Biology subjects

Gsaller, F.

Publications and source records attributed to Gsaller, F..

4 recordsLinked to original sources

bioRxiv2022

The fungal expel of 5-fluorocytosine derived fluoropyrimidines mitigates its antifungal activity and generates a cytotoxic environment

Invasive aspergillosis remains one of the most devastating fungal diseases and is predominantly linked to infections caused by the opportunistic human mold pathogen Aspergillus fumigatus. Major treatment regimens for the disease comprise the administration of antifungals belonging to the azole, polyene and echinocandin drug class. The prodrug 5-fluorocytosine (5FC), which is the only representative of a fourth class, the nucleobase analogs, shows unsatisfactory in vitro activities and is barely used for the treatment of aspergillosis. The main route of 5FC activation in A. fumigatus comprises its deamination into 5-fluorouracil (5FU) by FcyA, which is followed by Uprt-mediated 5FU phosphoribosylation into 5-fluorouridine monophosphate (5FUMP). In this study, we characterized and examined the role of a metabolic bypass that generates this nucleotide via 5-fluorouridine (5FUR) through uridine phosphorylase and uridine kinase activities. Resistance profiling of mutants lacking distinct pyrimidine salvage activities suggested a minor contribution of the alternative route in 5FUMP formation. We further analyzed the contribution of drug efflux in 5FC tolerance and found that A. fumigatus cells exposed to 5FC reduce intracellular fluoropyrimidine levels through their export into the environment. This release, which was particularly high in mutants lacking Uprt, generates a toxic environment for cytosine deaminase lacking mutants as well as mammalian cells. Employing the broad-spectrum fungal efflux pump inhibitor clorgyline, we demonstrate synergistic properties of this compound in combination with 5FC, 5FU as well as 5FUR.

molecular biology↗

bioRxiv2022

Azole resistance associated regulatory motifs within the promoter of cyp51A in Aspergillus fumigatus

Aspergillus fumigatus is one of the deadliest fungal species causing hundreds of thousands of deaths each year. As azoles provide the preferred first-line option for treatment of Aspergillosis, the increase in rates of resistance and the poor therapeutic outcomes for those infected with a resistant isolate constitutes a serious global health threat. Azole resistance is frequently associated with specific tandem repeat duplications of a promoter element upstream of cyp51A, the gene which encodes the target for this drug class in A. fumigatus. This promoter element is recognized by the activating transcription factors SrbA and AtrR. This region also provides a docking platform for the CCAAT-binding-complex (CBC) and HapX that cooperate in the regulation of genes involved in iron-consuming pathways including cyp51A. Here, we studied the regulatory contribution of SrbA, AtrR, CBC and HapX binding sites on cyp51A expression and azole resistance during different iron availability employing promoter mutational analysis and protein/DNA interaction analysis. This strategy revealed iron status-dependent and -independent roles of these regulatory elements. We show that promoter occupation by both AtrR and SrbA is required for iron-independent steady-state transcriptional activation of cyp51A and its induction during short-term iron exposure relies on HapX binding. We further uncover the HapX binding site as repressor element the disruption of which elevates cyp51A expression and azole resistance regardless of iron availability.

molecular biology↗

bioRxiv2021

Antifungal siderophore conjugates for theranostic applications in invasive pulmonary aspergillosis using low molecular TAFC scaffolds

Invasive pulmonary aspergillosis (IPA) is a life-threatening form of fungal infection, primarily in immunocompromised patients and associated with a significant mortality. Diagnostic procedures are often invasive and/or time consuming and existing antifungals can be constrained by dose limiting toxicity and drug interaction. In this study, we modified triacetylfusarinine C (TAFC), the main siderophore produced by the opportunistic pathogen Aspergillus fumigatus, with antifungal molecules to perform antifungal susceptibility tests and molecular imaging. MethodsA variation of small organic molecules (eflornithine, fludioxonil, thiomersal, fluoroorotic acid (FOA), cyanine 5 (Cy5)) with antifungal activity were coupled to TAFC, resulting in a "Trojan horse" to deliver antifungal compounds specifically into Aspergillus fumigatus hyphae by the major facilitator transporter MirB. Radioactive labelling with gallium-68 allowed to perform in vitro characterization (LogD, stability, uptake assay) as well as biodistribution experiments and PET/CT imaging in an IPA rat infection model. Compounds labelled with stable gallium were used for antifungal susceptibility tests. Results[Ga]DAFC-fludioxonil, -FOA and Cy5 revealed a MirB dependent active uptake with fungal growth inhibition at 16 g/mL after 24 h. Visualization of an Aspergillus fumigatus infection in lungs of a rat was possible with gallium-68 labelled compounds using PET/CT. Heterogeneous biodistribution patterns revealed the immense influence of the antifungal moiety conjugated to DAFC. ConclusionOverall, novel antifungal siderophore conjugates with promising fungal growth inhibition and the possibility to perform PET-imaging, combine both therapeutic and diagnostic potential in a theranostic compound for IPA caused by Aspergillus fumigatus.

microbiology↗

bioRxiv2020

Multiplex genetic engineering exploiting pyrimidine salvage pathway-based self-encoded selectable markers

Selectable markers are indispensable for genetic engineering, yet their number and variety is limited. Most selection procedures for prototrophic cells rely on the introduction of antibiotic resistance genes. New minimally invasive tools are needed to facilitate sophisticated genetic manipulations. Here, we characterized three endogenous genes in the human fungal pathogen Aspergillus fumigatus for their potential as markers for targeted genomic insertions of DNAs-of-interest (DOIs). Since these genes are involved in uptake and metabolization of pyrimidines, resistance to the toxic effects of prodrugs 5-fluorocytosine and 5-fluorouracil can be used to select successfully integrated DOIs. We show that DOI integration, resulting in the inactivation of these genes, caused no adverse effects with respect to nutrient requirements, growth or virulence. Beside the individual use of markers for site-directed integration of reporter cassettes including the 17-kb penicillin biosynthetic cluster, we demonstrate their sequential use inserting three fluorescent protein encoding genes into a single strain for simultaneous multicolor localization microscopy. In addition to A. fumigatus, we validated the applicability of this novel toolbox in Penicillium chrysogenum and Fusarium oxysporum. Enabling multiple targeted insertions of DOIs without the necessity for exogenous markers, this technology has the potential to significantly advance genetic engineering.

genetics↗