Biology subjects

Bilitewski, U.

Publications and source records attributed to Bilitewski, U..

5 recordsLinked to original sources

bioRxiv2025

Metabolic reprogramming of methylthioadenosine-dependent sulfur recycling is a major driver of CHIKV infection

The supply of key metabolites into viral replication compartments must be assured through a coordinated reprogramming of host metabolic pathways. For chikungunya virus (CHIKV), the cellular metabolites required for a successful infection are largely unknown. We show that CHIKV reprograms sulfur-dependent pathways. To maintain the resupply of thiols, the methionine (Met) salvage players, 5'-methylthioadenosine (MTA) and methionine adenosyltransferase-2a (Mat2a) are co-induced specifically. Under sulfur-depleted conditions, exogenously added MTA restores CHIKV replication more efficiently than its precursor S-adenosylmethionine, while inhibitions of Mat2a or de novo cysteine (Cys) biosynthesis reduce viral infectivity. Upon sulfur insufficiency CHIKV upregulates the U34-tRNA methyltransferase ALKBH8, and when ALKBH8 is deleted, virus replication is reduced by impairing sulfur relay, recapitulating Met-Cys deprivation effects. We found that MTA-mediated CHIKV replication occurs via m6A-independent priming, and that the S-adenosylhomocysteine hydrolase inhibitors DzNep and Adox, inhibited CHIKV replication with nanomolar potency. Our findings uncovered a major pro-CHIKV metabolic rheostat regulating tRNA modifications that can be targeted with host-directed antiviral agents.

microbiology↗

bioRxiv2024

Predicting antimicrobial class specificity of small molecules using machine learning

Whilst the useful armory of antibiotic drugs is continually depleted due to the emergence of drug-resistant pathogens, the development of novel therapeutics has also slowed down. In the era of advanced computational methods, approaches like machine learning (ML) could be one potential solution to help reduce the high costs and complexity of antibiotic drug discovery and attract collaboration across organizations. In our work, we developed a large antimicrobial knowledge graph (AntiMicrobial-KG) as a repository for collecting and visualizing public in-vitro antibacterial assay. Utilizing this data, we build ML models to efficiently scan compound libraries to identify compounds with the potential to exhibit antimicrobial activity. Our strategy involved training seven classic ML models across six compound fingerprint representations, of which the Random Forest trained on the MHFP6 fingerprint outperformed, demonstrating an accuracy of 75.9% and Cohens Kappa score of 0.68. Finally, we illustrated the models applicability for predicting the antimicrobial properties of two small molecule screening libraries. Firstly, the EU-OpenScreen library was tested against a panel of Gram-positive, Gram-negative, and Fungal pathogens. Here, we unveiled that the model was able to correctly predict more than 30% of active compounds for Gram-positive, Gram-negative, and Fungal pathogens. Secondly, with the Enamine library, a commercially available HTS compound collection with claimed antibacterial properties, we predicted its antimicrobial activity and pathogen class specificity. These results may provide a means for accelerating research in AMR drug discovery efforts by carefully filtering out compounds from commercial libraries with lower chances of being active.

bioinformatics↗

bioRxiv2021

Repurposing screen highlights broad-spectrum coronavirus antivirals and their host targets

Libraries composed of licensed drugs represent a vast repertoire of molecules modulating physiologic processes in humans, thus providing unique opportunities for discovery of host targeting antivirals. We interrogated the ReFRAME repurposing library with 12,993 molecules for broad-spectrum coronavirus antivirals and discovered 134 compounds inhibiting an alphacoronavirus, mapping to 59 molecular target categories. Dominant targets included the 5-hydroxytryptamine receptor and dopamine receptor and cyclin-dependent kinase inhibitors. Counter-screening with SARS-CoV-2 and validation in primary cells identified Phortress, an aryl hydrocarbon receptor (AHR) ligand, Bardoxolone and Omaveloxolone, two nuclear factor, erythroid 2 like 2 (NFE2L2) activators as inhibitors of both alpha- and betacoronaviruses. The landscape of coronavirus targeting molecules provides important information for the development of broad-spectrum antivirals reinforcing pandemic preparedness.

microbiology↗

bioRxiv2021

Identification of translocation inhibitors targeting the type III secretion system of enteropathogenic E. coli

Infections with enteropathogenic E. coli (EPEC) cause severe diarrhea in children. The non-invasive bacteria adhere to enterocytes of the small intestine and use a type III secretion system (T3SS) to inject effector proteins into host cells to modify and exploit cellular processes in favor of bacterial survival and replication. Several studies have shown that the T3SSs of bacterial pathogens are essential for virulence. Furthermore, the loss of T3SS-mediated effector translocation results in increased immune recognition and clearance of the bacteria. The T3SS is, therefore, considered a promising target for antivirulence strategies and novel therapeutics development. Here, we report the results of a high-throughput screening assay based on the translocation of the EPEC effector protein Tir. Using this assay, we screened more than 13,000 small molecular compounds of six different compound libraries and identified three substances which showed a significant dose-dependent effect on translocation without adverse effects on bacterial or eukaryotic cell viability. Additionally, these substances reduced bacterial binding to host cells, effector-dependent cell detachment and abolished A/E lesion formation without affecting the expression of components of the T3SS or associated effector proteins. Moreover, no effects of the inhibitors on bacterial motility or Shiga-toxin expression were observed. In summary, we have identified three new compounds that strongly inhibit T3SS-mediated translocation of effectors into mammalian cells, which could be valuable as lead substances for treating EPEC and EHEC infections.

microbiology↗

bioRxiv2019

A NanoLuc luciferase-based assay enabling the real-time analysis of protein secretion and injection by bacterial type III secretion systems

The elucidation of the molecular mechanisms of secretion through bacterial protein secretion systems is impeded by a lack of assays to quantitatively assess secretion kinetics. Also the analysis of the biological role of these secretion systems as well as the identification of inhibitors targeting these systems would greatly benefit from the availability of a simple, quick and quantitative assay to monitor principle secretion and injection into host cells. Here we present a versatile solution to this need, utilizing the small and very bright NanoLuc luciferase to assess secretion and injection through the type III secretion system encoded by Salmonella pathogenicity island 1. The NanoLuc-based secretion assay features a very high signal-to-noise ratio and sensitivity down to the nanoliter scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384-well microplates. We further developed NanoLuc and split-NanoLuc-based assays that enable the monitoring of type III secretion-dependent injection of effector proteins into host cells.\n\nImportanceThe ability to secrete proteins to the bacterial cell surface, to the extracellular environment, or even into target cells is one of the foundations of interbacterial as well as pathogen-host interaction. While great progress has been made in elucidating assembly and structure of secretion systems, our understanding of their secretion mechanism often lags behind, not last because of the challenge to quantitatively assess secretion function. Here, we developed a luciferase-based assay to enable the simple, quick, quantitative, and high throughput-compatible assessment of secretion and injection through virulence-associated type III secretion systems. The assay allows detection of minute amounts of secreted substrate proteins either in the supernatant of the bacterial culture or within eukaryotic host cells. It thus provides an enabling technology to elucidate the mechanisms of secretion and injection of type III secretion systems and is likely adaptable to assay secretion through other bacterial secretion systems.

microbiology↗