Biology subjects

Rupp, B.

Publications and source records attributed to Rupp, B..

3 recordsLinked to original sources

bioRxiv2024

Polybacterial Intracellular Macromolecules Shape Single-Cell Epikine Profiles in Upper Airway Mucosa

The upper airway, particularly the nasal and oral mucosal epithelium, serves as a primary barrier for microbial interactions throughout life. Specialized niches like the anterior nares and the tooth are especially susceptible to dysbiosis and chronic inflammatory diseases. To investigate host-microbial interactions in mucosal epithelial cell types, we reanalyzed our single-cell RNA sequencing atlas of human oral mucosa, identifying polybacterial signatures (20% Gram-positive, 80% Gram-negative) within both epithelial- and stromal-resident cells. This analysis revealed unique responses of bacterial-associated epithelia when compared to two inflammatory disease states of mucosa. Single-cell RNA sequencing, in situ hybridization, and immunohistochemistry detected numerous persistent macromolecules from Gram-positive and Gram-negative bacteria within human oral keratinocytes (HOKs), including bacterial rRNA, mRNA and glycolipids. Epithelial cells with higher concentrations of 16S rRNA and glycolipids exhibited enhanced receptor-ligand signaling in vivo. HOKs with a spectrum of polybacterial intracellular macromolecular (PIM) concentrations were challenged with purified exogenous lipopolysaccharide, resulting in the synergistic upregulation of select innate (CXCL8, TNFSF15) and adaptive (CXCL17, CCL28) epikines. Notably, endogenous lipoteichoic acid, rather than lipopolysaccharide, directly correlated with epikine expression in vitro and in vivo. Application of the Drug2Cell algorithm to health and inflammatory disease data suggested altered drug efficacy predictions based on PIM detection. Our findings demonstrate that PIMs persist within mucosal epithelial cells at variable concentrations, linearly driving single-cell effector cytokine expression and influencing drug responses, underscoring the importance of understanding host-microbe interactions and the implications of PIMs on cell behavior in health and disease at single-cell resolution. One-sentence summaryThis study reveals how persistent intracellular bacterial macromolecules in mucosal epithelial cells drive inflammatory signaling, offering new insights into microbial-host interactions and their potential impact on inflammatory disease treatment and drug efficacy.

cell biology↗

bioRxiv2023

Identification of key residues in MERS-CoV and SARS-CoV-2 main proteases for resistance against clinically applied inhibitors nirmatrelvir and ensitrelvir

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an epidemic, zoonotically emerging pathogen initially reported in Saudi Arabia in 2012. MERS-CoV has the potential to mutate or recombine with other coronaviruses, thus acquiring the ability to efficiently spread among humans and become pandemic. Its high mortality rate of up to 35 % and the absence of effective targeted therapies call for the development of antiviral drugs for this pathogen. Since the beginning of the SARS-CoV-2 pandemic, extensive research has focused on identifying protease inhibitors for the treatment of SARS-CoV-2. Our intention was therefore to assess whether these protease inhibitors are viable options for combating MERS-CoV. To that end, we used previously established protease assays to quantify inhibition of the SARS-CoV-2 and MERS-CoV main proteases. Furthermore, we selected MERS-CoV-Mpro mutants resistant against nirmatrelvir, the most effective inhibitor of this protease, with a safe, surrogate virus-based system, and suggest putative resistance mechanisms. Notably, nirmatrelvir demonstrated effectiveness against various viral proteases, illustrating its potential as a broad-spectrum coronavirus inhibitor. To adress the inherent resistance of MERS-CoV-Mpro to ensitrelvir, we applied directed mutagenesis to a key ensitrelvir-interacting residue and provided structural models. One-Sentence SummaryWe investigate antivirals for MERS-CoV with a pool of SARS-CoV-2 antiviral drugs and study potential resistances developing against those drugs.

molecular biology↗

bioRxiv2019

Controlled dehydration, structural flexibility, and Gadolinium MRI contrast compound binding in human plasma glycoprotein afamin

Afamin, a human blood plasma glycoprotein, a putative multi-functional transporter of hydrophobic molecules and a marker for metabolic syndrome, poses multiple challenges for crystallographic structure determination, both practically and in analysis of the models. Several hundred crystals were analysed, and unusual variability in cell volume and difficulty solving the structure despite a ~34% sequence identity with non-glycosylated human serum albumin indicated that the molecule exhibits variable and context-sensitive packing, despite greatly simplified glycosylation in insect cell expressed recombinant afamin. Controlled dehydration of the crystals was able to stabilise the orthorhombic crystal form reducing the number of molecules in the asymmetric unit from the monoclinic form and changing the conformational states of the protein. An iterative strategy, using fully automatic experiments available on MASSIF-1, was used to quickly determine the optimal protocol to achieve the phase transition that should be readily applicable to many types of sample. The study also highlights the drawback of using a single crystallographic structure model for computation modelling purposes given that conformational state of the binding sites and electron density in the binding site, likely resulting from PEGs, greatly varies between models. This also holds for the analysis of unspecific low-affinity ligands, where often a variety fragments with similar uncertainty can be modelled, inviting interpretative bias. As a promiscuous transporter, afamin also seems to bind Gadoteridol, a magnetic resonance imaging contrast compound, in at least two sites. One pair of Gadoteridol molecules is located near the human albumin Sudlow-site, and a second Gadoteridol at an intermolecular site in proximity of domain IA. The data from the co-crystals provide an opportunity to evaluate modern metrics of data quality in the context of the information that can be gleaned from data sets that would be abandoned on classical measures.\n\nSynopsisControlled dehydration experiments have revealed a new crystal form of afamin, a human blood plasma glycoprotein and transporter of hydrophobic molecules. The comparison shows substantial molecular plasticity and amplifies the necessity to examine multiple crystal forms and to refine multiple models, while at the same time the new structure cautions against interpretation of fatty acid ligand density in crystals derived from PEG as major precipitants. An isomorphic low-resolution structure model suggests that afamin is capable of transporting Gadolinium-DO3A, a magnetic resonance imaging compound.

biochemistry↗