Biology subjects

Briegel, A.

Publications and source records attributed to Briegel, A..

At least 19 recordsLinked to original sources

bioRxiv2025

Nanoscale imaging of native symbiotic animal tissue using amultimodal large volume imaging pipeline for cryo-electrontomography

The field of cryo-EM offers the possibility to gain high-resolution structural information of biomolecules in their native state. Advances in sample thinning of cryo-EM samples allows the study of proteins inside intact cells using tomography, opening the door for visual proteomics. However, thicker samples such as tissues or entire organisms are still largely unsuitable for cryo-electron tomography (cryo-ET). Therefore, significant efforts are directed toward developing and improving preparation methods to enable cryo-ET of such complex samples. We focused on the binary association between the Hawaiian bobtail squid Euprymna scolopes and the luminous bacteria Vibrio fischeri. The Squid-Vibrio system has long been studied to understand host-symbiont interactions. Our goal is to study the bacterial-host interface using cryo-ET, at a resolution previously unattainable by conventional EM methods. Here, we present a multi-modal preparation and correlative imaging workflow--including cryo- fluorescence microscopy, microCT, freeze-substitution electron tomography (FS-ET), and serial blockface SEM--to localize and prepare specific regions of the dissected symbiotic light organs for cryo-ET. This approach enabled us to directly visualize symbiotic V. fischeri within the internal host crypts at macromolecular resolution, revealing spatial organization, physical contact, and putative exchange interfaces between host and microbe. Our findings provide structural insights into a foundational model of host-microbe symbiosis and demonstrate the feasibility of cryo-ET for investigating intact tissues at the nanoscale.

microbiology↗

bioRxiv2025

ICP1 bacteriophage treatment antagonizes colonization of the zebrafish larval intestine by Vibrio cholerae

Outbreaks of cholera pose a major threat to human health. Currently, antibiotics are the most effective treatment against the causative agent, the bacterium Vibrio cholerae. However, the use of antibiotics eventually leads to the emergence of resistant strains, which necessitates the need for alternative approaches. The use of bacteriophages to target the infection by antibiotic resistant bacteria is one promising alternative. While clearance of Vibrio cholerae with the use of phages has been performed on several animal models, none of these models are natural hosts of V. cholerae. Therefore, we set out to investigate the interaction between V. cholerae and bacteriophage ICP1 both in vitro and in vivo in a natural host, the zebrafish model, Danio rerio. To study the interplay between host, bacteria and phages we used a combination of light and ultrastructural imaging techniques, including confocal fluorescence microscopy, serial block face scanning electron microscopy (EM) imaging and cryogenic EM, which allowed us to investigate both the colonization process by V. cholerae and clearance by the ICP1 bacteriophage. In addition, we determined the effects of the microbiome on this treatment by using germ-free, conventionalized and monoassociated zebrafish larvae as a host. Independent of the presence and composition of microbiomes used here, V. cholerae efficiently colonized the larval intestine. Finally, we demonstrate significant in vivo clearance of V. cholerae N16961-dsRED by ICP1, underscoring the role of phage-bacteria dynamics in shaping pathogen colonization within the zebrafish larval host. SIGNIFICANCECholera remains a life-threatening disease that causes recurring outbreaks and significant mortality, particularly in developing and conflict-affected regions. As antimicrobial resistance continues to rise, there is an urgent need to better understand the ecological and microbial dynamics that govern Vibrio cholerae colonization and persistence. This research investigates how V. cholerae interacts with bacteriophages, the host environment, and the resident microbiota within a natural vertebrate host, offering new insights into the factors that influence pathogen clearance and shaping of the gut ecosystem during infection. The powerful combination of serial block-face scanning and cryogenic electron microscopy, fluorescence microscopy, and traditional colony/plaque counting methods revealed previously unobserved aspects of the interplay between host, pathogen, phages, and the microbiome, highlighting phage-driven clearance of V. cholerae during colonization.

microbiology↗

bioRxiv2025

Morphological plasticity of endophytic Chitinophaga pinensis

Environmental changes, whether due to climate change or human influences, compromise the resilience of plants to biotic and abiotic stresses, such as pathogens, drought and heat. Plant microbiota are known to promote plant resilience. To be able to harness the power of the plant microbiome we need to identify microbiota with health-promoting properties. Recent studies have demonstrated that the bacterium Chitinophaga pinensis enhances plant health and increases resistance to fungal infections. Here, we show that C. pinensis exhibits an unusually high morphological plasticity, switching between a filamentous and a spherical cell state, each of which is characterized by a distinct transcriptional profile. Despite these transcriptional differences, spherical cells remained metabolically active and replicating, while lacking structural characteristics typically associated with dormant states. Furthermore, the spherical cell morphology of C. pinensis facilitates hitchhiking behaviour and motility via surfactin cheating, potentially influencing its dispersal and interactions within the plant microbiome. To investigate the structural dynamics and transcriptional adaptation of this plant endophyte, we applied a combination of microscopy and culture-based techniques. Taken together, our study provides new insights into the morphological flexibility and transcriptional regulation of the plant-beneficial C. pinensis.

microbiology↗

bioRxiv2025

Defining the minimal structural requirements of DivIVA in filamentous Actinomycetota

The morphogenetic protein DivIVA exhibits diverse functions across bacterial phyla. In Bacillota, DivIVA is primarily involved in cell division, whereas in Actinomycetota, it plays a central role in coordinating polar growth. Due to its essential nature, gaining insight into DivIVA function is challenging. Here we report on the functionality of truncated DivIVA proteins, using a unique divIVA deletion mutant created in cell wall-deficient Kitasatospora viridifaciens L-forms. DivIVA comprises an N-terminal domain, two coiled-coil regions separated by an intercoil linker, and a C-terminal domain. Deleting either the intercoil or the C-terminal region impacted branching dynamics. We also created a minimized variant wherein both were deleted simultaneously, containing the N-terminus and fused coiled-coils, resembling DivIVA from unicellular bacteria. Expression of this minimized variant resulted in severe growth defects. Cells exhibited a strong increase in hyphal width and cell wall thickness, accompanied by frequent tip bursting. Finally, we successfully introduced chimeric DivIVA from the unicellular actinobacterium Mycolicibacterium smegmatis with an N-terminal domain of Kitasatospora viridifaciens, demonstrating functional conservation within the phylum. In contrast, a chimeric DivIVA from Bacillus subtilis could not support growth, underscoring that polar growth is encoded within Actinomycetota-specific amino acid motifs encoded in the first and second coiled-coil. These findings enhance our understanding of the structure-function relationship for DivIVA and present new opportunities to study polar growth. ImpactDivIVA is essential for polar growth in Actinomycetota. In Streptomycetaceae, this membrane-binding protein localizes at growing hyphal tips and along lateral hyphal walls where new branches emerge. Due to its essentiality, the structural relationship of DivIVA between unicellular and multicellular species remains elusive. Using a Kitasatospora viridifaciens L-form divIVA deletion mutant, we expressed truncated DivIVA variants to identify essential regions. Deleting two large unstructured domains produced a minimized variant containing the N-terminus and fused coiled-coils, resembling DivIVA from unicellular bacteria. This strongly impacted morphogenesis, increasing hyphal width and cell wall thickness, and leading to hyphal tip bursting. Finally, we successfully substituted DivIVA of K. viridifaciens with that from Mycolicibacterium smegmatis. Furthermore, Bacillus subtilis DivIVA could not facilitate reversion, showing that polar growth depends on amino acid motifs unique to Actinomycetota. These findings enhance our understanding of the structure-function relationship of DivIVA and offer new opportunities to study polar growth.

microbiology↗

bioRxiv2025

Using cryogenic electron microscopy methods to gain insight into structure and initial host attachment of the flagellotropic bacteriophage 7-7-1

Understanding the structural and functional mechanisms of bacteriophage 7-7-1, the flagellotropic phage infecting Agrobacterium sp. H13-3, offers promising insights into phage-host interactions. Using single particle analysis (SPA) cryo-electron microscopy (cryo-EM), we determined the capsid and tail structure, and built atomic models of capsid hexamers, pentamers and tail. Combined with cryo-electron tomography (cryo-ET) and machine learning methodologies, our findings indicate that phage 7-7-1 uses capsid fibers to establish initial contact with the host flagellum, followed by subsequent attachment to cell surface receptors. Proteinase K treatment confirmed the time-dependent degradation of capsid fibers. The study also demonstrated that capsid fibers are flexible and can interact with other phages and host flagella, suggesting a cooperative infection strategy. These results provide crucial structural insights and may open avenues for developing phage-based therapeutics against resistant bacterial pathogens.

microbiology↗

bioRxiv2024

Gene therapy for targeting a prenatally enriched potassium channel associated with severe childhood epilepsy and premature death

Dysfunction of the sodium-activated potassium channel KNa1.1 (encoded by KCNT1) is associated with a severe condition characterized by frequent seizures (up to hundreds per day) and is often fatal by age three years. We defined the early developmental onset of KNa1.1 channels in prenatal and neonatal brain tissue, establishing a timeline for pathophysiology and a window for therapeutic intervention. Using patch-clamp electrophysiology, we observed age-dependent increases in KNa1.1 K+conductance. In neurons derived from a child with a gain-of-function KCNT1 pathogenic variant (p.R474H), we detected abnormal excitability and action potential afterhyperpolarization kinetics. In a clinical trial, two individuals with the p.R474H variant showed dramatic reductions in seizure occurrence and severity with a first-in-human antisense oligonucleotide (ASO) RNA therapy. ASO-treated p.R474H neurons in vitro exhibited normalized spiking and burst properties. Finally, we demonstrated the feasibility of ASO knockdown of KNa1.1 in midgestation human neurons, suggesting potential for early therapeutic intervention before the onset of epileptic encephalopathy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/620125v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@eaf38corg.highwire.dtl.DTLVardef@11b7e3corg.highwire.dtl.DTLVardef@1fcce20org.highwire.dtl.DTLVardef@40d364_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

A family of lanthipeptides with anti-phage function

Bacteria synthesize natural products to adapt to their environment, where phage-bacteria interactions play a crucial role in bacterial ecology. Although a few natural products have been shown to protect bacteria from phage infection, the prevalence and diversity of chemical anti-phage defense remain largely unexplored. Here, we uncover a novel family of over 2000 lanthipeptide biosynthetic gene clusters from Actinobacteria that participate in anti-phage defense, which we named lanthiphages. Lanthiphages colocalize with other anti-phage systems in defense islands. We demonstrate that native lanthiphage expression protects the model strain Streptomyces coelicolor against diverse phages. Heterologous expression of four additional lanthiphage pathways shows that the anti-phage function is conserved across this family of biosynthetic gene clusters. Finally, we demonstrate that lanthiphage expression leads to the production of a novel compound and alters phage transcription. Our findings highlight that biosynthetic gene clusters with anti-phage functions can be successfully identified through genomic analysis. This work paves the way for the systematic mining of anti-phage natural products, which could constitute a novel reservoir of antiviral drugs.

microbiology↗

bioRxiv2023

CslA and GlxA from Streptomyces lividans form a functional cellulose synthase complex

Filamentous growth of streptomycetes coincides with the synthesis and deposition of an uncharacterized protective glucan at hyphal tips. Synthesis of this glucan depends on the integral membrane protein CslA and the radical copper oxidase GlxA, which are part of a presumably large multiprotein complex operating at growing tips. Here, we show that CslA and GlxA interact by forming a protein complex that is sufficient to synthesize cellulose in vitro. Mass spectrometry analysis revealed that the purified complex produces cellulose chains with a degree of polymerization of at least 80 residues. Truncation analyses demonstrated that the removal of a significant extracellular segment of GlxA had no impact on complex formation, but significantly diminished activity of CslA. Altogether, our work demonstrates that CslA and GlxA form the active core of the cellulose synthase complex and provides molecular insights into a unique cellulose biosynthesis system that is conserved in streptomycetes. SignificanceCellulose stands out as the most abundant polysaccharide on Earth. While the synthesis of this polysaccharide has been extensively studied in plants and Gram-negative bacteria, the mechanisms in Gram-positive bacteria have remained largely unknown. Our research unveils a novel cellulose synthase complex formed by the interaction between the cellulose synthase-like protein CslA and the radical copper oxidase GlxA from Streptomyces lividans, a soil-dwelling Gram-positive bacterium. This discovery provides molecular insights into the distinctive cellulose biosynthesis machineries. Beyond expanding our understanding of cellulose biosynthesis, this study also opens avenues for exploring biotechnological applications and ecological roles of cellulose in Gram-positive bacteria, thereby contributing to the broader field of microbial cellulose biosynthesis and biofilm research.

microbiology↗

bioRxiv2022

Mycobacteria form viable cell wall-deficient cells that are undetectable by conventional diagnostics

The cell wall is a unifying trait in bacteria and provides protection against environmental insults. Therefore, the wall is considered essential for most bacteria. Despite this critical role, many bacteria can transiently shed their cell wall and recent observations suggest a link of such wall-deficient cells to chronic infections. Whether shedding the cell wall also occurs in mycobacteria has not been established unambiguously. Here we provide compelling evidence that a wide range of mycobacterial species, including clinical and non-clinical isolates, form viable cell wall-deficient cells in response to environmental stressors. Using cryo-transmission electron micrography we show that the complex multi-layered wall is largely lost in such cells. Notably, we show that their formation in Mycobacterium marinum and BCG vaccine strains of Mycobacterium bovis is stimulated by exposure to cell wall-targeting antibiotics. Given that these wall-deficient mycobacteria are undetectable using conventional diagnostic methods, such cells have likely been overlooked in clinical settings. Altogether, these results indicate that mycobacteria can readily switch between a walled and wall-deficient lifestyle, which provides a plausible explanation for enabling persistence of infections caused by members of this genus.

microbiology↗

bioRxiv2022

A flexible and efficient microfluidics platform for the characterization and isolation of novel bacteriophages

Bacteriophages are viruses that infect bacteria. This property makes them highly suitable for varied uses in industry or in the development of the treatment of bacterial infections. However, the conventional methods that are used to isolate and analyze these bacteriophages from the environment are generally cumbersome and time-consuming. Here, we adapted a high-throughput microfluidic setup for long-term analysis of bacteriophage-bacteria interaction and demonstrate isolation of phages from environmental samples.

microbiology↗

bioRxiv2022

High resolution reconstruction of a Jumbo bacteriophage infecting capsulated bacteria using hyperbranched tail fibers

The Klebsiella jumbo myophage {phi}Kp24 displays an unusually complex arrangement of tail fibers interacting with a host cell. In this study, we combined cryo-electron microscopy methods, protein structure prediction methods, molecular simulations, and machine learning approaches to explore the capsid, tail, and tail fibers of this phage at high resolution. We determined the structure of the capsid and tail at 4.3[A] and 4.1[A] resolution. We observed that the tail fibers were highly branched and rearranged dramatically upon cell surface attachment. This complex configuration involves fourteen putative tail fibers with depolymerase activity that provide {phi}Kp24 with the ability to infect a broad panel of capsular polysaccharide (CPS) types of Klebsiella pneumoniae. Taken together, our study provides structural and functional insight into how {phi}Kp24 adapts to the highly variable surfaces of capsulated bacterial pathogens, which will be useful for the development of phage therapy approaches against pan-drug resistant K. pneumoniae strains.

microbiology↗

bioRxiv2022

A new class of protein sensor links spirochete pleomorphism, persistence, and chemotaxis

Pathogenic spirochetes can alter their morphologies and behaviors to infect and survive within their hosts. Previous reports demonstrate that the formation of so-called round bodies and biofilms, and chemotaxis are involved in spirochete pathogenesis. Here, in the spirochete Treponema denticola, we report a direct link between these cellular states that involves a new class of protein sensor (CheWS) with hitherto unclear function. Using cryo-EM methods, protein modeling, bioinformatics, genetics methods, and behavioral assays we demonstrate that spirochetes regulate these behaviors in response to the small molecule s-adenosylmethionine (SAM) via a SAM sensor that is anchored to chemotaxis arrays. CheWS influences chemotaxis, biofilm and round body formation under nonstressed conditions by a novel sporulation-like mechanism. Taken together, we establish an improved model for round body formation, we discovered a direct link between this SAM sensor and changes in cellular states, as well as characterized a new sensor class involved in chemotaxis.

microbiology↗

bioRxiv2022

Antibiotic action revealed by real-time imaging of the mycobacterial membrane

The current understanding of mycobacterial cell envelope remodeling in response to antibiotics is limited. Chemical tools that report on phenotypic changes with minimal cell wall perturbation are critical to understanding such time-dependent processes. We employed a fluorogenic chemical probe to image how antibiotics perturb mycobacterial cell envelope assembly in real-time. Time-lapse microscopy revealed that differential antibiotic treatment elicited unique cellular phenotypes, providing a platform for simultaneously monitoring cell envelope construction and remodeling responses. Our data show that rifampicin, which does not directly inhibit cell wall biosynthesis, affords a readily detected mycomembrane phenotype. The fluorogenic probe revealed the production of extracellular vesicles in response to antibiotics, and analyses of these vesicles indicate that antibiotic treatment elicits the release of agents that attenuate macrophage activation.

microbiology↗

bioRxiv2021

Reversible bacteriophage resistance by shedding the bacterial cell wall

Phages are highly abundant in the environment and a major threat for bacteria. Therefore, bacteria have evolved sophisticated defense systems to withstand phage attacks. Here, we describe a previously unknown mechanism by which mono- and diderm bacteria survive infection with diverse lytic phages. Phage exposure leads to a rapid and near complete conversion of walled cells to a cell wall-deficient state, which remain viable in osmoprotective conditions and can revert to the walled state. While shedding the cell wall dramatically reduces the number of progeny phages produced by the host, it does not always preclude phage infection. Altogether, these results show that the formation of cell wall-deficient cells prevents complete eradication of the bacterial population and suggest that cell wall-deficiency may limit the efficacy of phage therapy, especially in highly osmotic environments or when used together with antibiotics that target the cell wall.

microbiology↗

bioRxiv2021

Transition of Vibrio cholerae through a natural host induces resistance to environmental changes

The pandemic-related strains of Vibrio cholerae are known to cause diarrheal disease in animal hosts. These bacteria must overcome rapid changes in their environment, such as the transition from fresh water to the gastrointestinal system of their host. To study the morphological adjustments during environmental transitions, we used zebrafish as a natural host. Using a combination of fluorescent light microscopy, cryogenic electron tomography and serial block face scanning electron microscopy, we studied the structural changes that occur during the infection cycle. We show that the transition from an artificial nutrient-rich environment to a nutrient-poor environment has a dramatic impact on the cell shape, most notably membrane dehiscence. In contrast, excreted bacteria from the host retain a uniform distance between the membranes as well as their vibrioid shape. Inside the intestine, V. cholerae cells predominantly colonized the anterior to mid-gut, forming micro-colonies associated with the microvilli as well as within the lumen. The cells retained their vibrioid shape but changed their cell-length depending on their localization. Our results demonstrate dynamic changes in morphological characteristics of V. cholerae during the transition between the different environments, and we propose that these structural changes are critical for the pathogens ability to colonize host tissues.

microbiology↗

bioRxiv2021

The VarA-CsrA regulatory pathway influences cell shape in Vibrio cholerae

Despite extensive studies on the curve-shaped bacterium Vibrio cholerae, the causative agent of the diarrheal disease cholera, its virulence-associated regulatory two-component signal transduction system VarS/VarA is not well understood. This pathway, which mainly signals through the downstream protein CsrA, is highly conserved among gamma-proteobacteria, indicating there is likely a broader function of this system beyond virulence regulation. In this study, we investigated the VarA-CsrA signaling pathway and discovered a previously unrecognized link to the shape of the bacterium. We observed that varA-deficient V. cholerae cells showed an abnormal spherical morphology during late-stage growth. Through peptidoglycan (PG) composition analyses, we discovered that these mutant bacteria contained an increased content of disaccharide dipeptides and reduced peptide crosslinks, consistent with the atypical cellular shape. The spherical shape correlated with the CsrA-dependent overproduction of aspartate ammonia lyase (AspA) in varA mutant cells, which likely depleted the cellular aspartate pool; therefore, the synthesis of the PG precursor amino acid meso-diaminopimelic acid was impaired. Importantly, this phenotype, and the overall cell rounding, could be prevented by means of cell wall recycling. Collectively, our data provide new insights into how V. cholerae use the VarA-CsrA signaling system to adjust its morphology upon unidentified external cues in its environment. Significance StatementResponsible for the diarrheal disease cholera, the bacterium Vibrio cholerae tightly regulates its virulence program according to external stimuli. Here, we discovered that a sensing-response mechanism involved in the regulation of virulence also controls bacterial shape. We show that V. cholerae lacking this system lose their normal comma shape and become spherical due to an abnormal cell wall composition caused by metabolic changes that reduce available cell wall building blocks. Our study therefore sheds new light on how V. cholerae modulates its morphology based on environmental changes.

microbiology↗

bioRxiv2021

A novel widespread bacterial structure related to the flagellar type III secretion system

The flagellar type III secretion system (fT3SS) is a suite of membrane-embedded and cytoplasmic proteins responsible for building the bacterial flagellar motility machinery. Homologous proteins form the injectisome machinery bacteria use to deliver effector proteins into eukaryotic cells, and other family members have recently been reported to be involved in the formation of membrane nanotubes. Here we describe a novel, ubiquitous and evolutionarily widespread hat-shaped structure embedded in the inner membrane of bacteria, of yet-unidentified function, that is related to the fT3SS, adding to the already rich repertoire of this family of nanomachines.

microbiology↗

bioRxiv2021

In situ imaging of bacterial membrane projections and associated protein complexes using electron cryo-tomography

The ability to produce membrane projections in the form of tubular membrane extensions (MEs) and membrane vesicles (MVs) is a widespread phenomenon among bacteria. Despite this, our knowledge of the ultrastructure of these extensions and their associated protein complexes remains limited. Here, we surveyed the ultrastructure and formation of MEs and MVs, and their associated protein complexes, in tens of thousands of electron cryo-tomograms of [~] 90 bacterial species that we have collected for various projects over the past 15 years (Jensen lab database), in addition to data generated in the Briegel lab. We identified MEs and MVs in 13 species and classified several major ultrastructures: 1) tubes with a uniform diameter (with or without an internal scaffold), 2) tubes with irregular diameter, 3) tubes with a vesicular dilation at their tip, 4) pearling tubes, 5) connected chains of vesicles (with or without neck-like connectors), 6) budding vesicles and nanopods. We also identified several protein complexes associated with these MEs and MVs which were distributed either randomly or exclusively at the tip. These complexes include a secretin-like structure and a novel crown-shaped structure observed primarily in vesicles from lysed cells. In total, this work helps to characterize the diversity of bacterial membrane projections and lays the groundwork for future research in this field.

molecular biology↗