Biology subjects

Papagiannidis, D.

Publications and source records attributed to Papagiannidis, D..

8 recordsLinked to original sources

bioRxiv2026

Bacterial metabolism of synthetic steroids across ecosystems reveals diverse biotransformation products, reactions, and enzymes

Biotransformation of synthetic steroid drugs by gut and environmental bacteria affects their activity within the host, persistence, and environmental fate of these widely used pharmaceuticals. However, the spectrum of bacterial transformation of synthetic steroids, the enzymes involved, and the role of cooperative microbial metabolism in these processes remain poorly understood. We systematically mapped the biotransformation of 22 steroids, comprising 20 synthetic and 2 natural compounds and spanning clinically used estrogens, progestogens, corticosteroids, and prodrugs, across 12 bacterial species (8 intestinal and 4 environmental), yielding 264 steroid-bacteria combinations. We identified 97 unique biotransformation products and found that the tested bacteria catalyze diverse reactions including ester hydrolysis, oxidation-reduction chemistry, and steroid side-chain cleavage. Notably, the environmental bacterium Sphingobium herbicidovorans catalyzed desmolase-like steroid side-chain cleavage under aerobic conditions, a transformation previously reported solely for anaerobic bacteria. Combining sequence homology searches, a genetic gain-of-function screen, and expression proteomics we identified six steroid-transforming enzymes in S. herbicidovorans. We further demonstrate that distinct bacterial species cooperatively metabolize synthetic steroids through cross-feeding, enabling sequential activation and metabolism of corticosteroids across microbial communities. Together, our findings uncover previously unrecognized bacterial enzymes and community-level interactions involved in synthetic steroid metabolism. By directly linking steroid metabolites, enzymes, and microbial community interactions, this study provides molecular-level mechanistic insights into microbial steroid biotransformation. Such insights are essential for ultimately predicting metabolic interactions within and across microbial communities, as well as their interactions with the host and the environment.

microbiology↗

bioRxiv2026

ESCRT proteins organise stress-induced organelle contact between endoplasmic reticulum and Golgi

ESCRT proteins remodel membranes at many cell organelles, including the endoplasmic reticulum (ER). Here, we investigate whether ESCRTs in budding yeast participate in stress-induced ER reorganisation. We find that ER stress triggers the formation of tubular ER subdomains that recruit various ESCRT proteins. Recruitment of the major ESCRT-III protein Snf7 is mediated by the ESCRT-associated protein Bro1, a homologue of human ALIX, in a manner that is mechanistically distinct from Bro1 function at endosomes. ESCRT-containing ER subdomains are derived from ceramide-rich ER exit sites and form contacts with the Golgi. Furthermore, Bro1 helps to concentrate the tethering and lipid transfer protein Tcb3, a homologue of human extended synaptotagmins, at these organelle contacts and contributes to cellular fitness when lipid metabolism is perturbed. These results indicate that specialised ER exit sites can be repurposed for contacting the Golgi directly and uncover ESCRTs as organisers of stress-inducible ER-Golgi contacts that help maintain cell homeostasis.

cell biology↗

bioRxiv2026

Personalized microbiotas (counter-)select for antibiotic resistant strains

Antibiotic resistant pathogens are an increasing public health threat, as development of novel therapeutics is outpaced by resistance emergence and dissemination. Approaches to slow down or even revert antibiotic resistance are necessary to maintain efficacy of both existing and new antibiotics. Such approaches exploit the fitness cost of resistance elements, but have largely relied on assessing this cost in laboratory conditions that poorly reflect the native context in which pathogens reside. Here we present a method that allows to investigate the influence of personalized human gut microbiota compositions on the competitive fitness of antibiotic resistant pathogens. Using fecal matter-derived microbiomes we identified a specific community that selected for a carbapenem-resistant Klebsiella pneumoniae strain. This selective advantage was due to mutations arising in a LacI-type transcriptional regulator, GlyR, which upregulated expression of the downstream glycoporin GlyP, causing the effect. By deconvoluting the microbiome composition, we identified a focal E. coli strain as a central driver of the selection, which was further modulated by other microbiota members. We further demonstrate that the selective advantage was due to carbohydrate competition, and in particular for glycerol-containing compounds. Importantly, glyR mutations are under strong positive but conditional selection in clinical K. pneumoniae isolates. This implies a reduced competitiveness in other environments, which we experimentally validated in vitro. Overall, this study offers a path to identify microbiome-specific interactions that modulate the competitiveness of antibiotic resistant pathogens.

microbiology↗

bioRxiv2026

Multi-omics and biophysical phosphoproteomics upon BRAF inhibition uncover functional networks of BRAFV600E-driven signaling

Dysregulated kinase activity drives oncogenic signaling, disrupts cellular homeostasis, and promotes tumour progression. The BRAFV600E mutation constitutively activates the MAPK pathway and is a key therapeutic target in melanoma and other cancers, but the functional relevance of most downstream phosphorylation events and mechanisms of drug resistance remain unclear. To address this, a global multi-omic model of BRAF inhibition response was established in BRAFV600E-mutant cells by integrating time-resolved and biophysical phosphoproteomics, transcriptomics, and thermal proteome profiling. Ultradeep phosphoproteomics revealed extensive phosphorylation changes upon BRAF inhibitor treatment, while biophysical phosphoproteomics identified phosphorylation events linked to altered protein solubility and subcellular localization, suggesting changes in nucleic acid interactions and nuclear reorganisation. Network-based integration of these datasets prioritized functionally relevant phosphorylation sites and kinases. Experimental validation identified CDK9, CLK3, and TNIK as critical regulators of BRAFV600E signaling and candidate targets for combinatorial inhibition capable of re-sensitising resistant cells. The transcription factor ETV3 emerged as a previously unrecognised effector of BRAF signaling. Biophysical proteomics data confirmed that ETV3 phosphorylation modulates DNA-binding, while functional assays combining knockdown, metabolomics, and drug screening demonstrated its role in coordinating transcriptional and metabolic adaptations to BRAF inhibition. This study provides a systems-level framework linking phosphorylation dynamics to protein function and phenotype, identifies ETV3 as a new node in oncogenic BRAF signaling, and illustrates how integrated, site-resolved models can reveal mechanisms of kinase-driven oncogenesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=43 SRC="FIGDIR/small/704793v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@1df1401org.highwire.dtl.DTLVardef@9a77a5org.highwire.dtl.DTLVardef@124f819org.highwire.dtl.DTLVardef@1c6b57_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LITime- and cell type-resolved phosphoproteomics maps BRAF inhibition dynamics C_LIO_LIBiophysical phosphoproteomics, combining quantitative phosphoproteomics with solubility profiling or nuclear fractionation, reveals phosphorylation-driven changes of protein solubility and localization C_LIO_LIIntegration of abundance and biophysical phosphoproteomics data identifies functionally relevant phosphorylation events of BRAFV600E signaling C_LIO_LINetwork integration of multimodal phosphoproteomic, transcriptomic and thermal proteome profiling data links signaling to protein function and cellular phenotypes C_LIO_LIBiophysical evidence improves models and identifies non-canonical kinases driving BRAF signaling as well as novel downstream regulators such as ETV3 C_LIO_LIFollow-up experiments reveal a ETV3-GLUT3-mediated metabolic adaptation in BRAFV600E cells C_LI

systems biology↗

bioRxiv2024

Extensive PFAS accumulation by human gut bacteria

Per- and polyfluoroalkyl Substances (PFAS) - the so-called forever chemicals - are a major cause of environmental and health concern due to their toxicity and long-term persistence1,2. Yet, no efficient mechanisms for their removal have been identified. Here we report bioaccumulation of PFAS by several gut bacterial species over a wide range of concentrations from nanomolar up to 500 M. For bioaccumulating Bacteroides uniformis, a highly prevalent species, we estimate intracellular PFAS concentration in the mM range - above that of most native metabolites. Despite this high bioaccumulation, B. uniformis cells could grow appreciably up to 250 M perfluorononanoic acid (PFNA) exposure. Escherichia coli, which accumulated PFAS to a much lesser extent, substantially increased PFAS bioaccumulation when lacking TolC efflux pump indicating trans-membrane transport in PFAS bioaccumulation. Electron microscopy and cryogenic Focused Ion Beam-Secondary Ion Mass-spectrometry revealed distinct morphological changes and intracellular localisation of PFNA aggregates. Bioaccumulation of PFAS and transmembrane transport is also evident in proteomics, metabolomics, thermal proteome profiling, and mutations following adaptive laboratory evolution. In an in vivo context, mice colonized with human gut bacteria showed, compared to germ-free controls or those colonized with low-bioaccumulating bacteria, higher PFNA levels in excreted feces. As the gut microbiota is a critical interface between exposure and human body, our results have implications for understanding and utilizing microbial contribution to PFAS clearance.

microbiology↗

bioRxiv2024

RNA-binding proteins identified by R-DeeP/TripepSVM are involved in heterocyst differentiation

RNA-binding proteins (RBPs) are central components of gene regulatory networks. The differentiation of heterocysts in filamentous cyanobacteria is an example of cell differentiation in prokaryotes. Although multiple non-coding transcripts are involved in this process, no RBPs have been implicated thus far. Here we used quantitative mass spectrometry to analyze the differential fractionation of RNA-protein complexes after RNase treatment in density gradients yielding 333 RNA-associated proteins, while a bioinformatic prediction yielded 311 RBP candidates in Nostoc sp. PCC 7120. We validated in vivo the RNA-binding capacity of 6 RBP candidates. Some participate in essential physiological aspects, such as photosynthesis (Alr2890), thylakoid biogenesis (Vipp1) or heterocyst differentiation (PrpA, PatU3), but their association with RNA was unknown. Validated RBPs Asl3888 and Alr1700 were not previously characterized. Alr1700 is an RBP with two OB-fold domains that is differentially expressed in heterocysts. Deletion of alr1700 led to complete deregulation of the cell differentiation process, a striking increase in the number of heterocyst-like cells, and was ultimately lethal in the absence of combined nitrogen. These observations characterize this RBP as a master regulator of the heterocyst patterning and differentiation process, leading us to rename Alr1700 to PatR. The data can be accessed at https://sunshine.biologie.uni-freiburg.de/R-DeeP-Nostoc/.

molecular biology↗

bioRxiv2020

Control of endoplasmic reticulum membrane biogenesis by regulators of lipid metabolism

Cells dynamically adapt organelle size to current physiological demand. Organelle growth requires membrane biogenesis and therefore needs to be coordinated with lipid metabolism. The endoplasmic reticulum (ER) can undergo massive expansion, but the regulatory mechanisms that govern ER membrane biogenesis are largely unclear. Here, we conduct a genetic screen for factors involved in ER membrane expansion in budding yeast and identify the ER transmembrane protein Ice2 as a strong hit. We show that Ice2 promotes ER membrane biogenesis by opposing the phosphatidic acid phosphatase Pah1, which is called lipin in metazoa. Ice2 inhibits the conserved Nem1-Spo7 complex, which dephosphorylates and thus activates Pah1. Furthermore, Ice2 cooperates with the transcriptional regulation of lipid synthesis genes and helps to maintain ER homeostasis during ER stress. These findings establish inhibition of the lipin phosphatase complex as an important mechanism for controlling ER membrane biogenesis.

cell biology↗

bioRxiv2019

ESCRT machinery mediates selective microautophagy of endoplasmic reticulum

ER-phagy, the selective autophagy of endoplasmic reticulum (ER), safeguards organelle homeostasis by eliminating misfolded proteins and regulating ER size. ER-phagy can occur by macroautophagic and microautophagic mechanisms. While dedicated machinery for macro-ER-phagy has been discovered, the molecules and mechanisms mediating micro-ER-phagy remain unknown. Here, we first show that micro-ER-phagy in yeast involves the conversion of stacked cisternal ER into multilamellar ER whorls during microautophagic uptake into lysosomes. Second, we identify the conserved Nem1-Spo7 phosphatase complex and ESCRT proteins as key components for micro-ER-phagy. Third, we demonstrate that macro- and micro-ER-phagy are parallel pathways with distinct molecular requirements. Finally, we provide evidence that ESCRT proteins directly function in scission of the lysosomal membrane to complete the microautophagic uptake of ER. These findings establish a framework for a mechanistic understanding of micro-ER-phagy and, thus, a comprehensive appreciation of the role of autophagy in ER homeostasis.

cell biology↗