Biology subjects

Pfeffer, K.

Publications and source records attributed to Pfeffer, K..

11 recordsLinked to original sources

bioRxiv2026

APEX2 proximity labeling maps the mGBP2-proximal proteome at the Toxoplasma gondii parasitophorous vacuole

Interferon-inducible guanylate binding proteins (GBPs) are large GTPases crucial for the host immune response against Toxoplasma gondii, a globally prevalent intracellular apicomplexan parasite. Upon infection, T. gondii forms a parasitophorous vacuole (PV) that protects the parasite from host intracellular defense mechanisms. Murine GBPs (mGBPs) can recognize these PVs and assemble into supramolecular homo- and heterocomplexes, which is an essential step for PV membrane disruption, ultimately enabling cell-autonomous immune control of vacuole-resident pathogens. Our previous work has shown that mGBP2 is an important factor in the immune control of T. gondii. To further elucidate the functions of mGBP2, we employed an APEX2-based proximity labeling strategy to biotinylate proteins in the vicinity of mGBP2 during T. gondii infection. Mass spectrometry analysis of the biotinylated proteins revealed several novel PV-associated proteins such as Cyclooxygenase 2 (COX2/ Ptgs2), Caspase 12 (Cas12), Annexin 11 (Anxa11), Calnexin (Canx), Galectin 3 (Gal3), and Transgelin (Tagln) and valosin-containing protein (VCP) that co-recruit with mGBP2 at T. gondii PVs. In addition, we investigated their regulation by IFN-{gamma} and determined that inhibition of COX2 severely impairs T. gondii growth, highlighting a critical role for COX2 for parasite survival.

immunology↗

bioRxiv2026

Characterization of a fungal mixed-linkage glucan synthase

Mixed-linkage (1,3;1,4)-{beta}-glucan (MLG) is a cell wall polysaccharide found in fungi, bacteria, and plants, yet the enzymatic machinery and structural determinants governing fungal MLG biosynthesis remain poorly understood. In Aspergillus fumigatus, TFT1 (AfTFT1) has previously been implicated in MLG biosynthesis, but its direct biochemical activity remained unresolved. The structural characterization of A. fumigatus wall MLG revealed a distinct polymer profile. Heterologous expression of AfTFT1 in Komagataella phaffii enabled MLG production, generating an oligosaccharide profile that closely matched that of the native fungal polymer and demonstrating that AfTFT1 functions as an MLG synthase. Phylogenetic analysis placed AfTFT1 within a distinct glycosyltransferase family 2 (GT2) fungal lineage containing related candidate MLG synthases across diverse filamentous Ascomycota and separate from the major plant and bacterial synthase lineages. Structure-guided mutagenesis showed that individual substitutions within the transmembrane pore and switch motif altered the lichenase-derived oligosaccharide profile while retaining detectable MLG production, whereas replacement of the entire switch motif with the corresponding HvCSLF6 sequence resulted in no detectable MLG production. Together, these findings establish AfTFT1 as a fungal MLG synthase and identify the switch motif and adjacent transmembrane region as important determinants of MLG synthase function and product fine structure, providing insight into the structural and evolutionary diversification of MLG biosynthesis.

synthetic biology↗

bioRxiv2026

Barcode Crosstalk in ONT Multiplex Sequencing: Quantification and Mitigation Strategies

Multiplexing samples in long-read sequencing with Oxford Nanopore Next Generation Sequencing Technology (ONT) by ligating specific native barcodes to individual DNA samples enables significant increases of high throughput sequencing combined with a significant reduction of sequencing costs. However, this advantage carries the risk of barcode misassignment / crosstalk. Employing ONT multiplex sequencing with samples, we observed misassigned barcodes so called barcode crosstalk, after ONT library preparation according to the standard protocol, particularly in samples with low input DNA concentrations. We assumed that these barcode misassignments are largely due to misligation of remaining native barcodes during subsequent the subsequent sequencing adapter ligation. To systematically investigate and quantify barcode crosstalk, genomic DNA (gDNA) from four bacterial type strains with different DNA input concentrations was prepared using three protocols for library preparation: the Nanopore standard protocol (protocol A: version valid until July 2, 2025) the new Nanopore protocol (protocol B: version from July 2, 2025), and an in house protocol with pooling of the barcoded samples only after the sequencing adapter ligation step (protocol C: in house). All samples were sequenced on a Nanopore PromethIon device. The results clearly showed that the use of protocol A resulted in a pronounced barcode crosstalk especially detectable in samples with low DNA input concentrations (up to 2.4% misassigned reads). The ONT adjustment in protocol B (altered washing buffer vs. protocol A) significantly alleviated the barcode crosstalk to below 0.01%, whereas protocol C eliminated barcode crosstalk virtually completely. These observations emphasize that sequencing results obtained with older ONT native barcoding protocol variants should be critically reviewed. The newer ONT barcoding protocol is preferable for sequencing, but it does not completely eliminate the barcode crosstalk effect. In conclusion, for low DNA input and high accuracy sequencing, protocol C is recommended.

molecular biology↗

bioRxiv2025

Intra-Strain Genetic Heterogeneity in Toxoplasma gondii ME49: Oxford Nanopore Long-Read Sequencing Reveals Copy Number Variation in the ROP8-ROP2A Locus

BackgroundToxoplasma gondii is an important pathogen and model organism for studying mechanisms of immune evasion and defense. Within the same strain, model organisms are typically assumed to be isogenic; for T. gondii, within-strain genetic divergence has been detected based on phenotypic changes and older molecular techniques but not characterized at the genomic level. We therefore used Oxford Nanopore long-read sequencing to characterize three independently maintained T. gondii ME49 isolates: 2015T and 2020T (obtained from ATCC and propagated in cell culture), and 2000B (propagated in mice). ResultsWe de novo assembled a new T. gondii ME49 reference genome and, using state of-the-art variant calling combined with pangenomic genotyping, detected variants between the sequenced isolates. Our new reference genome exceeded existing reference genomes in continuity (NG50 = 6.68 Mb versus 1.2 Mb in RefSeq) and structural accuracy, resolving all chromosomes except for a single break in the ribosomal DNA region. For isolates 2000B and 2020T, we identified 106 and 128 variants, respectively, across a final call set of 79 SNVs, 93 INDELs, and five structural variants; 18 small non-synonymous variants included genes associated with T. gondii life cycle (AP2X-8) and virulence in vivo (6-phosphogluconate dehydrogenase). A 13 kb expansion in the ROP8-ROP2A virulence locus increased the copy number of ROP2A-ROP8 genes in isolates 2000B and 2020T from three to six. ConclusionsWe provide an improved T. gondii ME49 reference genome and demonstrate the potentially confounding effect of intra-strain genetic heterogeneity, highlighting the need for continuous genomic monitoring for long-term genetic identity.

genomics↗

bioRxiv2024

TNF Superfamily Member 14 Drives Post-Influenza Depletion of Alveolar Macrophages Enabling Secondary Pneumococcal Pneumonia

Secondary bacterial infection, often caused by Streptococcus pneumoniae (Spn), is one of the most frequent and severe complications of influenza A virus (IAV)-induced pneumonia. Phenotyping of the pulmonary innate immune landscape after IAV infection revealed a significant depletion of the tissue-resident alveolar macrophage (TR-AM) population at day 7, which was associated with increased susceptibility to Spn outgrowth. To elucidate the molecular mechanisms underlying TR-AM depletion, and to define putative targets for treatment, we combined single-cell transcriptomics and cell-specific PCR profiling in an unbiased manner, using in vivo models of IAV infection and IAV/Spn co-infection. The TNF superfamily 14 (TNFSF14) ligand-receptor axis was revealed as the driving force behind post-influenza TR-AM death during the early infection phase, enabling the transition to pneumococcal pneumonia, while intrapulmonary transfer of genetically modified TR-AMs and antibody-mediated neutralization of specific pathway components alleviated disease severity. With a mainly neutrophilic expression and a high abundance in the bronchoalveolar fluid (BALF) of patients with severe virus-induced ARDS, TNFSF14 emerged as a novel determinant of virus-driven lung injury. Targeting the TNFSF14-mediated intercellular communication network in the virus-infected lung can, therefore, improve host defense, minimizing the risk of subsequent bacterial pneumonia, and ameliorating disease outcome.

molecular biology↗

bioRxiv2023

Dual role of Toxoplasma gondii ROP5 and ROP18 for NLRP3 inhibition

Inflammasome activation leads release of IL-1{beta}, a proinflammatory cytokine that drives antimicrobial immune responses. Toxoplasma gondii has been shown to activate the NLRP3 inflammasome but the trigger has not yet been identified. Here we provide evidence that vacuolar disruption is a prerequisite for NLRP3 activation. T. gondii type I ROP5 and ROP18 protect the parasitophorous vacuolar membrane (PVM) and thereby inhibit inflammasome activation and IL-1{beta} release. Besides protection of the PVM, we demonstrate an additional function of ROP5 and ROP18 for NLRP3 inhibition. We demonstrate the molecular mechanism of this inhibition includes direct interaction with GBP5. In conclusion, T. gondii ROP5 and ROP18 inhibit IL-1{beta} production by protection of the intracellular replicative niche of the parasite and by actively subverting NLRP3 activation. Our findings provide further insight into the intricate mechanisms governing inflammasome activation and inhibition, enhancing our understanding of the complex dynamics during T. gondii infection. O_FIG O_LINKSMALLFIG WIDTH=155 HEIGHT=200 SRC="FIGDIR/small/558105v1_ufig1.gif" ALT="Figure 1"> View larger version (78K): org.highwire.dtl.DTLVardef@10a71b2org.highwire.dtl.DTLVardef@ae753eorg.highwire.dtl.DTLVardef@19365e7org.highwire.dtl.DTLVardef@74d9c_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

MetaGut: Insights into gut microbiomes in stem cell transplantation by comprehensive shotgun long-read sequencing

The gut microbiome is a diverse ecosystem, dominated by bacteria; however, fungi, phages/viruses, archaea, and protozoa are also important members of the gut microbiota. Up to recently, exploration of taxonomic compositions beyond bacteria as well as an understanding of the interaction between the bacteriome with the other members was limited due to 16S rDNA sequencing. Here, we developed MetaGut, a method enabling the simultaneous interrogation of the gut microbiome (bacteriome, mycobiome, archaeome, eukaryome, DNA virome) and of antibiotic resistance genes based on optimized long-read shotgun metagenomics protocols and custom bioinformatics. Using MetaGut we investigated the longitudinal composition of the gut microbiome in an exploratory clinical study in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT; n = 31). Pre-transplantation microbiomes exhibited a 3-cluster structure, associated with Bacteroides/Phocaeicola, mixed composition and Enterococcus abundances. MetaGut revealed substantial inter-individual and temporal variabilities of microbial domain compositions, human DNA, and antibiotic resistance genes during the course of alloHSCT. Interestingly, viruses and fungi accounted for substantial proportions of microbiome content in individual samples (up to >50% and >20%, respectively). After leukopenia, strains were stable or newly acquired. Our results demonstrate the disruptive effect of alloHSCT on the gut microbiome and pave the way for future studies based on long-read metagenomics.

bioinformatics↗

bioRxiv2022

Integrative modeling of guanylate binding protein dimers

Guanylate binding proteins (GBPs) are interferon-{gamma}-activated large GTPases, effective against intracellular pathogens like Toxoplasma gondii. Their host-protective functions require oligomerization, however, the oligomer structures have not been completely resolved yet. Here, we provide dimer models for hGBP1 and the murine GBPs 2 and 7 (mGBP2 and mGBP7) based on integrative modeling that involves the crystal structure of the G domain dimer of hGBP1, cross-linking mass spectrometry (XL-MS), small angle X-ray scattering (SAXS), protein-protein docking, and molecular dynamics (MD) simulations of hGBP1, mGBP2 and mGBP7. We first compare the sequences and protein dynamics of the monomeric hGBP1, mGBP2, and mGBP7, finding that the M/E domain of all three proteins is highly mobile featuring a hinge movement, yet this motion is less pronounced in mGBP7 while its GTPase (G) domain is more flexible. These differences can be explained by the variations in the sequences between mGBP7 and hGBP1/mGBP2 and extend to their dimers. While hGBP1 and its close orthologue mGBP2 dimerize via their G domains, mGBP7 shows a variety of possible dimer structures, among them parallel and crossed-stalk conformations. The G domain is only partly involved in mGBP7 dimerization, which provides a rational why mGBP7, unlike hGBP1 and mGBP2, can dimerize in the absence of GTP. The different GBP dimer structures, which still exhibit hinge movements to certain degrees, are expected to encode diverging functions, such as a destabilization of pathogenic membranes or fusion of the parasitophorous vacuole membrane with the autophagic machinery.

biochemistry↗

bioRxiv2022

Macrophages inhibit Coxiella burnetii by the ACOD1-itaconate pathway for containment of Q fever

Infection with the intracellular bacterium Coxiella (C.) burnetii can cause chronic Q fever with severe complications and limited treatment options. Here, we identify the enzyme cis- aconitate decarboxylase 1 (ACOD1 or IRG1) and its product itaconate as protective host immune pathway in Q fever. Infection of mice with C. burnetii induced expression of several anti-microbial candidate genes, including Acod1. In macrophages, Acod1 was essential for restricting C. burnetii replication, while other antimicrobial pathways were dispensable. Intratracheal or intraperitoneal infection of Acod1-/- mice caused increased C. burnetii burden, significant weight loss and stronger inflammatory gene expression. Exogenously added itaconate restored pathogen control in Acod1-/- mouse macrophages and blocked replication in human macrophages. In axenic cultures, itaconate directly inhibited growth of C. burnetii. Finally, treatment of infected Acod1-/-mice with itaconate efficiently reduced the tissue pathogen load. Thus, ACOD1-derived itaconate is a key factor in the macrophage-mediated defense against C. burnetii and may be exploited for novel therapeutic approaches in chronic Q fever.

immunology↗

bioRxiv2021

mGBP2 engages Galectin-9 and Cytoskeleton-associated Protein 4 for immunity against Toxoplasma gondii

Guanylate binding proteins (GBPs) are large interferon-inducible GTPases, executing essential host defense activities against Toxoplasma gondii, an invasive intracellular apicomplexan protozoan parasite of global importance. T. gondii establishes a parasitophorous vacuole (PV) which shields the parasite from the hosts intracellular defense mechanisms. Murine GBPs (mGBPs) recognize T. gondii PVs and assemble into supramolecular mGBP homo- and heterocomplexes that are required for the disruption of the membrane of PVs eventually resulting in the cell-autonomous immune control of vacuole-resident pathogens. We have previously shown that mGBP2 plays an important role in T. gondii immune control. Here, to unravel mGBP2 functions, we report Galectin-9 (Gal9) as a critical mGBP2 interaction partner engaged for immunity to T. gondii. Interestingly, Gal9 also accumulates and colocalizes with mGBP2 at the T. gondii PV. Furthermore, we could prove the requirement of Gal9 for growth control of T. gondii by CRISPR/Cas9 mediated gene editing. These discoveries clearly indicate that Gal9 is a crucial factor for the mGBP2 coordinated cell autonomous host defense mechanism against T. gondii.

immunology↗

bioRxiv2021

Lymphotoxin β receptor: A crucial role in innate and adaptive immune responses against Toxoplasma gondii

The LT{beta}R plays an essential role in the initiation of immune responses to intracellular pathogens. In mice, the LT{beta}R is crucial for surviving acute toxoplasmosis, however, up to now a functional analysis is largely incomplete. Here, we demonstrate that the LT{beta}R is a key regulator required for the intricate balance of adaptive immune responses. T. gondii infected LT{beta}R-/- mice show globally altered IFN{gamma} regulation, reduced IFN{gamma}-controlled host effector molecule expression, impaired T cell functionality and an absent anti-parasite specific IgG response resulting in a severe loss of immune control of the parasites. Reconstitution of LT{beta}R-/- mice with toxoplasma immune serum significantly prolongs the survival following T. gondii infection. Notably, analysis of RNAseq data clearly indicates a specific effect of T. gondii infection on the B cell response and isotype switching. This study unfolds the decisive role of the LT{beta}R in cytokine regulation and adaptive immune responses to control T. gondii.

immunology↗