Biology subjects

Haas, H.

Publications and source records attributed to Haas, H..

10 recordsLinked to original sources

bioRxiv2025

Lipid monolayers adsorbing mRNA as models for mRNA enclosed in lipid nanoparticles for transfection

In lipid-based mRNA pharmaceuticals applicable for vaccination, cancer therapy, and other types of therapeutics, the negatively charged RNA is embedded in nanoparticles containing positively charged or ionizable lipids. The local cohesion at the lipid-RNA interface plays an important role for the stability and biological activity of the resulting nanoparticles. Ions and other buffer components in the RNAs immediate surroundings may affect RNA stability against hydrolysis and its binding strength to the oppositely charged lipid layers, which is relevant for release after cellular uptake and endosomal processing. Here, we use Langmuir monolayers at the air/water interface as well-defined experimental models to study the local molecular organization of mRNA adsorbed to lipid layers. Binding of mRNA in the presence of monovalent and divalent ions from the aqueous phase to monolayers consisting of cationic transfection lipids and zwitterionic phospholipids is investigated with synchrotron-based x-ray fluorescence and scattering techniques. The experiments provide detailed insights into the structure of the adsorbed layers as well as the fractions of all molecular moieties contributing to the interfacial electrostatic balance, specifically phosphates from RNA, phosphates from phospholipids in the monolayer, and elemental counterions. The mRNA forms discrete electron-dense layers tightly bound to the lipid membrane, where cationic ions accumulate at the interface together with the mRNA. This leads to an excess of anionic nucleotides relative to cationic lipids at the interface, which is more pronounced in the presence of divalent compared to monovalent cations. The quantitative information on local interfacial moieties obtained here may constitute a valuable basis for the evaluation of quality aspects of nanoparticles comprising mRNA in the bulk phase.

biophysics↗

bioRxiv2024

Cooperative cluster-binding regulates the functional transitions of the Aspergillus fumigatus iron regulator HapX for adaptation to iron starvation, sufficiency and excess

Accurate sensing of cellular iron levels is vital, as this metal is essential but toxic in excess. The iron-sensing transcription factor HapX is crucial for virulence of Aspergillus fumigatus, the predominant human mold pathogen. Its absence impairs growth under iron limitation and excess, but not under moderate iron availability, suggesting that HapX switches between three states to adapt to varying iron availability. This study suggests that the HapX state transitions are regulated by the different propensities of four phylogenetically conserved cysteine-rich regions (CRRs) to coordinate [2Fe-2S] clusters resulting in cumulative occupancies that depend on iron availability. In the iron starvation state, CRR-B and -C lack [2Fe-2S] clusters, the iron sufficiency/"neutral" state features clusters in CRR-B and/or -C and the iron excess state has clusters in all CRR-A, B, and -C, while CRR-D plays a minor role. Combinatorial mutation of CRR-B and -C blocked growth by locking HapX in the iron starvation state, leading to uncontrolled iron uptake, iron accumulation, repression of iron-consuming pathways and impaired iron detoxification. Loss of the C-terminal 27 amino acid region of HapX, which is crucial for the iron starvation state and was found to contain a degron, rescued the severe growth defect. Noteworthy, the - Fe state of HapX induced several gene clusters encoding secondary metabolites.

molecular biology↗

bioRxiv2024

Extensive transmission and variation in a functional receptor for praziquantel resistance in endemic Schistosoma mansoni

Mass-drug administration (MDA) of human populations using praziquantel monotherapy has become the primary strategy for controlling and potentially eliminating the major neglected tropical disease schistosomiasis. To understand how long-term MDA impacts schistosome populations, we analysed whole-genome sequence data of 570 Schistosoma mansoni samples (and the closely related outgroup species, S. rodhaini) from eight countries incorporating both publicly-available sequence data and new parasite material. This revealed broad-scale genetic structure across countries but with extensive transmission over hundreds of kilometres. We characterised variation across the transient receptor potential melastatin ion channel, TRPMPZQ, a target of praziquantel, which has recently been found to influence praziquantel susceptibility. Functional profiling of TRPMPZQ variants found in endemic populations identified four mutations that reduced channel sensitivity to praziquantel, indicating standing variation for resistance. Analysis of parasite infrapopulations sampled from individuals pre- and post-treatment identified instances of treatment failure, further indicative of potential praziquantel resistance. As schistosomiasis is targeted for elimination as a public health problem by 2030 in all currently endemic countries, and even interruption of transmission in selected African regions, we provide an in-depth genomic characterisation of endemic populations and an approach to identify emerging praziquantel resistance alleles. One Sentence SummaryPopulation genomics and functional genetics of praziquantel resistance in Schistosoma mansoni

genomics↗

bioRxiv2024

Aspergillus fumigatus SidF is a dual substrate acyltransferase involved in biosynthesis of both fusarinine- and ferrichrome-type siderophores

The human pathogen Aspergillus fumigatus produces fusarinine-type (FusTS) and ferrichrome-type siderophores (FchTS), both of which have been shown to be crucial for virulence of this mold. After the common first siderophore biosynthetic step, SidA-catalyzed hydroxylation of ornithine, the pathway splits. For FusTS biosynthesis, SidF incorporates an anhydromevalonyl group, while for FchTS biosynthesis, SidL and an as yet unknown enzyme incorporate an acetyl group. The transacylases SidF and SidL share only limited similarity in their C-terminal GNAT (Gcn5-related N-acetyltransferases) motif-containing domains. SidF is transcriptionally induced by iron limitation and localizes to peroxisomes, whereas SidL is a cytosolic enzyme with largely iron-independent expression. Here, we discovered that simultaneous inactivation of both SidF and SidL abolished the biosynthesis of both FusTS and FchTS and caused a growth defect under iron limitation, similar to the inactivation of SidA. Biosynthesis of both FusTS and FchTS depended on both the unique N-terminal and the GNAT motif-containing C-terminal SidF domains. In conclusion, SidF is the hitherto unknown FchTS biosynthetic enzyme: in contrast to SidL, SidF is a bifunctional enzyme accepting acetyl-CoA and anhydromevalonyl-CoA as substrates for biosynthesis of both FusTS and FchTS. Furthermore, this study revealed interdependence of FusTS and FchTS production, and that the peroxisomal localization of FusTS enzymes is important for optimizing FusTS production at the expense of FchTS. Phylogenetic analyses supported the relevance of these findings to other fungal species and revealed overlapping but distinct consensus sequences for the GNAT motifs of SidL and SidF, most likely reflecting their different substrate specificities. IMPORTANCEAdaptation to the host niche is key for any pathogenic organism. Aspergillus fumigatus is a major fungal pathogen causing 90% of invasive aspergillosis cases, which is associated with a high mortality rate. Siderophore-mediated iron acquisition has been shown to be essential for virulence of A. fumigatus and other fungal pathogens. In recent years, the hyphal siderophore biosynthetic pathway has been largely elucidated with exception of a single unknown enzyme, which we identified here as SidF. In contrast to another siderophore biosynthetic acyltransferase, SidL, SidF is a bifunctional enzyme accepting different substrates. As simultaneous inactivation of SidF and SidL, which share a common protein domain and a common substrate, blocks the biosynthesis of all siderophores, simultaneous targeting of SidF and SidL may allow development of new antifungal drugs. Phylogenetic analyses supported the relevance of these findings to other fungal species Moreover, this study clarified the rational for partial peroxisomal localization of siderophore biosynthesis and their metabolic interdependence. The human pathogen Aspergillus fumigatus produces fusarinine-type (FusTS) and ferrichrome-type siderophores (FchTS), both of which have been shown to be crucial for virulence of this mold. After the common first siderophore biosynthetic step, SidA-catalyzed hydroxylation of ornithine, the pathway splits. For FusTS biosynthesis, SidF incorporates an anhydromevalonyl group, while for FchTS biosynthesis, SidL and an as yet unknown enzyme incorporate an acetyl group. The transacylases SidF and SidL share only limited similarity in their C-terminal GNAT (Gcn5-related N-acetyltransferases) motif-containing domains. SidF is transcriptionally induced by iron limitation and localizes to peroxisomes, whereas SidL is a cytosolic enzyme with largely iron-independent expression. Here, we discovered that simultaneous inactivation of both SidF and SidL abolished the biosynthesis of both FusTS and FchTS and caused a growth defect under iron limitation, similar to the inactivation of SidA. Biosynthesis of both FusTS and FchTS depended on both the unique N-terminal and the GNAT motif-containing C-terminal SidF domains. Taken together, SidF is the hitherto unknown FchTS biosynthetic enzyme: in contrast to SidL, SidF is a bifunctional enzyme accepting acetyl-CoA and anhydromevalonyl-CoA as substrates for biosynthesis of both FusTS and FchTS. Moreover, this study revealed interdependence of FusTS and FchTS production, and that peroxisomal localization of FusTS enzymes is important for optimizing FusTS production at the expense of FchTS.

microbiology↗

bioRxiv2024

Structural and functional characterization of SidF, a possible dual substrate Aspergillus fumigatus N5-acetyl-N5-hydroxy-L-ornithine transacetylase

Siderophore-mediated iron acquisition is essential for the virulence of Aspergillus fumigatus, a fungus causing life-threatening aspergillosis. Developing drugs targeting the siderophore biosynthetic pathway could help improve disease management. The transacetylases SidF and SidL generate intermediates for different siderophores in A. fumigatus. A. fumigatus has a yet unidentified transacetylase that complements SidL during iron deficiency in SidL-lacking mutants. We present the first X-ray structure of SidF, revealing a conserved two-domain architecture with tetrameric assembly. Importantly, the N-terminal domain contributes to protein solubility and oligomerization, while the C-terminal domain containing the GCN5-related N-acetyltransferase (GNAT) motif is crucial for the enzymatic activity and mediates oligomer formation. Notably, AlphaFold modelling demonstrated structural similarity between SidF and SidL. Enzymatic assays showed that SidF can utilize acetyl-CoA as a donor, previously thought to be a substrate of SidL but not SidF, and selectively uses N5-hydroxy-L-ornithine as an acceptor. Based on these findings, we propose SidF as the unknown transacetylase complementing SidL activity, highlighting its central role in A. fumigatus siderophore biosynthesis. This study elucidates the structure of SidF and reveals a novel role in siderophore biosynthesis. Investigation of this uncharacterized GNAT protein enhances our understanding of fungal virulence and holds promise for its potential application in developing antifungal therapies.

biochemistry↗

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

Cooperative DNA base and shape recognition by the CCAAT-binding complex and its bZIP transcription factor HapX

The heterotrimeric CCAAT-binding complex (CBC) is a master regulator of transcription. It specifically recognizes the CCAAT-box, a fundamental eukaryotic promoter element. Certain fungi, like Aspergilli, encode a fourth CBC-subunit, HapX, to fine-tune expression of genes involved in iron metabolism. Although being a basic region leucine zipper with its own DNA recognition motif, HapX function strictly relies on the CBC. We here report two crystal structures of the CBC-HapX complex bound to DNA duplexes with distinct sequence and position of HapX sites. In either structure, a HapX dimer targets the nucleic acid downstream of the CCAAT-box and the leash-like N-terminus of the distal HapX subunit interacts with CBC and DNA. In vitro and in vivo analyses of HapX mutants support the structures, highlight the complex as an exceptional major and minor groove DNA binder, and enrich our understanding of the functional as well as structural plasticity of related complexes across species.

biochemistry↗

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

Pou5f3 and Sox19b select gene expression repertoire at Zygotic Genome Activation

Awakening of zygotic transcription in animal embryos relies on maternal pioneer transcription factors. The interplay of global and specific functions of these proteins remains poorly understood. Here, we analyze chromatin accessibility and time-resolved transcription in single and double mutant zebrafish embryos lacking pluripotency factors Pou5f3 and Sox19b. We show that two factors modify chromatin in a largely independent manner. We distinguish four types of direct enhancers by differential requirements for Pou5f3 or Sox19b. We demonstrate that changes in chromatin accessibility of enhancers underlie the changes in zygotic expression repertoire in the double mutants. Pou5f3 or Sox19b promote chromatin accessibility of enhancers linked to the genes involved in gastrulation and ventral fate specification. The genes regulating mesendodermal and dorsal fates are primed for activation independently of Pou5f3 and Sox19b. Strikingly, simultaneous loss of Pou5f3 and Sox19b leads to premature expression of genes, involved in regulation of organogenesis and differentiation.

developmental biology↗

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↗