Biology subjects

Binder, U.

Publications and source records attributed to Binder, U..

3 recordsLinked to original sources

bioRxiv2023

H3K4 methylation regulates development, DNA repair, and virulence in Mucorales

Mucorales are basal fungi that opportunistically cause a fatal infection known as mucormycosis (black fungus disease), which poses a significant threat to human health due to its high mortality rate and its recent association with SARS-CoV-2 infections. On the other hand, histone methylation is a regulatory mechanism with pleiotropic effects, including the virulence of several pathogenic organisms. However, the role of epigenetic changes at the histone level never has been studied in Mucorales. Here, we dissected the functional role of Set1, a histone methyltransferase that catalyzes the methylation of H3K4, which is associated with the activation of gene transcription and virulence. A comparative analysis of the Mucor lusitanicus genome (previously known as Mucor circinelloides f. lusitanicus) identified only one homolog of Set1 from Candida albicans and Saccharomyces cerevisiae that contains the typical SET domain. Knockout strains in the gene set1 lacked H3K4 monomethylation, dimethylation, and trimethylation enzymatic activities. These strains also showed a significant reduction in vegetative growth and sporulation. Additionally, set1 null strains were more sensitive to SDS, EMS, and UV light, indicating severe impairment in the repair process of the cell wall and DNA lesions and a correlation between Set1 and these processes. During pathogen-host interactions, strains lacking the set1 gene exhibited shortened polar growth within the phagosome and attenuated virulence both in vitro and in vivo. Our findings suggest that the histone methyltransferase Set1 coordinates several cell processes related to the pathogenesis of M. lusitanicus and may be an important target for future therapeutic strategies against mucormycosis. Author SummaryThe knowledge regarding the role of epigenetic modification in regulating gene expression in early diverging fungi is scarce, despite they represent an important fraction of the fungal kingdom. The order Mucorales, which causes the lethal infection known as mucormycosis, is not an exception. There is an urgent need to enhance our understanding of the biology of these fungi to develop effective treatments for mucormycosis, which are currently absent due to the natural resistance of Mucorales to most antifungal drugs. This work represents the first investigation into the role of the methylation of lysine 4 on histone 3 (H3K4) in a mucoralean fungus. This was accomplished by the generation of deletion mutants in the set1 gene, which encodes the specific H3K4 methyltransferase. Phenotypic analyses of these mutants suggest that H3K4 methylation regulates physiology, development, cell wall integrity, and DNA repair. Furthermore, our findings indicate that it also contributes to the virulence of M. lusitanicus, as strains lacking the set1 gene exhibited shortened polar growth within the phagosome and attenuated virulence both in vitro and in vivo.

genetics↗

bioRxiv2022

CotH genes are necessary for normal spore formation and virulence in Mucor lusitanicus

Mucormycosis is an invasive fungal infection caused by certain members of the fungal order of Mucorales. The species most frequently identified as the etiological agents of mucormycosis belong to the genera Rhizopus, Lichtheimia and Mucor. The frequency of systemic mucormycosis has been increasing, mainly because of the elevating numbers of susceptible patients. Furthermore, Mucorales display intrinsic resistance to the majority of routinely used antifungal agents (e.g., echinocandins and short-tailed azoles), which limits the number of possible therapeutic options. All the above-mentioned issues urge the improvement of molecular identification methods and the discovery of new antifungal targets and strategies. Spore coat proteins (CotH) constitute a kinase family present in many pathogenic bacteria and fungi and participate in the spore formation in these organisms. Moreover, some of them can act as virulence factors being receptors of the human GRP78 protein during Rhizopus delemar-induced mucormycosis. We identified 17 cotH-like genes in the Mucor lusitanicus genome database. Successful disruption of five cotH genes was performed using the CRISPR-Cas9 system in Mucor. The CotH3 and CotH4 proteins play a role in adaptation to different temperatures as well as in developing the cell wall structure. We also show CotH4 protein is involved in spore wall formation by affecting the total chitin content and thus, the composition of the spore wall. Furthermore, we demonstrate that spore size is dependent on the cotH4 gene. The role of CotH3 and CotH4 proteins in virulence was confirmed in two invertebrate models and DKA mouse model. IMPORTANCECurrent treatment options for mucormycosis are inadequate resulting in high mortality rates especially among immunosuppressed patients. The development of novel therapies for mucormycosis has been hampered by lack of understanding of the pathogenetic mechanisms. The importance of the cell surface CotH proteins in the pathogenesis of Rhizopus-mediated mucormycosis has been recently described. However, the contribution of this family of proteins to the virulence of other mucoralean fungi and their functionality in vital processes remain undefined. Through the use of CRISPR-Case9 gene disruption system, we demonstrate the importance of several of the CotH proteins to the virulence of Mucor lusitanicus by using three infection models. We also report on the importance of one of these proteins, CotH4, to the spore wall formation through affecting the chitin content. Therefore, our studies extend the importance of CotH proteins to Mucor and identifies the mechanism by which one of the CotH proteins contributes to the development of a normal fungal cell wall, thereby indicating that this family of proteins can be targeted for future development of novel therapeutic strategies of mucormycosis.

microbiology↗

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↗