Biology subjects

Lehmann, A.

Publications and source records attributed to Lehmann, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Human metabolites modulate antifungal efficacy and reveal creatinine-mediated antagonism of flucytosine

Clearing fungal infections can fail, even when the causative pathogen is susceptible to antifungal drugs in vitro. The reasons for this discrepancy often remain unclear. Here, we report that human metabolites affect antifungal drug efficacy. Studying Saccharomyces cerevisiae, we observed that human serum and common growth supplements alter the minimal inhibitory concentrations (MIC) across major antifungal classes, including fluconazole (FLC) and flucytosine (5-FC). To identify causal metabolites, we generated a library reflecting the physiological concentrations of 67 abundant blood metabolites. Assessing their impact on fungal fitness in the presence of antifungals revealed that creatinine, owing to its structural similarity to 5-FC, reduced the uptake and efficacy of 5-FC in S. cerevisiae, Candida albicans, and Nakaseomyces glabratus. Notably, 5-FC efficacy in urinary tract infections can be limited, and creatinine is highly concentrated in urine. We show that creatinine levels in urine increase the MIC of 5-FC in N. glabratus by approximately 100-fold, while reducing creatinine concentration in urine enhances 5-FC efficacy. Together, our findings highlight that drug interactions with creatinine and other human metabolites can strongly influence the activity of antifungals.

biochemistry↗

bioRxiv2025

Oncogene Silencing via ecDNA Micronucleation

Extrachromosomal DNA (ecDNA) contributes to cancer genome instability by enabling high-copy oncogene amplification, intratumoural heterogeneity and rapid genetic change. Micronuclei (MN) are frequently observed in chromosomally unstable cancers, yet their origins and relevance in ecDNA-driven tumours remain incompletely understood. Here, we investigate the relationship between ecDNA segregation errors and MN formation. We find that ecDNA frequently localizes to MN and represents a prominent source of MN content in ecDNA-positive cancer cells. Mitotic clustering of oncogene-bearing ecDNAs is associated with asymmetric inheritance and mis-segregation into MN. Transfer of oncogenes from ecDNA into MN is accompanied by reduced transcriptional output. Single-MN sequencing shows that individual MN are enriched for ecDNA to a degree that exceeds expectations from stochastic mis-segregation and that in MN oncogenes originating from multiple distinct ecDNAs coalesce. Using live-cell imaging, we observe that cells inheriting ecDNA-positive MN show limited proliferative capacity and an increased likelihood of cell death. In neuroblastoma patients with MYCN-amplified ecDNA, higher frequencies of ecDNA-positive MN at diagnosis are associated with improved event-free and overall survival. Together, these findings link ecDNA mis-segregation to MN formation and reduced cellular fitness, suggesting that ecDNA-positive MN may reflect a state of impaired oncogenic ecDNA function with potential relevance for clinical outcome.

cancer biology↗

bioRxiv2025

Sequencing by Expansion (SBX) -- a novel, high-throughput single-molecule sequencing technology

Remarkable advances in high-throughput sequencing have enabled major biological discoveries and clinical applications, but achieving wider distribution and use depends critically on further improvements in scale and cost reduction. Nanopore sequencing has long held the promise for such progress, but has had limited market penetration. This is because efficient and accurate nanopore sequencing of nucleic acids has been challenged by fundamental signal-to-noise limitations resulting from the poor spatial resolution and molecular distinction of nucleobases. Here, we describe Sequencing by Expansion (SBX), a single-molecule sequencing technology that overcomes these limitations by using a biochemical conversion process to encode the sequence of a target nucleic acid molecule into an Xpandomer, a highly measurable surrogate polymer. Expanding over 50 times longer than the parent DNA templates, Xpandomers are engineered with high signal-to-noise reporter codes to enable facile, high-accuracy nanopore sequencing. We demonstrate the performance of SBX and present the specialized molecular structures, chemistries, enzymes and methods that enable it. The innovative molecular and systems engineering in SBX create a transformative technology to address the needs of existing and emerging sequencing applications.

genomics↗

bioRxiv2025

Vertical RAS-pathway inhibition in pancreatic cancer drives therapeutically exploitable mitochondrial alterations

Background & AimsOncogenic KRAS mutations drive metabolic rewiring in pancreatic ductal adenocarcinoma (PDAC). Src-homology 2 domain-containing phosphatase 2 (SHP2) is essential for full KRAS activity and promising dual SHP2/mitogen-activated protein kinase (MAPK) inhibition is currently being tested in clinical trials. Exploitable metabolic adaptations may contribute to an invariably evolving resistance. MethodsTo understand the metabolic changes induced by dual inhibition, we comprehensively tested cell lines, endogenous tumor models, and patient-derived organoids representing the full spectrum of PDAC molecular subtypes. ResultsWe find that dual SHP2/mitogen-activated protein kinase kinase (MEK1/2) inhibition induces major mitochondrial alterations, elevates reactive oxygen species (ROS) levels and triggers a lipid peroxidase dependency. While anabolic pathways, glycolysis and autophagy were also affected, mitochondrial alterations persisted longterm into a therapy resistant state. ConclusionsThe resulting vulnerability to induction of ferroptotic cell death via combined SHP2/MEK1/2 and glutathione peroxidase (GPX4) inhibition provides a metabolic lever to reinforce RAS-pathway inhibition for targeted PDAC treatment.

cancer biology↗

bioRxiv2024

Psilocybin prevents habituation to familiar stimuli and preserves sensitivity to sound following repeated stimulation in mouse primary auditory cortex.

Psilocybin, a psychoactive substance derived from fungi, has been utilized historically by diverse cultures for both medicinal and non-medicinal purposes, owing to its ability to elicit profound sensory and cognitive alterations and sustain long-term changes in mood and cognition. Promising results from recent clinical studies have generated a wave of interest in employing psilocybin to treat neuropsychiatric and neuro-degenerative conditions. How psychedelics cause acute perceptual effects, and how these relate to long-lasting alterations is still debated. Whereas it is thought that perceptual disturbances may be caused by disrupted flow of information between sensory and higher order areas, in vivo studies have focused mostly on the latter. In particular, there has been little study of how psilocybin affects sensory representations in primary auditory cortex (A1). We used two-photon microscopy and wide field calcium imaging to examine how psilocybin affects A1 neuron response properties in the mouse. Administration of 1 mg/kg psilocybin prevented habituation of sound-evoked responses to repeated stimuli, maintaining overall responsiveness, bandwidth, and sound-level response thresholds after repeated stimulation. This was in contrast to marked habituation of responses and narrowing of tuning in controls. We observed no effect on overall distribution of best frequencies at the cortical level, suggesting psilocybin in A1 disrupts normal sensory gating, rather than tonotopic organization. This supports models of psychedelic action in which perceptual disturbances are driven by disrupted hierarchical sensory gating. With further research, influences of psychedelics on sensory representations could be harnessed to target maladaptive sensory processing in conditions such as tinnitus. Significance StatementDespite its role in altering auditory sensory perception, the impact of psilocybin on modulating neuronal activity in the auditory cortex remains understudied. This study is the first to identify an inhibition of normal auditory habituation to repeated stimuli with single-neuron resolution. We identify a role for psilocybin in the targeted, context-dependent modulation of auditory sensory neural tuning properties, which may help to explain how disruption of hierarchical control of sensory representations leads to perceptual disturbances. With further work, this influence on sensory representations could be used to target conditions where maladaptive sensory processing leads to deleterious health outcomes.

neuroscience↗

bioRxiv2023

Surfactant-mediated effects on hydrological and physical soil properties: a data synthesis

Soils are under threat of a multitude of anthropogenic factors affecting the complex interplay of various physical and hydrological soil processes and properties. One such factor is the group of surface-active compounds. Surfactants have a broad range of applications, and can reduce solid-liquid interfacial forces and increase wettability and dispersion of particles. Surfactant effects are context-dependent, giving rise to a wide range of reported effects on different soil processes and properties. Here, we evaluate the evidence base of surfactant research on 11 hydrological and physical soil variables. Our goal was to identify knowledge gaps and to test the robustness of proposed surfactant effects. We found that the current knowledge base is insufficient to reach strong data-backed conclusions about effects of surfactants in soils. We identified a unique case of bias in the data as a result of conflated patterns of lab and field studies. We could not support the hypothesis that surfactant charge determines soil effects for any of the tested soil variables. We believe that further experiments on surfactant mediated effects on soil properties and processes are urgently required, paying attention in particular to improving experimental design and data reporting standards. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/547370v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@13e7953org.highwire.dtl.DTLVardef@8f5fc3org.highwire.dtl.DTLVardef@d81c52org.highwire.dtl.DTLVardef@2b4dfa_HPS_FORMAT_FIGEXP M_FIG C_FIG

ecology↗

bioRxiv2022

Fungal traits help to understand the decomposition of simple and complex plant litter

Litter decomposition is a key ecosystem process, responsible for the release and storage of nutrients and carbon. Soil fungi are one of the dominant drivers of organic matter decomposition, but fungal taxa differ substantially in their functional ability to decompose plant litter. We used a trait-based approach to better understand functional differences among saprotrophic soil fungi (originating from a natural grassland ecosystem) in decomposing leaf and wood litter. Decomposition strongly varied among phyla and isolates, with Ascomycota decomposing the most and Mucoromycota decomposing the least. In this study, the phylogeny of the fungi in our dataset, but also the ability of fungi to use more complex carbon were important predictors for decomposition. While some enzymes (e.g. laccase and cellulase) influenced decomposition, the majority of enzyme activities was not correlated with decomposition. Thus, we suggest using more directly assessed traits as predictors for decomposition, such as the ability to use carbon substrates, rather than a single enzyme activity, which could misrepresent the degradation potential of certain isolates. The findings of our study offer important new insights for the trait-based prediction of fungal litter decomposition in grassland soils.

ecology↗

bioRxiv2022

Analysis of Skin Cancers from Xeroderma Pigmentosum Patients Reveals Heterogeneous UV-Induced Mutational Profiles Shaped by DNA Repair

Xeroderma pigmentosum (XP) is a genetic disorder caused by mutations in genes of the Nucleotide Excision Repair (NER) pathway (groups A-G) or in Translesion Synthesis (TLS) DNA polymerase {eta} (V). XP is associated with an increased skin cancer risk, reaching, for some groups, several thousand-fold compared to the general population. Here, we analyzed 38 skin cancer genomes from five XP groups. We found that the activity of NER determines heterogeneity of the mutation rates across skin cancer genomes and that transcription-coupled NER extends beyond the gene boundaries reducing the intergenic mutation rate. Mutational profile in XP-V tumors and experiments with POLH-KO cell line revealed the role of polymerase {eta} in the error-free bypass of (i) rare TpG and TpA DNA lesions, (ii) 3 nucleotides in pyrimidine dimers, and (iii) TpT photodimers. Our study unravels the genetic basis of skin cancer risk in XP and provides insights into the mechanisms reducing UV-induced mutagenesis in the general population.

cancer biology↗

bioRxiv2022

PI3K/AKT signaling allows for MAPK/ERK pathway independency mediating dedifferentiation-driven treatment resistance in melanoma

Current therapeutic management of advanced melanoma patients largely depends on their BRAF mutation status. However, the vast heterogeneity of the tumors hampers the success of therapies targeting the MAPK/ERK pathway alone. Dissecting this heterogeneity will contribute to identifying key players in the oncogenic progression to tailor more effective therapies. We performed a comprehensive molecular and phenotypic characterization of a panel of patient-derived BRAFV600E-positive melanoma cell lines. Transcriptional profiling was used to identify groups of coregulated genes whose expression relates to an increased migratory potential and a higher resistance. A decrease in sensitivity to MAPK/ERK pathway inhibition with vemurafenib or trametinib corresponded with an increasing quiescence and migratory properties of the cells. This was accompanied by the loss of transcriptional signatures of melanocytic differentiation, and the gain of stem cell features that conferred highly-resistant/mesenchymal-like cells with increased xenobiotic efflux capacity. Nevertheless, targeting of the implicated ABC transporters did not improve the response to vemurafenib, indicating that incomplete BRAF inhibition due to reduced drug uptake is not a main driver of resistance. Rather, indifference to MAPK/ERK pathway inhibition arose from the activation of compensatory signaling cascades. The PI3K/AKT pathway in particular showed a higher activity in mesenchymal-like cells, conferring a lower dependency on MAPK/ERK signaling and supporting stem-like properties that could be reverted by dual PI3K/mTOR inhibition with dactolisib. In case of MAPK/ERK independency, therapeutic focus may be shifted to the PI3K/AKT pathway to overcome late-stage resistance in melanoma tumors that have acquired a mesenchymal phenotype.

cancer biology↗

bioRxiv2022

The natural diversity of the yeast proteome reveals chromosome-wide dosage compensation in aneuploids

Aneuploidy, an imbalance in chromosome copy numbers, causes genetic disorders, and drives cancer progression, drug tolerance, and antimicrobial resistance. While aneuploidy can confer stress resistance, it is not well understood how cells overcome the fitness burden caused by aberrant chromosomal copy numbers. Studies using both systematically generated 1-5 and natural aneuploid yeasts 6-8 triggered an intense debate about the role of dosage compensation, concluding that aneuploidy is transmitted to the transcriptome and proteome without significant buffering at the chromosome-wide level, and is, at least in lab strains, associated with significant fitness costs. Conversely, systematic sequencing and phenotyping of large collections of natural isolates revealed that aneuploidy is frequent and has few - if any - fitness costs in nature 9. To address these discrepant findings at the proteomic level, we developed a platform that yields highly precise proteomic measurements across large numbers of genetically diverse samples, and applied it to natural isolates collected as part of the 1011 genomes project 9. For 613 of the isolates, we were able to match the proteomes to their corresponding transcriptomes and genomes, subsequently quantifying the effect of aneuploidy on gene expression by comparing 95 aneuploid with 518 euploid strains. We find, as in previous studies, that aneuploid gene dosage is not buffered chromosome-wide at the transcriptome level. Importantly, in the proteome, we detect an attenuation of aneuploidy by about 25% below the aneuploid gene dosage in natural yeast isolates. Furthermore, this chromosome-wide dosage compensation is associated with the ubiquitin-proteasome system (UPS), which is expressed at higher levels and has increased activity across natural aneuploid strains. Thus, through systematic exploration of the species-wide diversity of the yeast proteome, we shed light on a long-standing debate about the biology of aneuploids, revealing that aneuploidy tolerance is mediated through chromosome-wide dosage compensation at the proteome level.

systems biology↗

bioRxiv2022

Inorganic sulfur fixation via a new homocysteine synthase allows yeast cells to cooperatively compensate for methionine auxotrophy

The assimilation, incorporation, and metabolism of sulfur is a fundamental process across all domains of life, yet how cells deal with varying sulfur availability is not well understood. We studied an unresolved conundrum of sulfur fixation in yeast, in which an organosulfur-auxotrophy caused by deletion of homocysteine synthase Met17p is overcome when cells are inoculated at high cell density. We discovered that an uncharacterized gene YLL058Wp, herein named Hydrogen sulfide utilizing-1 (HSU1), acts as a homocysteine synthase and allows the cells to substitute for Met17p by re-assimilating hydrosulfide ions leaked from met17{Delta} cells into O-acetyl-homoserine and forming homocysteine. Our results show that cells can cooperate to achieve sulfur fixation, indicating that the collective properties of microbial communities facilitate their basic metabolic capacity. SummarySulfur limitation activates a dormant hydrogen sulfide fixation route via a novel homocysteine synthase Hsu1p (YLL058Wp).

molecular biology↗

bioRxiv2021

Effects of perfluoroalkyl and polyfluoroalkyl substances on soil structure and function

Soils are impacted at a global scale by several anthropogenic factors, including chemical pollutants. Among those, perfluoroalkyl and polyfluoroalkyl substances (PFAS) are of concern due to their high environmental persistence, and as they might affect soil health and functions. However, data on impacts of PFASs on soil structure and microbially-driven processes are currently lacking. This study explored the effects of perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA) and perfluorobutanesulfonic acid (PFBS) at environmental-relevant nominal concentrations (1 ~ 1000 ng g-1) on soil functions, using a 6-week microcosm experiment. We measured soil respiration, litter decomposition, enzyme and microbial activities, soil aggregates, and bacterial abundance. PFAS (even at 1 ng g-1 for PFBS) significantly increased litter decomposition, associated with positive effects on bacterial abundance, and {beta}-glucosidase activities. This effect increased with PFAS concentrations. Soil respiration was significantly inhibited by PFAS in the 3rd week, while this effect was more variable in week 6. Water-stable aggregates were negatively affected by PFOS and PFOA, possibly related to microbial shifts. The general microbial activities and {beta}-D-cellobiosidase and phosphatase activities were barely affected by PFAS treatments. Our work highlights the potential effects of PFAS on soil health, and we argue that this substance class could be a factor of environmental change of potentially broad relevance in terrestrial ecosystem functioning. SynopsisPFAS are likely to affect soil health. Abstract Art O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/465889v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@88c630org.highwire.dtl.DTLVardef@18b985corg.highwire.dtl.DTLVardef@1bfdc81org.highwire.dtl.DTLVardef@125b09e_HPS_FORMAT_FIGEXP M_FIG C_FIG

ecology↗

bioRxiv2020

Single-cell RNA sequencing reveals distinct tumor microenvironmental patterns in lung adenocarcinoma

Recent developments in immuno-oncology demonstrate that not only cancer cells, but also features of the tumor microenvironment guide precision medicine. Still, the relationship between tumor and microenvironment remains poorly understood. To overcome this limitation and identify clinically relevant microenvironmental and cancer features, we applied single-cell RNA sequencing to lung adenocarcinomas. While the highly heterogeneous carcinoma cell transcriptomes reflected histological grade and activity of relevant oncogenic pathways, our analysis revealed two distinct microenvironmental patterns. We identified a prognostically unfavorable group of tumors with a microenvironment composed of cancer-associated myofibroblasts, exhausted CD8+ T cells, proinflammatory monocyte-derived macrophages and plasmacytoid dendritic cells (CEP2 pattern) and a prognostically favorable group characterized by myeloid dendritic cells, anti-inflammatory monocyte-derived macrophages, normal-like myofibroblasts, NK cells and conventional T cells (MAN2C pattern). Our results show that single-cell gene expression profiling allows to identify patient subgroups based on the tumor microenvironment beyond cancer cell-centric profiling.

cancer biology↗

bioRxiv2020

Deconvoluting the T cell response to SARS-CoV-2: specificity versus chance- and cognate cross-reactivity

SARS-CoV-2 infection takes a mild or clinically inapparent course in the majority of humans who contract this virus. After such individuals have cleared the virus, only the detection of SARS-CoV-2-specific immunological memory can reveal the exposure, and hopefully the establishment of immune protection. With most viral infections, the presence of specific serum antibodies has provided a reliable biomarker for the exposure to the virus of interest. SARS-CoV-2 infection, however, does not reliably induce a durable antibody response, especially in sub-clinically infected individuals. Consequently, it is plausible for a recently infected individual to yield a false negative result within only a few months after exposure. Immunodiagnostic attention has therefore shifted to studies of specific T cell memory to SARS-CoV-2. Most reports published so far agree that a T cell response is engaged during SARS-CoV-2 infection, but they also state that in 20-81% of non-SARS-CoV-2-exposed individuals, T cells respond to SARS-CoV-2 antigens (mega peptide pools), allegedly due to T cell cross-reactivity with coronaviruses causing Common Cold (CCC), or other antigens. Here we show that by introducing irrelevant mega peptide pools as negative controls to account for chance cross-reactivity, and by establishing the antigen dose-response characteristic of the T cells, one can clearly discern between cognate T cell memory induced by SARS-CoV-2 infection vs. cross-reactive T cell responses in individuals who had not been infected with SARS-CoV-2.

immunology↗

bioRxiv2020

Microplastic fibers affect dynamics and intensity of CO2 and N2O fluxes from soil differently

Microplastics may affect soil ecosystem functioning in critical ways, with previously documented effects including changes in soil structure and water dynamics; this suggests that microbial populations and the processes they mediate could also be affected. Given the importance for global carbon and nitrogen cycle and greenhouse warming potential, we here experimentally examined potential effects of plastic microfiber additions on CO2 and N2O greenhouse gas fluxes. We carried out a fully factorial laboratory experiment with the factors presence of microplastic fibers (0.4% w/w) and addition of urea fertilizer (100 mg N kg-1). The conditions in an intensively N-fertilized arable soil were simulated by adding biogas digestate at the beginning of the incubation to all samples. We continuously monitored CO2 and N2O emissions from soil before and after urea application using a custom-built flow-through steady-state system, and we assessed soil properties, including soil structure. Microplastics affected soil properties, notably increasing soil aggregate water-stability and pneumatic conductivity, and caused changes in the dynamics and overall level of emission of both gases, but in opposite directions: overall fluxes of CO2 were increased by microplastic presence, whereas N2O emission were decreased, a pattern that was intensified following urea addition. This divergent response is explained by effects of microplastic on soil structure, with the increased air permeability likely improving O2 supply: this will have stimulated CO2 production, since mineralization benefits from better aeration. Increased O2 would at the same time have inhibited denitrification, a process contributing to N2O emissions, thus likely explaining the decrease in the latter. Our results clearly suggest that microplastic consequences for greenhouse gas emissions should become an integral part of future impact assessments, and that to understand such responses, soil structure should be assessed.

ecology↗

bioRxiv2020

Highly potent anti-SARS-CoV-2 multi-DARPin therapeutic candidates

Globally accessible therapeutics against SARS-CoV-2 are urgently needed. Here, we report the generation of the first anti-SARS-CoV-2 DARPin molecules with therapeutic potential as well as rapid large-scale production capabilities. Highly potent multivalent DARPin molecules with low picomolar virus neutralization efficacies were generated by molecular linkage of three different monovalent DARPin molecules. These multivalent DARPin molecules target various domains of the SARS-CoV-2 spike protein, thereby limiting possible viral escape. Cryo-EM analysis of individual monovalent DARPin molecules provided structural explanations for the mode of action. Analysis of the protective efficacy of one multivalent DARPin molecule in a hamster SARS-CoV-2 infection model demonstrated a significant reduction of pathogenesis. Taken together, the multivalent DARPin molecules reported here, one of which has entered clinical studies, constitute promising therapeutics against the COVID-19 pandemic.

immunology↗

bioRxiv2020

Microplastic shape, concentration and polymer type affect soil properties and plant biomass

Microplastics are an increasing concern in terrestrial systems. These particles can be incorporated into the soil in a wide range of shapes and polymers, reflecting the fact that manufacturers produce plastics in a variety of physical and chemical properties matching their intended use.Despite of this, little is known about the effects that the addition into the soil of microplastics of different shapes, polymer type and concentration levels may have on soil properties and plant performance. To fill this gap, we selected four microplastic shapes: fibers, films, foams and fragments; and for each shape we selected three microplastics made of one of the following polymers: polyester, polyamide, polypropylene, polyethylene, polyethylenterephthalat, polyurethane, polystyrene and polycarbonate. In a glasshouse experiment, each microplastic was added to a soil from a dry grassland at a concentration of 0.1%, 0.2%, 0.3% and 0,4%. A carrot (Daucus carota) plant grew in each pot during four weeks. At harvest, shoot and root mass, soil aggregation and microbial activity were measured. Our results showed that all microplastic shapes increased shoot and root masses. As concentration increased, microfibers increased plant biomass probably as fibers may hold water in the soil for longer. In contrast, microfilms decreased biomass with concentration, likely because they can create channels in the soil that promote water evaporation affecting plant performance. All microplastic shapes decreased soil aggregation, probably since microplastics may introduce fracture points in the aggregates affecting their stability and also due to potential negative effects on soil biota. The latter may also explain the decrease in microbial activity with, for example, polyethylene films. Similar to plant biomass, microfilms decreased soil aggregation with increasing concentration. Our study tested the microplastic shape mediation and dissimilarity hypotheses, highlighting the importance of microplastic shape, polymer type and concentration when studying the effects of microplastics on terrestrial systems.

ecology↗

bioRxiv2020

The Gb3-enriched CD59/flotillin plasma membrane domain regulates host cell invasion by Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa is responsible for a high number of acute and chronic hospital-acquired infections. As it develops more and more resistances against existing antibiotics, P. aeruginosa has been placed highest on the global priority list of antibiotic-resistant bacteria for which alternative treatments are urgently needed. Former studies have highlighted the crucial role of the bacterial lectin LecA and the host cell glycosphingolipid globotriaosylceramide (Gb3) for the cellular uptake of P. aeruginosa into epithelial cells via the lipid zipper mechanism. To further characterize the host cell plasma membrane domain for LecA-driven attachment and invasion, we analyzed the protein and lipid composition of pulled-down membrane domains for novel interaction partners of LecA by mass spectrometry. We unraveled a predilection of LecA for binding to saturated Gb3 species in the extracellular membrane leaflet and an induction of dynamic phosphatidylinositol (3,4,5)-trisphosphate clusters at the intracellular leaflet co-localizing with sites of LecA binding. Moreover, we identified the GPI-anchored protein CD59 and flotillins, known as cargo and eponymous component of flotillin-assisted endocytosis, as LecA interaction partners. Depletion of each of these host cell proteins resulted in more than 50% of reduction in invasiveness of the P. aeruginosa strain PAO1 highlighting the importance of this LecA-induced plasma membrane domain. Our strategy to reduce the complexity of host-pathogen interactions by first identifying interaction partners of a single virulence factor and subsequently transferring these findings to the bacterium has been proven to be a successful approach in elucidating the molecular mechanisms of bacterial infections.

cell biology↗