Biology subjects

Hedberg, C.

Publications and source records attributed to Hedberg, C..

5 recordsLinked to original sources

bioRxiv2026

The Legionella phosphocholinase AnkX modulates IMPDH2 regulation and cytoophidia dynamics

Legionella pneumophila, the causative agent of Legionnaires disease, secretes more than 300 different effector proteins into the host cell to modulate processes such as signal transduction, membrane dynamics, and metabolism. A key regulator of cellular metabolism and proliferation is inosine 5-monophosphate dehydrogenase type II (IMPDH2), which catalyzes the rate-limiting step of de novo GTP biosynthesis. IMPDH2 assembles into filaments and larger structures known as cytoophidia, which enable fine-tuned regulation of enzymatic activity in response to changing nucleotide demands. Here, we identify human IMPDH2 as a previously unrecognized host target of the Legionella effector AnkX. We show that AnkX post-translationally modifies IMPDH2 within its regulatory domain by covalently attaching a phosphocholine moiety. While this post-translational modification does not alter the catalytic activity of IMPDH2, it disrupts filament formation, thereby impairing nucleotide-dependent regulation of enzyme activity. Consequently, AnkX-mediated phosphocholination affects IMPDH2 filament assembly, cytoophidia formation, and subcellular localization to the specific Legionella-containing vacuole (LCV). Thus, L. pneumophila subverts host GTP metabolism by posttranslationally modifying a central enzyme of the GTP biosynthetic pathway. Graphical abstract description O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC="FIGDIR/small/701698v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@18f7724org.highwire.dtl.DTLVardef@1d70e86org.highwire.dtl.DTLVardef@1fa8890org.highwire.dtl.DTLVardef@11bc5ba_HPS_FORMAT_FIGEXP M_FIG C_FIG The Legionella effector AnkX post-translationally modifies the human host enzyme IMPDH2 by transferring a phosphocholine group using CDP-choline as co-substrate. We identified the modification site and discovered that this post-translational modification disrupts the ability of IMPDH2 to assemble into filaments and cytoophidia. By shifting the equilibrium toward free octamers, AnkX-mediated PCylation perturbs the structural regulation of IMPDH2 and alters GTP metabolism in L. pneumophila-infected host cells. This figure was inspired by (https://www.biorxiv.org/content/10.1101/2024.07.29.605679v2).

microbiology↗

bioRxiv2023

The Legionella autoinducer LAI-1 is delivered by outer membrane vesicles to promote inter-bacterial and inter-kingdom signaling

Legionella pneumophila is an environmental bacterium, which replicates in amoeba but also in macrophages, and causes a life-threatening pneumonia called Legionnaires disease. The opportunistic pathogen employs the -hydroxyketone compound LAI-1 (Legionella autoinducer-1) for intra-species and inter-kingdom signaling. LAI-1 is produced by the autoinducer synthase LqsA, but it is not known, how LAI-1 is released by the pathogen. Here, we use a V. cholerae luminescence reporter strain and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect bacteria-produced and synthetic LAI-1. Ectopic production of LqsA in E. coli generated LAI-1, which partitions to outer membrane vesicles (OMVs), and slightly reduces OMV size. These E. coli OMVs trigger luminescence of the V. cholerae reporter strain and inhibit the migration of Dictyostelium discoideum amoeba. Overexpression of lqsA in L. pneumophila under the control of strong stationary phase promoters (PflaA or P6SRNA), but not under control of its endogenous promoter (PlqsA), produces LAI-1, which is detected in purified OMVs. These L. pneumophila OMVs trigger luminescence of the Vibrio reporter strain and inhibit D. discoideum migration. L. pneumophila OMVs are smaller upon overexpression of lqsA or upon addition of LAI-1 to growing bacteria, and therefore, LqsA affects OMV production. The overexpression of lqsA but not a catalytically inactive mutant promotes intracellular replication of L. pneumophila in macrophages, indicating that intracellularly produced LA1-1 modulates the interaction in favour of the pathogen. Taken together, we provide evidence that L. pneumophila LAI-1 is secreted through OMVs and promotes inter-bacterial communication as well as interactions with eukaryotic host cells. Originality - Significance StatementInter-kingdom signaling involving low molecular weight bacterial compounds that are detected by eukaryotic cells represents an important, yet incompletely understood aspect of pathogen-host interactions. In many cases, the small signaling molecules are produced in only little amounts, their secretion mechanism is not known, and their effects on eukaryotic host cells are barely studied. Here, we reveal that the -hydroxyketone compound LAI-1 of L. pneumophila is released from the bacteria by outer membrane vesicles, which promote inter-bacterial communication as well as inter-kingdom signaling.

microbiology↗

bioRxiv2022

Morphological Subprofile Analysis for Bioactivity Annotation of Small Molecules

Fast prediction of mode of action for bioactive compounds would immensely foster bioactivity annotation in compound collections and may early on reveal off-targets in chemical biology research and drug discovery. A variety of target-based assays is available for addressing the modulation of druggable proteins. However, they cannot precisely predict how a compound would influence cellular processes due to polypharmacology. Furthermore, non-protein targets are often not considered. Morphological profiling, e.g., using the Cell Painting assay that monitors hundreds of morphological features upon compound perturbation and staining of cellular components, offers a fast, unbiased assessment of compound activity on various targets and cellular processes in one single experiment. However, due to incomplete bioactivity annotation and unknown activities of reference (landmark) compounds, prediction of bioactivity is not straightforward. Here we introduce the concept of subprofile analysis to map the mode of action for both reference and unexplored compounds. We defined mode-of-action clusters for a group of reference compounds and extracted cluster subprofiles that contain only a subset of morphological features (i.e., subprofiles) to represent a consensus profile. Subprofile analysis allows for assignment of compounds to, currently, ten different targets or modes of action in one single assay and bypasses the need of exploring all biosimilar reference compounds for the generation of target hypothesis. This approach will enable rapid bioactivity annotation of compound collections, particularly of uncharacterized small molecules, and will be extended to further bioactivity clusters in future. The data is public accessible via https://github.com/mpimp-comas/2022_pahl_ziegler_subprofiles and the web app tool http://cpcse.pythonanywhere.com/.

cell biology↗

bioRxiv2020

Monoclonal anti-AMP-antibodies reveal broad and diverse AMPylation patterns in cancer cells

AMPylation is a post-translational modification that modifies amino acid side chains with adenosine monophosphate (AMP). Recent progress in the field reveals an emerging role of AMPylation as a universal regulatory mechanism in infection and cellular homeostasis, however, generic tools to study AMPylation are required. Here, we describe three monoclonal anti-AMP antibodies (mAbs) from mouse which are capable of protein backbone independent recognition of AMPylation, in denatured (Western Blot) as well as native (ELISA, IP) applications, thereby outperforming previously reported tools. These antibodies are highly sensitive and specific for AMP modifications, highlighting their potential as tools for new target identification, as well as for validation of known targets. Interestingly, applying the anti-AMP mAbs to various cancer cell lines reveals a previously undescribed broad and diverse AMPylation pattern. In conclusion, the anti-AMP mABs will aid the advancement of understanding AMPylation and the spectrum of modified targets.

biochemistry↗

bioRxiv2019

Intravital multi-modal flux analysis reveals the transport mechanism of bile acids through hepatic microconduits

Small-molecule flux in tissue-microdomains is essential for organ function, but knowledge of this process is scant due to the lack of suitable methods. We developed two independent techniques that allow the quantification of advection (flow) and diffusion in individual bile canaliculi and in interlobular bile ducts of intact livers in living mice, namely Fluorescence Loss After Photoactivation (FLAP) and Intravital Arbitrary Region Image Correlation Spectroscopy (IVARICS). The results challenge the prevailing ‘mechano-osmotic’ theory of canalicular bile flow. After active transport across hepatocyte membranes bile acids are transported in the canaliculi primarily by diffusion. Only in the interlobular ducts, diffusion is augmented by regulatable advection. Photoactivation of fluorescein bis-(5-carboxymethoxy-2-nitrobenzyl)-ether (CMNB-caged fluorescein) in entire lobules demonstrated the establishment of diffusive gradients in the bile canalicular network and the sink function of interlobular ducts. In contrast to the bile canalicular network, vectorial transport was detected and quantified in the mesh of interlobular bile ducts. In conclusion, the liver consists of a diffusion dominated canalicular domain, where hepatocytes secrete small molecules and generate a concentration gradient and a flow-augmented ductular domain, where regulated water influx creates unidirectional advection that augments the diffusive flux.One Sentence Summary/Keywords Bile flux proceeds by diffusion in canaliculi, augmented by advection in ducts.Competing Interest StatementThe authors have declared no competing interest.View Full Text

physiology↗