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Biology subjects

Hipp, K.

Publications and source records attributed to Hipp, K..

7 recordsLinked to original sources

Dynamic co-existence of bacteriophages and their hosts in the Arabidopsis thaliana phyllosphere

Bacterial communities and the bacteriophages infecting them are the basis of every ecosystem, including holobionts. The various ways in which these microorganisms interact with each other in complex communities over the life of the host affects the holobiont fitness. Despite being ubiquitous and environmentally relevant, plant-associated microbial communities remain understudied, especially in the phyllosphere, mainly because of the low abundance of microbes and the complexity of the system. In this work we followed bacteria and phage community dynamics in the phyllosphere over a growing cycle of Arabidopsis thaliana, to understand the ecology and relevance of bacteriophages in complex bacterial communities. We focused on Pseudomonas, a common plant pathogen and commensal, and the phages infecting them, in three setups of increasing complexity: in vitro, controlled experiments in planta and in wild populations of A. thaliana. We found that bacterial communities are resilient to phage infection, and more dynamic than the phages infecting them over the growing season, suggesting that although ubiquitous and abundant, bacteriophages exert selective pressures on leaf bacterial communities only intermittently.

microbiology↗

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↗

Latent endogenous viral elements drive active infection and inheritance in a multicellular host

Endogenous viral elements (EVEs) inserted in host genomes are often regarded as inert relics of past infections. Whether they can retain infective potential and contribute to active viral cycles has remained unresolved. Here, we demonstrate that EVEs in the brown alga Ectocarpus can reactivate and drive productive viral infections. Using long-read sequencing and transcriptomics, we identify full-length, transcriptionally active giant viruses integrated within the host genome. Reactivation of these elements is specific to reproductive cells, transforming gametangia into virus-producing structures, and viral symptoms strictly correlate to the presence of active EVEs. Genetic analyses show that these elements are stably inherited, while their activation is precisely regulated by developmental and environmental cues. By resolving the genomic integration sites, we propose a mechanism for Phaeovirus integration and replication. This work provides the first direct evidence that giant EVEs can reactivate, replicate, and transmit both horizontally and vertically in a multicellular eukaryote, establishing a new model to explore latency, inheritance and the evolutionary impact of large dsDNA viruses.

genetics↗

Kinetic and structure-based comparisons of silent and stimulatory flagellin interactions with TLR5

The bacterial protein flagellin is the sole ligand of the innate immune receptor Toll- like receptor 5 (TLR5). Flagellins with strong agonism bind TLR5 at their D1 and D0 domains, while poor agonist "silent" flagellins bind with D1 only. However, D0-TLR5 interactions insufficiently explain the silent phenotype. Here, we characterize the D1 domain binding kinetics of the silent flagellin RhFlaB compared to the strong canonical agonist StFliC. Using Surface Plasmon Resonance, we show that the RhFlaB-D1 binds more strongly than StFliC-D1, but forms shorter-lived complexes. Cryo-EM analysis of RhFlaB showed its D1 primary interface (PI) contains two distinct hydrophobic pockets, which should facilitate rapid association, while its D1s secondary interface (SI) is dominated by negatively charged residues, which likely impede residue-residue interactions. Comparisons with an existing StFliC-D1 structure suggest that strong agonism requires strong binding at both the PI and SI of the D1, and silent flagellins evade detection partly through weaker SI interactions. These findings offer a foundation for designing targeted interventions that can modulate TLR5 activity to influence immunity, inflammation, or even gut microbiome composition.

biochemistry↗

The baseless mutant links protein phosphatase 2A with basal cell identity in the brown alga Ectocarpus

The first mitotic division of the initial cell is a key event in all multicellular organisms and is usually concomitant with the establishment of major developmental axes and cell fates. The brown alga Ectocarpus has a haploid-diploid life cycle that involves the development of two multicellular and independent generations, the sporophyte and the gametophyte. Each generation deploys a distinct developmental program autonomously from an initial cell, whose first cell division sets up the future body pattern. Here, we show that mutations in the BASELESS (BAS) gene result in multiple cellular defects during the first division of the initial cell and subsequently failure to produce basal structures (rhizoids and prostrate filaments) during both generations of the life cycle. Cloning-by-sequencing revealed that BAS encodes a type B" regulatory subunit of protein phosphatase 2A, and transcriptomic analysis of early developmental stages uncovered potential effector genes involved in setting up basal cell fate in this organism. The bas mutant phenotype is very similar to that observed in the distag (dis) mutants, which lack a functional TBCCd1 protein, at both the cellular and morphological levels. The high level of similarity of the dis and bas mutant phenotypes indicate that TBCCd1 and PP2A are two critical components of the cellular machinery that regulates the division of the initial cell and mediates the establishment of basal cell fate in the developing thallus.

developmental biology↗

naRNA is a canonical neutrophil extracellular trap (NET) component and novel inflammation-amplifying composite DAMP

Neutrophil extracellular traps (NETs) are a key antimicrobial feature of cellular innate immunity mediated by polymorphonuclear neutrophils (PMNs), the primary human leukocyte population. NETs trap and kill microbes but have also been linked to inflammation, e.g. atherosclerosis, arthritis or psoriasis by unknown mechanisms. We here characterize naRNA (NET-associated RNA), as a new canonical, abundant, and unexplored inflammatory NET component. naRNA, upon release by NET formation, drove further NET formation in naive PMN, and induced macrophage and keratinocyte activation via TLR8 in humans and Tlr13 in mice, in vitro and in vivo. Importantly, in vivo naRNA strongly drove skin inflammation, whereas genetic ablation of RNA sensing drastically ameliorated psoriatic skin inflammation. Rather than accidentally assembling with LL37 on the NET, naRNA was intracellularly pre-associated in resting neutrophils as a composite DAMP, thus highlighting NET formation as a DAMP release process. This re-defines sterile NETs as an intentionally inflammatory agent, signaling and amplifying neutrophil activation. Moreover, in the many conditions previously linked to NETs and extracellular RNA, TLR-mediated naRNA sensing emerges as both potential cause and new intervention target. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/499571v4_ufig1.gif" ALT="Figure 1"> View larger version (94K): org.highwire.dtl.DTLVardef@11b866org.highwire.dtl.DTLVardef@16139ceorg.highwire.dtl.DTLVardef@1a2905org.highwire.dtl.DTLVardef@17b0e7e_HPS_FORMAT_FIGEXP M_FIG C_FIG Created with biorender.com

immunology↗

Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation

Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge-and column-based protocols require specialised equipment, often use toxic reagents and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA and total RNA from different sources, as well as environmental TNA and PCR amplicons. We also provide a bead-based protocol for bisulfite conversion, and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility and extreme cost-effectiveness of using magnetic beads. These open source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.

molecular biology↗