Biology subjects

Stehle, T.

Publications and source records attributed to Stehle, T..

10 recordsLinked to original sources

bioRxiv2026

Conserved cis and trans communication domains mediate module interaction in glycopeptide antibiotic NRPS assembly lines

Non-ribosomal peptide synthetases (NRPSs) assemble structurally complex and clinically important natural products, yet the mechanisms coordinating communication between multiple enzymes remain incompletely understood. Here, we dissected the NRPS system underlying biosynthesis of the glycopeptide antibiotic (GPA) balhimycin in Amycolatopsis balhimycina to elucidate principles of inter-enzyme communication. Genetic perturbation of short terminal structural elements markedly reduced balhimycin production, demonstrating their critical role in biosynthesis. AlphaFold3 predictions identified these elements as trans-COM domains mediating specific NRPS interactions, primarily through hydrophobic contacts. Quantitative binding studies using microscale thermophoresis confirmed the importance of the trans-COM domains for multiprotein interaction, extending current models of NRPS communication. Comparative structural analysis further uncovered a conserved class of cis-COM domains within condensation domains across GPA NRPS assembly lines. Together, our findings establish a unified model in which trans- and cis-interfaces cooperatively maintain assembly line fidelity, redefining NRPS architecture and enabling rational engineering strategies.

microbiology↗

bioRxiv2026

Crystal structure of E. coli Nissle 1917 flagellin reveals novel features that modulate bacterial motility but not TLR5 recognition

The probiotic E. coli Nissle 1917 (EcN) strain is known to promote intestinal homeostasis via flagellin, the protomer of its motility apparatus, the flagellum. The flagellin of EcN shows atypical features, namely a hypervariable region (HVR), whose structure and significance have remained elusive. We therefore determined the crystal structure of the E. coli Nissle 1917 flagellin FliC at a resolution of 1.2 [A] which revealed an unusual domain architecture: the canonical D1 domain was found connected by an extended linker to an extensive HVR whose D2, D3 and D4 domains form an outer domain (OD) which surrounds the filament core comprised of conserved domains D0-D1. Using both recombinant proteins and gene-edited EcN strains expressing mutant flagellins, the functional requirement for these unique features was subsequently studied for effects on immune recognition on intestinal epithelial and immune cells, as well as on flagellar protein expression, assembly and bacterial motility. While human and mouse TLR5 immune recognition of flagellar proteins or intact bacteria was only moderately affected by removal of linker or D4, especially linker removal reduced protein stability and bacterial motility in both soft agar and liquid media swimming assays. Interestingly, depending on the environment, D4 or HVR removal had different effects on motility and surface structure. Finally, a site-directed mutagenesis approach highlighted that loss of TLR5 recognition strictly entails loss of motility but not vice versa. Our data indicate that specific HVRs/OD might be relevant for motility of E. coli Nissle 1917 in specialized environments, but not for immune recognition. Moreover, we find mutational tolerance is greater for immune recognition than for motility, providing new insights into bacterial adaptation to the host environment.

microbiology↗

bioRxiv2023

Novel multifunctional glycan probes to elucidate specificities of diverse glycan recognition systems

Glycan microarrays of sequence-defined glycans are the most widely used approach for high-throughput studies of interactions of glycans with glycan binding proteins. Currently nitrocellulose (NC) coated or N-hydroxysuccinimide (NHS) functionalized glass slides are the two most commonly used surfaces for immobilizing glycan probes as noncovalent and covalent arrays, respectively. The mode of glycan presentation can have an influence on the microarray readouts and is an important consideration in the glycan recognition knowledgebase. Here we present the development of glycan probes, tagged with a novel type of tri-functional Fmoc-amino-azido (FAA) linker, which can be readily converted into lipid-tagged or amino-terminating glycan probes for generating neoglycolipid (NGL)-based noncovalent arrays as well as covalent arrays. The azido functionality in the FAA glycan probes provides a means to light up the glycans via biorthogonal Click chemistry so that they become scanner-readable on the covalent arrays, which represents an advance in array quality control. Here analyses were carried out with a diverse set of 36 glycan binding proteins (GBPs) to compare the performance of 44 glycans presented in a liposomal formulation as noncovalent arrays on NC coated slides and on two types of NHS slides as covalent arrays. With most of anti-glycan antibodies and plant lectins investigated, there were negligible or subtle differences in the binding detected in different arrays. However, there were some striking differences observed between the covalent and noncovalent arrays. These include binding of Lotus Tetragonolobus Lectin (LTL) on the covalent but not the noncovalent arrays, and a clear preference observed for glycans on the noncovalent array in the binding of adhesins (VP1 proteins) of two human polyomaviruses JCPyV and BKPyV, and the human immune lectins Siglecs 7 and 9. Subtle yet significant differences in the binding to low affinity glycan ligands were also observed with three bacterial toxins in different arrays. These results revealed interesting insights into the binding behaviour of different GBPs on the noncovalent and covalent arrays and highlight the importance to consider different array platforms in elucidating glycan mediated interactions. The FAA glycan probes can be readily rendered fluorescent via Click chemistry in solution, enabling the detection of GBPs at the surface of cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/548266v1_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@8f2a49org.highwire.dtl.DTLVardef@1212409org.highwire.dtl.DTLVardef@c4ea39org.highwire.dtl.DTLVardef@1ed6a88_HPS_FORMAT_FIGEXP M_FIG C_FIG A new trifunctional linker can present glycans on different array surfaces for analyses of soluble glycan binding proteins or as fluorescent probes for binding by proteins at the surface of cells.

biochemistry↗

bioRxiv2023

A novel and highly specific Forssman antigen-binding protein from sheep polyomavirus

Polyomaviruses are small, non-enveloped double-stranded DNA viruses of humans and other mammals, birds, and fish. Infections are usually asymptomatic and result in latency, however, some polyomaviruses can induce severe diseases, including cancer, in immunocompromised individuals. Established cellular receptors for polyomavirus infection are sialylated glycolipids (such as gangliosides), membrane proteins, and glycosaminoglycans. Polyomaviruses are usually highly host specific but the exact principles that govern host tropism remain unknown in many cases. Here, glycan array screening shows that the major capsid protein VP1 of sheep polyomavirus (ShPyV) binds to the Forssman glycolipid, an antigen of many vertebrates and a potential tumor marker in humans. Following closer investigation, we can report for the first time that a neutral, non-sialylated glycolipid acts as a polyomavirus receptor. Concurrently, we present the first report of a viral protein that specifically engages the Forssman antigen. We demonstrate that ShPyV VP1 binds to Forssman-positive erythrocytes but not those of human A, B and O blood groups, which is a clear distinction from features thus far described for Forssman lectins. X-ray crystallography and structural analysis of the VP1-Forssman glycan complex define the terminal Forssman disaccharide as the determinant of this protein-receptor interaction. These results strongly suggest that the sheep polyomavirus can use Forssman antigen for infectious cell entry. Furthermore, the ability of ShPyV VP1 to distinguish Forssman-positive from -negative cells may prove useful for monitoring the Forssman- status of normal, preneoplastic and neoplastic cells and tissues and establishing the antigen level as a biomarker. Author summaryElucidation of host cell receptor specificities of viral infection is crucial to understand the pathobiology of associated diseases and develop treatments. However, for many polyomaviruses the receptor engagement as the initial event in infection is poorly understood. In only a few cases polyomavirus tropism has been pinned down to a single type of glycan receptor. While many polyomaviruses utilize sialyl glycans to attach to host cells, the role of non-sialylated glycans as receptors is so far underestimated. Here, we show for the first time that a glycan of neutral charge, in this case the carbohydrate portion of the Forssman antigen, acts as a ligand for a polyomavirus capsid protein and may thus contribute to host tropism and infective cell entry. These results represent a significant addition to knowledge on polyomavirus-glycan interactions and complement general principles of carbohydrate engagement by viruses. Furthermore, as a specific binding protein of Forssman antigen, VP1 may help to determine levels of this antigen in healthy and malignant tissues in humans.

biochemistry↗

bioRxiv2022

Structural and functional analysis of natural capsid variants reveals sialic-acid independent entry of BK polyomavirus

BK Polyomavirus (BKPyV) is an opportunistic pathogen that causes nephropathy in kidney transplant recipients. The BKPyV major capsid protein, VP1, engages gangliosides, lipid-linked sialylated glycans at the cell surface, to gain entry into cells. Here, we characterise the influence of VP1 mutations observed in patients with persistent post-transplant BKPyV replication on ganglioside binding, VP1 protein structure, and the tropism of the virus in two renal cell lines: 293TT and immortalised renal tubular epithelial (RS) cells. Infectious entry of single mutants E73Q, E73A and the triple mutant A72V-E73Q-E82Q (VQQ) remained sialic acid-dependent. These three variants acquired binding to a-series gangliosides, including GD1a, although only E73Q was able to infect GD1a-supplemented LNCaP or GM95 cells. Crystal structures of the three mutants showed a clear shift of the BC2 loop in mutants E73A and VQQ that correlated with the inability of these VP1 variants to infect ganglioside complemented cells. On the other hand, the double mutant K69N-E82Q lost the ability to bind sialic acid, with the K69N mutation leading to a steric clash which precludes sialic acid binding. Nevertheless, this mutant retained significant infectivity in 293TT cells that was not dependent on heparan sulphate proteoglycans, implying that an unknown sialic acid-independent entry receptor for BKPyV exists.

microbiology↗

bioRxiv2022

Genetic diversity of Staphylococcus aureus wall teichoic acid glycosyltransferases affects immune recognition

Staphylococcus aureus (S. aureus) is a leading cause of skin and soft tissue infections and (hospital-acquired) systemic infections. Wall teichoic acids (WTAs) are cell wall-anchored glycopolymers that are important for S. aureus nasal colonization, endocarditis, and antibiotic resistance. WTAs consist of a polymerized ribitol phosphate (RboP) chain that can be glycosylated with N-acetylglucosamine (GlcNAc) by three glycosyltransferases: TarS, TarM, and TarP. TarS and TarP modify WTA with {beta}-linked GlcNAc on the C-4 ({beta}1,4-GlcNAc) and the C-3 position ({beta}1,3-GlcNAc) of the RboP subunit, respectively, whereas TarM modifies WTA with -linked GlcNAc at the C-4 position (1,4-GlcNAc). Importantly, these WTA glycosylation patterns impact immune recognition and clearance of S. aureus. Previous studies suggest that tarS is near-universally expressed within the S. aureus population, whereas a smaller proportion co-express either tarM or tarP. To gain more insight in the presence and genetic variation of tarS, tarM, and tarP in the S. aureus population, we analyzed a collection of 25,652 S. aureus genomes within the PubMLST database. Over 99% of isolates contained tarS. Co-expression of tarS/tarM or tarS/tarP occurred in 37% and 7% of isolates, respectively, and was associated to specific S. aureus clonal complexes. We also identified 26 isolates (0.1%) that contained all three glycosyltransferase genes. At sequence level, we identified tar alleles with amino acid substitutions in critical enzymatic residues or with premature stop codons. Several tar variants were expressed in a S. aureus tar-negative strain. Analysis using specific monoclonal antibodies and human langerin showed that WTA glycosylation was severely attenuated or absent. Overall, our data provide a broad overview of the genetic diversity of the three WTA glycosyltransferases in the S. aureus population and the functional consequences for immune recognition.

microbiology↗

bioRxiv2022

Optimization of the precursor supply for an enhanced FK-506 production in Streptomyces tsukubaensis

Tacrolimus (FK-506) is a macrolide widely used as immunosuppressant to prevent transplant rejection. Synthetic production of FK-506 is not efficient and costly, whereas the biosynthesis of FK-506 is complex and the level produced by the wild type strain, Streptomyces tsukubaensis, is very low. We therefore engineered FK-506 biosynthesis and the supply of the precursor L-lysine to generate strains with improved FK-506 yield. To increase FK-506 production, first the intracellular supply of the essential precursor lysine was improved in the native host S. tsukubaensis by engineering the lysine biosynthetic pathway. Therefore, a feedback deregulated aspartate kinase AskSt* of S. tsukubaensis was generated by site directed mutagenesis. Whereas overexpression of AskSt* resulted only in a 17% increase in FK-506 yield, heterologous overexpression of a feedback deregulated AskCg* from Corynebacterium glutamicum was proven to be more efficient. Combined overexpression of AskCg* and DapASt, showed a strong enhancement of the intracellular lysine pool following increase in the yield by approximately 73% compared to the wild type. Lysine is coverted into the FK-506 building block pipecolate by the lysine cyclodeaminase FkbL. Construction of a {Delta}fkbL mutant led to a complete abolishment of the FK-506 production, confirming the indispensability of this enzyme for FK-506 production. Chemical complementation of the {Delta}fkbL mutant by feeding pipecolic acid and genetic complementation with fkbL as well as with other lysine cyclodeaminase genes (pipAf, pipASt, originating from Actinoplanes friuliensis and Streptomyces pristinaespiralis, respectively) completely restored FK-506 production. Subsequently, FK-506 production was enchanced by heterologous overexpression of PipAf and PipASp in S. tsukubaensis. This resulted in a yield increase by 65% compared to the WT in the presence of PipAf from A. friuliensis. For further rational yield improvement, the crystal structure of PipAf from A. friuliensis was determined at 1.3 [A] resolution with the cofactor NADH bound and at 1.4 [A] with its substrate lysine. Based on the structure the Ile91 residue was replaced by Val91 in PipAf, which resulted in an overall increase of FK-506 production by approx. 100% compared to the WT.

bioengineering↗

bioRxiv2021

Ex Vivo and In Vivo CD46 Receptor Utilization by Species D Human Adenovirus Serotype 26 (HAdV26)

Human adenovirus serotype 26 (Ad26) is used as a gene-based vaccine against SARS-CoV-2 and HIV-1. Yet, its primary receptor portfolio remains controversial, potentially including sialic acid, CAR, integrins, and CD46. We and others have shown that Ad26 can use CD46, but these observations were questioned by the inability to co-crystallize Ad26 fiber with CD46. Recent work demonstrated that Ad26 binds CD46 with its hexon protein rather than its fiber. We examined the functional consequences of Ad26 for infection in vitro and in vivo. Ectopic expression of human CD46 on Chinese hamster ovary cells increased Ad26 infection significantly. Deletion of the complement control protein domains CCP1 or CCP2 or the serine-threonine-proline (STP) region of CD46 reduced infection. Comparing wild type and sialic acid-deficient CHO cells, we show that the usage of CD46 is independent of its sialylation status. Ad26 transduction was increased in CD46 transgenic mice after intramuscular (IM) injection, but not after intranasal (IN) administration. Ad26 transduction was 10-fold lower than Ad5 after intratumoral (IT) injection of CD46-expressing tumors. Ad26 transduction of liver was 1000-fold lower than Ad5 after intravenous (IV) injection. These data demonstrate the use of CD46 by Ad26 under certain situations, but also show that the receptor has little consequence by other routes of administration. Finally, IV injection of high doses of Ad26 into CD46 mice induced release of liver enzymes in the bloodstream and reduced white blood cell counts, but did not induce thrombocytopenia. This suggests that Ad26 virions do not induce direct clotting side effects seen during COVID-19 vaccination with this serotype of adenovirus. IMPORTANCEHuman species D Ad26 is being pursued as a low seroprevalence vector for oncolytic virotherapy and gene-based vaccination against HIV-1 and SARS-CoV-2. However, there is debate in the literature about its tropism and receptor utilization, which directly influence its efficiency for certain applications. This work was aimed at determining which receptor(s) this virus uses for infection, and its role in virus biology, vaccine efficacy, and importantly, in vaccine safety.

pharmacology and toxicology↗

bioRxiv2021

The human neuronal receptor NgR1 bridges reovirus capsid proteins to initiate infection

Human Nogo-66 receptor 1 (NgR1) is a receptor for mammalian orthoreoviruses (reoviruses), but the mechanism of virus-receptor engagement is unknown. NgR1 binds a variety of structurally dissimilar ligands in the adult central nervous system (CNS) to inhibit axon outgrowth. Disruption of ligand binding to NgR1 and subsequent signaling can improve neuron regrowth, making NgR1 an important therapeutic target for diverse conditions such as spinal crush injuries and Alzheimer disease. To elucidate how NgR1 mediates cell binding and entry of reovirus, we defined the affinity of interaction between virus and receptor, determined the structure of the virus-receptor complex, and identified residues in the receptor required for virus binding and infection. These studies revealed that NgR1 sequences in a central concave region of the molecule establish a bridge between two copies of the viral capsid protein, {sigma}3. This unusual binding interface produces high-avidity interactions between virus and receptor and likely primes early entry steps. NgR1 sequences engaged by reovirus also are required for NgR1 binding to ligands expressed by neurons and oligodendrocytes. These studies redefine models of reovirus cell-attachment and highlight the evolution of viruses to engage multiple receptors using distinct capsid components.

microbiology↗

bioRxiv2020

Reovirus σ1 conformational flexibility modulates the efficiency of host cell attachment

Reovirus attachment protein {sigma}1 is a trimeric molecule containing tail, body, and head domains. During infection, {sigma}1 engages sialylated glycans and junctional adhesion molecule-A (JAM-A), triggering uptake into the endocytic compartment, where virions are proteolytically converted to infectious subvirion particles (ISVPs). Further disassembly allows {sigma}1 release and escape of transcriptionally active reovirus cores into the cytosol. Electron microscopy has revealed a distinct conformational change in {sigma}1 from a compact form on virions to an extended form on ISVPs. To determine the importance of {sigma}1 conformational mobility, we used reverse genetics to introduce cysteine mutations that can crosslink {sigma}1 by establishing disulfide bonds between structurally adjacent sites in the tail, body, and head domains. We detected phenotypic differences among the engineered viruses. A mutant with a cysteine pair in the head domain replicates with enhanced kinetics, forms large plaques, and displays increased avidity for JAM-A relative to the parental virus, mimicking properties of ISVPs. However, unlike ISVPs, particles containing cysteine mutations that crosslink the head domain uncoat and transcribe viral positive-sense RNA with kinetics similar to the parental virus and are sensitive to ammonium chloride. Together, these data suggest that {sigma}1 conformational flexibility modulates the efficiency of reovirus host cell attachment. IMPORTANCENonenveloped virus entry is an incompletely understood process. For reovirus, the functional significance of conformational rearrangements in the attachment protein, {sigma}1, that occur during entry and particle uncoating are unknown. We engineered and characterized reoviruses containing cysteine mutations that crosslink {sigma}1 monomers in non-reducing conditions. We found that the introduction of a cysteine pair in the receptor-binding domain of {sigma}1 yielded a virus that replicates with faster kinetics than the parental virus and forms larger plaques. Using functional assays, we found that crosslinking the {sigma}1 receptor-binding domain modulates reovirus attachment but not uncoating or transcription. These data suggest that {sigma}1 conformational rearrangements mediate the efficiency of reovirus host cell attachment.

microbiology↗