + V94L, and (E) D91N + A97V mutations. The minimum inhibitory concentrations (MIC) for LVX-sensitive (category A) and -resistant (categories B-E) strains were <1 mg/L and ≥32 mg/L, respectively. The binding mode of GyrA in category A with LVX identified G35/N87/Y90/D91/V94/G114/S115/I116/D117/G118/D119, as key residues, some residing outside the QRDR segment. Category B strains lost only one interaction (G35), which led to elevated binding free energy (∆G) and full LVX resistance. Categories C-E lost more contacts, with higher ∆G and again full LVX resistance. GXN bound to GyrA of categories A and B via a different set of key residues, while MXN retained the lost contact (G35) in LVX-resistant, category B strains. CONCLUSION: Using molecular docking tools, we identified the key residues responsible for interaction of GyrA with LVX, GXN, and MXN. In the presence of N87K-only mutation, the loss of one of these contacts (ie, G35) led to full LVX resistance. Yet, GXN and MXN overcame this mutation, by retaining all key contacts with GyrA.

Antibiotic hypersensitivity in MRSA induced by special protein aggregates.

Emergence of multidrug-resistant bacteria is a major global concern. According to WHO, methicillin-resistant Staphylococcus aureus (MRSA) is a threatening pathogen resistant to a wide spectrum of antibiotics. Herein, to overcome drug resistance in MRSA, we successfully integrated traditional antibacterial methods but with a novel trick that included use of hen egg-white lysozyme's special aggregates generated by fibrillization. The minimum inhibitory concentration of oxacillin (Ox) for MRSA declined from 600 µM to <20 µM when using aggregates. Scanning and transition electron micrographs showed completely disrupted cells when treated with aggregated protein/Ox (20 µM). The assisting role of aggregates to induce antibiotic hypersensitivity was continuous and stable, but sub-inhibitory antibiotic concentration (20 µM) was required again after 8 h. Investigations regarding mechanism of antibiotic hypersensitivity revealed that aggregates were oligomers but not mature fibrils. Furthermore, reactive oxygen species levels rose significantly after treating bacteria with aggregated protein/Ox. Study of resistance mechanisms indicated that in response to wall structure alterations, mecA expression dropped significantly in the presence of aggregated protein/Ox (20 µM) relative to Ox (20 µM). This observation can be a breakthrough in finding alternatives where antibiotic dosage can be significantly reduced, thereby preventing emergence of new multidrug-resistant bacteria.

Characterization of a farnesyl diphosphate synthase gene from Penicillium brevicompactum MUCL 19011.

OBJECTIVES: Farnesyl diphosphate synthase is a critical enzyme in the isoprenoids biosynthesis pathway responsible for ergosterol and secondary metabolites biosynthesis in fungi. RESULTS: Characterization of fds from Penicillium brevicompactum (Pbfds) was performed using TAIL-PCR and RT-PCR followed by complementation tests in Saccharomyces cerevisiae and determination of its expression profile by semi-quantitative RT-PCR. Promoter analysis suggests some binding sites for transcription factors some of which are involved in fungal growth and response to environmental stress. The Pbfds ORF encodes a cytosolic 39.7 kDa protein with a high conservation among Eurotiomycetes and the highest identity (96 %) with Pen. chrysogenum. Homology-based structural modeling suggests that the PbFDS is formed by the arrangement of 15 core helices around a large central cavity where the catalytic reaction takes place. Superimposition of the predicted 3D structure of the enzyme on its ortholog in human reveals the same folding pattern in the counterparts. CONCLUSION: The Pbfds expression may be stimulated in response to the environmental stresses and fungal growth and encodes the PBFDS--a cytosolic enzyme which with a key role in ergosterol and secondary metabolites biosynthesis.