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Abstract The mesophilic methanogenic archaeal model organism Methanosarcina mazei strain Gö1 is crucial for climate and environmental research due to its ability to produce methane. Here, we establish a Ribo-seq protocol for M. mazei strain Gö1 under two growth conditions (nitrogen sufficiency and limitation). The translation of 93 previously annotated and 314 unannotated small ORFs, coding for proteins ≤ 70 amino acids, is predicted with high confidence based on Ribo-seq data. LC-MS analysis validates the translation for 62 annotated small ORFs and 26 unannotated small ORFs. Epitope tagging followed by immunoblotting analysis confirms the translation of 13 out of 16 selected unannotated small ORFs. A comprehensive differential transcription and translation analysis reveals that 29 of 314 unannotated small ORFs are differentially regulated in response to nitrogen availability at the transcriptional and 49 at the translational level. A high number of reported small RNAs are emerging as dual-function RNAs, including sRNA154, the central regulatory small RNA of nitrogen metabolism. Several unannotated small ORFs are conserved in Methanosarcina species and overproducing several (small ORF encoded) small proteins suggests key physiological functions. Overall, the comprehensive analysis opens an avenue to elucidate the function(s) of multitudinous small proteins and dual-function RNAs in M. mazei.

ABSTRACT Bacterial dormancy is a valuable strategy to survive stressful conditions. Toxins from chromosomal toxin-antitoxin systems have the potential to halt cell growth, induce dormancy, and eventually promote a stress-tolerant persister state. Due to their potential toxicity when overexpressed, sophisticated expression systems are needed when studying toxin genes. Here, we present a moderate expression system for toxin genes based on an artificial 5′ untranslated region. We applied the system to induce expression of the toxin gene tisB from the chromosomal type I toxin-antitoxin system tisB/istR-1 in Escherichia coli. TisB is a small hydrophobic protein that targets the inner membrane, resulting in depolarization and ATP depletion. We analyzed TisB-producing cells by RNA-sequencing and revealed several genes with a role in recovery from TisB-induced dormancy, including the chaperone genes ibpAB and spy. The importance of chaperone genes suggested that TisB-producing cells are prone to protein aggregation, which was validated by an in vivo fluorescent reporter system. We moved on to show that TisB is an essential factor for protein aggregation upon DNA damage mediated by the fluoroquinolone antibiotic ciprofloxacin in E. coli wild-type cells. The occurrence of protein aggregates correlates with an extended dormancy duration, which underscores their importance for the life cycle of TisB-dependent persister cells.IMPORTANCEProtein aggregates occur in all living cells due to misfolding of proteins. In bacteria, protein aggregation is associated with cellular inactivity, which is related to dormancy and tolerance to stressful conditions, including exposure to antibiotics. In Escherichia coli, the membrane toxin TisB is an important factor for dormancy and antibiotic tolerance upon DNA damage mediated by the fluoroquinolone antibiotic ciprofloxacin. Here, we show that TisB provokes protein aggregation, which, in turn, promotes an extended state of cellular dormancy. Our study suggests that protein aggregation is a consequence of membrane toxins with the potential to affect the duration of dormancy and the outcome of antibiotic therapy.

Background: Although belonging to different branches of the immune system, cytotoxic CD8+ αβ T cells and γδ T cells utilize common cytolytic effectors including FasL, granzymes, perforin and granulysin. The effector proteins are stored in different subsets of lysosome-related effector vesicles (LREVs) and released to the immunological synapse upon target cell encounter. Notably, in activated cells, LREVs and potentially other vesicles are continuously produced and released as extracellular vesicles (EVs). Presumably, EVs serve as mediators of intercellular communication in the local microenvironment or at distant sites. Methods: EVs of activated and expanded cytotoxic CD8+ αβ T cells or γδ T cells were enriched from culture supernatants by differential and ultracentrifugation and characterized by nanoparticle tracking analyses and Western blotting. For a comparative proteomic profiling, EV preparations from both cell types were isobaric labeled with tandem mass tags (TMT10plex) and subjected to mass spectrometry analysis. Results: 686 proteins were quantified in EV preparations of cytotoxic CD8+ αβ T cells and γδ T cells. Both populations shared a major set of similarly abundant proteins, while much fewer proteins presented higher abundance levels in either CD8+ αβ T cells or γδ T cells. To our knowledge, we provide the first comparative analysis of EVs from cytotoxic CD8+ αβ T cells and γδ T cells.

Background: Large granular lymphocyte leukemias (LGLLs) are rare lymphoproliferative malignancies caused by clonal expansion of granular lymphocytes. T-cell LGLL and natural killer (NK) cell LGLL are defined based on their cellular origin. Their clinical manifestation and pathophysiology vary depending on the subtype and include, e.g., neutropenia, anemia, recurrent infections, and autoimmunity. A limited number of available patient-derived cell lines are considered valuable tools to study the biology of these malignancies. They differ in the expression of lineage-specific surface markers, but generally contain cytotoxic effector molecules in characteristic granules. Methods: We investigated the presence and release of lysosome-associated effector proteins in patient-derived LGLL cell lines by flow and imaging cytometry, by Western blotting and by bottom–up proteomics profiling. Results: The tested cell lines did not express FasL (CD178), but did express CD26/DPP4+. Intracellularly, we detected major differences in the abundance and subcellular distribution of granzymes, perforin, and granulysin. Similar differences were seen in enriched lysosome-related effector vesicles (LREVs). The proteomics profiling of enriched EVs from an NK-LGLL line (NKL) and a T-LGLL line (MOTN-1), confirmed individual profiles of effector molecules. Conclusion: Our analyses underscore the individual distribution of effector proteins but also open new routes to define the role of intra- and extracellular granules in the disease manifestation or pathology of LGLLs.

Abstract In today’s post-genomic era, it is crucial to rethink the concept of model organisms. While a few historically well-established organisms, e.g. laboratory rodents, have enabled significant scientific breakthroughs, there is now a pressing need for broader inclusion. Indeed, new organisms and models, from complex microbial communities to holobionts, are essential to fully grasp the complexity of biological principles across the breadth of biodiversity. By fostering collaboration between biology, advanced molecular science and omics communities, we can collectively adopt new models, unraveling their molecular functioning, and uncovering fundamental mechanisms. This concerted effort will undoubtedly enhance human health, environmental quality, and biodiversity conservation.

ABSTRACTThe Caenorhabditis elegans natural microbiota isolates Pseudomonas lurida MYb11 and Pseudomonas fluorescens MYb115 protect the host against pathogens through distinct mechanisms. While P. lurida produces an antimicrobial compound and directly inhibits pathogen growth, P. fluorescens MYb115 protects the host without affecting pathogen growth. It is unknown how these two protective microbes affect host biological processes. We used a proteomics approach to elucidate the C. elegans response to MYb11 and MYb115. We found that both Pseudomonas isolates increase vitellogenin protein production in young adults, which confirms previous findings on the effect of microbiota on C. elegans reproductive timing. Moreover, the C. elegans responses to MYb11 and MYb115 exhibit common signatures with the response to other vitamin B12-producing bacteria, emphasizing the importance of vitamin B12 in C. elegans-microbe metabolic interactions. We further analyzed signatures in the C. elegans response specific to MYb11 or MYb115. We provide evidence for distinct modifications in lipid metabolism by both symbiotic microbes. We could identify the activation of host-pathogen defense responses as an MYb11-specific proteome signature and provide evidence that the intermediate filament protein IFB-2 is required for MYb115-mediated protection. These results indicate that MYb11 not only produces an antimicrobial compound but also activates host antimicrobial defenses, which together might increase resistance to infection. In contrast, MYb115 affects host processes such as lipid metabolism and cytoskeleton dynamics, which might increase host tolerance to infection. Overall, this study pinpoints proteins of interest that form the basis for additional exploration into the mechanisms underlying C. elegans microbiota-mediated protection from pathogen infection and other microbiota-mediated traits.IMPORTANCESymbiotic bacteria can defend their host against pathogen infection. While some protective symbionts directly interact with pathogenic bacteria, other protective symbionts elicit a response in the host that improves its own pathogen defenses. To better understand how a host responds to protective symbionts, we examined which host proteins are affected by two protective Pseudomonas bacteria in the model nematode Caenorhabditis elegans. We found that the C. elegans response to its protective symbionts is manifold, which was reflected in changes in proteins that are involved in metabolism, the immune system, and cell structure. This study provides a foundation for exploring the contribution of the host response to symbiont-mediated protection from pathogen infection.

ABSTRACT Short open reading frames (sORF) have the potential to encode small proteins, sORF-encoded peptides (SEP), which are less than or equal to 100 amino acids in length. These SEP have been associated with various physiological processes and are known for their potential roles in modulating immune responses, signaling pathways, and cellular functions. Using bottom-up and top-down proteomics, we identified 45 novel and formerly reported SEP under different growth conditions and stress exposure in single cultures of Blautia producta that plays a central role in mediating colonization resistance and dampening inflammation in the human gut microbiome. Top-down analysis improved identification confidence and allowed the detection of a number of full-length and N- or C-terminal truncated proteoforms of the SEP and of proteoforms carrying post-translational modifications such as disulfides, which provides hints for a wide functional potential of these molecules. The identification of the SEP in single cultures of this bacterium shows that, in contrast to previous results, the biosynthesis of specific SEP is not restricted to direct bacterial interactions within the microbiome but is dependent on growth and environmental conditions during cultivation. IMPORTANCE The identification of short open reading frame-encoded peptides (SEP) and different proteoforms in single cultures of gut microbes offers new insights into a largely neglected part of the microbial proteome landscape. This is of particular importance as SEP provide various predicted functions, such as acting as antimicrobial peptides, maintaining cell homeostasis under stress conditions, or even contributing to the virulence pattern. They are, thus, taking a poorly understood role in structure and function of microbial networks in the human body. A better understanding of SEP in the context of human health requires a precise understanding of the abundance of SEP both in commensal microbes as well as pathogens. For the gut beneficial B. producta, we demonstrate the importance of specific environmental conditions for biosynthesis of SEP expanding previous findings about their role in microbial interactions.

The human leukocyte antigen (HLA) proteins are an indispensable component of adaptive immunity because of their role in presenting self and foreign peptides to T cells. Further, many complex diseases are associated with genetic variation in the HLA region, implying an important role for specific HLA-presented peptides in the etiology of these diseases. Identifying the specific set of peptides presented by an individual’s HLA proteins in vivo, as a whole being referred to as the immunopeptidome, has therefore gathered increasing attention for different reasons. For example, identifying neoepitopes for cancer immunotherapy, vaccine development against infectious pathogens, or elucidating the role of HLA in autoimmunity. Despite the tremendous progress made during the last decade in these areas, several questions remain unanswered. In this perspective, we highlight five remaining key challenges in the analysis of peptide presentation and T cell immunogenicity and discuss potential solutions to these problems. We believe that addressing these questions would not only improve our understanding of disease etiology but will also have a direct translational impact in terms of engineering better vaccines and in developing more potent immunotherapies.

Summary: Microproteins and short open reading frame-encoded peptides (SEPs) can, like all proteins, carry numerous posttranslational modifications. Together with posttranscriptional processes, this leads to a high number of possible distinct protein molecules, the proteoforms, out of a limited number of genes. The identification, quantification, and molecular characterization of proteoforms possess special challenges to established, mainly bottom-up proteomics (BUP) based analytical approaches. While BUP methods are powerful, proteins have to be inferred rather than directly identified, which hampers the detection of proteoforms. An alternative approach is top-down proteomics (TDP) which allows to identify intact proteoforms. This perspective article provides a brief overview of modified microproteins and SEPs, introduces the proteoform terminology, and compares present BUP and TDP workflows highlighting their major advantages and caveats. Necessary future developments in TDP to fully accentuate its potential for proteoform-centric analytics of microproteins and SEPs will be discussed.

Proteolysis catalyzed by the major lysosomal aspartyl protease cathepsin-D (CTSD) appears to be of pivotal importance for proteostasis within the central nervous system and in neurodegeneration. Neuronal Ceroid Lipofuscinosis (NCL) type 10 is caused by a lack of CTSD leading to a defective autophagic flow and pathological accumulation of proteins. We previously demonstrated a therapeutic-relevant clearance of protein aggregates after dosing a NCL10 mouse model with recombinant human pro-cathepsin-D (proCTSD). Similar results could be achieved in cells and mice accumulating α-synuclein. Prompted by these positive effects and our in vitro findings showing that cathepsin-D can cleave the Alzheimer's Disease (AD)-causing amyloid beta peptides (Aβ), we envisaged that such a treatment with proCTSD could similarly be effective in clearance of potentially toxic Aβ species.We demonstrated that CTSD is able to cleave human Aβ1–42 by using liquid chromatography-mass spectrometry. Intracerebral dosing of proCTSD in a NCL10 (CTSD knockout) mouse model revealed uptake and processing of CTSD to its mature and active form. However, the re-addition of CTSD did not obviously affect intracellular APP processing or the generation of soluble APP and Aβ-species. ProCTSD treated HEK cells in comparison with untreated cells were found to contain comparable levels of soluble and membrane bound APP and Aβ-species. Also, the early intracranial application (P1 and P20) of proCTSD in the 5xFAD mouse model did not change Aβ pathology, plaque number and plaque composition and neuroinflammation, however we observed an increased level of Aβ1–42 in the CSF.Our data confirm proteolytic cleavage of human Aβ1–42 by CTSD but exclude a prominent role of CTSD in APP processing and Aβ degradation in our in vitro and in vivo models.

Abstract Background The human leukocyte antigen (HLA) proteins play a fundamental role in the adaptive immune system as they present peptides to T cells. Mass-spectrometry-based immunopeptidomics is a promising and powerful tool for characterizing the immunopeptidomic landscape of HLA proteins, that is the peptides presented on HLA proteins. Despite the growing interest in the technology, and the recent rise of immunopeptidomics-specific identification pipelines, there is still a gap in data-analysis and software tools that are specialized in analyzing and visualizing immunopeptidomics data. Results We present the IPTK library which is an open-source Python-based library for analyzing, visualizing, comparing, and integrating different omics layers with the identified peptides for an in-depth characterization of the immunopeptidome. Using different datasets, we illustrate the ability of the library to enrich the result of the identified peptidomes. Also, we demonstrate the utility of the library in developing other software and tools by developing an easy-to-use dashboard that can be used for the interactive analysis of the results. Conclusion IPTK provides a modular and extendable framework for analyzing and integrating immunopeptidomes with different omics layers. The library is deployed into PyPI at https://pypi.org/project/IPTKL/ and into Bioconda at https://anaconda.org/bioconda/iptkl , while the source code of the library and the dashboard, along with the online tutorials are available at https://github.com/ikmb/iptoolkit .

Summary: A 5,000-year-old Yersinia pestis genome (RV 2039) is reconstructed from a hunter-fisher-gatherer (5300–5050 cal BP) buried at Riņņukalns, Latvia. RV 2039 is the first in a series of ancient strains that evolved shortly after the split of Y. pestis from its antecessor Y. pseudotuberculosis ∼7,000 years ago. The genomic and phylogenetic characteristics of RV 2039 are consistent with the hypothesis that this very early Y. pestis form was most likely less transmissible and maybe even less virulent than later strains. Our data do not support the scenario of a prehistoric pneumonic plague pandemic, as suggested previously for the Neolithic decline. The geographical and temporal distribution of the few prehistoric Y. pestis cases reported so far is more in agreement with single zoonotic events.

Murine T cells express the GPI-anchored ADP-ribosyltransferase 2.2 (ARTC2.2) on the cell surface. In response to T cell activation or extracellular NAD+ or ATP-mediated gating of the P2X7 ion channel ARTC2.2 is shed from the cell surface as a soluble enzyme. Shedding alters the target specificity of ARTC2.2 from cell surface proteins to secreted proteins. Here we demonstrate that shed ARTC2.2 potently ADP-ribosylates IFN-γ in addition to other cytokines. Using mass spectrometry, we identify arginine 128 as the target site of ADP-ribosylation. This residue has been implicated to play a key role in binding of IFN-γ to the interferon receptor 1 (IFNR1). Indeed, binding of IFN-γ to IFNR1 blocks ADP-ribosylation of IFN-γ. Moreover, ADP-ribosylation of IFN-γ inhibits the capacity of IFN-γ to induce STAT1 phosphorylation in macrophages and upregulation of the proteasomal subunit ß5i and the proteasomal activator PA28-α in podocytes. Our results show that ADP-ribosylation inhibits the signaling functions of IFN-γ and point to a new regulatory mechanism for controlling signaling by IFN-γ.

Photoaffinity labeling is frequently employed for the investigation of ligand–receptor interactions in solution. We have employed an interdisciplinary methodology to achieve facile photolabeling of the lectin FimH, which is a bacterial protein, crucial for adhesion, colonization and infection. Following our earlier work, we have here designed and synthesized diazirine-functionalized mannosides as high-affinity FimH ligands and performed an extensive study on photo-crosslinking of the best ligand (mannoside 3) with a series of model peptides and FimH. Notably, we have employed high-performance mass spectrometry to be able to detect radiation results with the highest possible accuracy. We are concluding from this study that photolabeling of FimH with sugar diazirines has only very limited success and cannot be regarded a facile approach for covalent modification of FimH.

The biology of all organisms is influenced by the associated community of microorganisms. In spite of its importance, it is usually not well understood how exactly this microbiota affects host functions and what are the underlying molecular processes. To rectify this knowledge gap, we took advantage of the nematode Caenorhabditis elegans as a tractable, experimental model system and assessed the inducible transcriptome response after colonization with members of its native microbiota. For this study, we focused on two isolates of the genus Ochrobactrum. These bacteria are known to be abundant in the nematode’s microbiota and are capable of colonizing and persisting in the nematode gut, even under stressful conditions. The transcriptome response was assessed across development and three time points of adult life, using general and C. elegans-specific enrichment analyses to identify affected functions. Our assessment revealed an influence of the microbiota members on the nematode’s dietary response, development, fertility, immunity, and energy metabolism. This response is mainly regulated by a GATA transcription factor, most likely ELT-2, as indicated by the enrichment of (i) the GATA motif in the promoter regions of inducible genes and (ii) of ELT-2 targets among the differentially expressed genes. We compared our transcriptome results with a corresponding previously characterized proteome data set, highlighting a significant overlap in the differentially expressed genes, the affected functions, and ELT-2 target genes. Our analysis further identified a core set of 86 genes that consistently responded to the microbiota members across development and adult life, including several C-type lectin-like genes and genes known to be involved in energy metabolism or fertility. We additionally assessed the consequences of induced gene expression with the help of metabolic network model analysis, using a previously established metabolic network for C. elegans. This analysis complemented the enrichment analyses by revealing an influence of the Ochrobactrum isolates on C. elegans energy metabolism and furthermore metabolism of specific amino acids, fatty acids, and also folate biosynthesis. Our findings highlight the multifaceted impact of naturally colonizing microbiota isolates on C. elegans life history and thereby provide a framework for further analysis of microbiota-mediated host functions.

Spatial clustering of enzymes has proven an elegant approach to optimize metabolite transfer between enzymes in synthetic metabolic pathways. Among the multiple methods used to promote colocalisation, enzyme fusion is probably the simplest. Inspired by natural systems, we have explored the metabolic consequences of spatial reorganizations of the catalytic domains of Xanthophyllomyces dendrorhous carotenoid enzymes produced in Saccharomyces cerevisiae. Synthetic genes encoding bidomain enzymes composed of CrtI and CrtB domains from the natural CrtYB fusion were connected in the two possible orientations, using natural and synthetic linkers. A tridomain enzyme (CrtB, CrtI, CrtY) harboring the full β-carotene producing pathway was also constructed. Our results demonstrate that domain order and linker properties considerably impact both the expression and/or stability of the constructed proteins and the functionality of the catalytic domains, all concurring to either diminish or boost specific enzymatic steps of the metabolic pathway. Remarkably, the yield of β-carotene production doubled with the tridomain fusion while precursor accumulation decreased, leading to an improvement of the pathway efficiency, when compared to the natural system. Our data strengthen the idea that fusion of enzymatic domains is an appropriate technique not only to achieve spatial confinement and enhance the metabolic flux but also to produce molecules not easily attainable with natural enzymatic configurations, even with membrane bound enzymes. Keywords: Metabolic engineering, Synthetic biology, Metabolic flux, Enzyme spatial proximity, Carotenoids, Multidomain enzymes

ADAM17 is a member of the ‘Disintegrin and Metalloproteinase’ family of proteases, that cleaves transmembrane substrates from the surfaces of cells. Here the authors show that surface exposure of phosphatidylserine is required for ADAM17 sheddase activity, possibly by directing the protease to its substrates.

The hepatitis B virus (HBV) is one of the most widespread human pathogens known today, yet its origin and evolutionary history are still unclear and controversial. Here, we report the analysis of three ancient HBV genomes recovered from human skeletons found at three different archaeological sites in Germany. We reconstructed two Neolithic and one medieval HBV genome by de novo assembly from shotgun DNA sequencing data. Additionally, we observed HBV-specific peptides using paleo-proteomics. Our results demonstrated that HBV has circulated in the European population for at least 7000 years. The Neolithic HBV genomes show a high genomic similarity to each other. In a phylogenetic network, they do not group with any human-associated HBV genome and are most closely related to those infecting African non-human primates. The ancient viruses appear to represent distinct lineages that have no close relatives today and possibly went extinct. Our results reveal the great potential of ancient DNA from human skeletons in order to study the long-time evolution of blood borne viruses.

Microalga are of high relevance for the global carbon cycling and it is well-known that they are associated with a microbiota. However, it remains unclear, if the associated microbiota, often found in phycosphere biofilms, is specific for the microalga strains and which role individual bacterial taxa play. Here we provide experimental evidence that Chlorella saccharophila, Scenedesmus quadricauda, and Micrasterias crux-melitensis, maintained in strain collections, are associated with unique and specific microbial populations. Deep metagenome sequencing, binning approaches, secretome analyses in combination with RNA-Seq data implied fundamental differences in the gene expression profiles of the microbiota associated with the different microalga. Our metatranscriptome analyses indicates that the transcriptionally most active bacteria with respect to key genes commonly involved in plant–microbe interactions in the Chlorella (Trebouxiophyceae) and Scenedesmus (Chlorophyceae) strains belong to the phylum of the α-Proteobacteria. In contrast, in the Micrasterias (Zygnematophyceae) phycosphere biofilm bacteria affiliated with the phylum of the Bacteroidetes showed the highest gene expression rates. We furthermore show that effector molecules known from plant–microbe interactions as inducers for the innate immunity are already of relevance at this evolutionary early plant-microbiome level.

Signaling of the cytokine interleukin-6 (IL-6) via its soluble IL-6 receptor (sIL-6R) is responsible for the proinflammatory properties of IL-6 and constitutes an attractive therapeutic target, but how the sIL-6R is generated in vivo remains largely unclear. Here, we use liquid chromatography-mass spectrometry to identify an sIL-6R form in human serum that originates from proteolytic cleavage, map its cleavage site between Pro-355 and Val-356, and determine the occupancy of all O- and N-glycosylation sites of the human sIL-6R. The metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) uses this cleavage site in vitro, and mutation of Val-356 is sufficient to completely abrogate IL-6R proteolysis. N- and O-glycosylation were dispensable for signaling of the IL-6R, but proteolysis was orchestrated by an N- and O-glycosylated sequon near the cleavage site and an N-glycan exosite in domain D1. Proteolysis of an IL-6R completely devoid of glycans is significantly impaired. Thus, glycosylation is an important regulator for sIL-6R generation.

The production and consumption of mare's milk in Europe has gained importance, mainly based on positive health effects and a lower allergenic potential as compared to cows' milk. The allergenicity of milk is to a certain extent affected by different genetic variants. In classical dairy species, much research has been conducted into the genetic variability of milk proteins, but the knowledge in horses is scarce. Here, we characterize two major forms of equine αS2-casein arising from genomic 1.3 kb in-frame deletion involving two coding exons, one of which represents an equid specific duplication. Findings at the DNA-level have been verified by cDNA sequencing from horse milk of mares with different genotypes. At the protein-level, we were able to show by SDS-page and in-gel digestion with subsequent LC-MS analysis that both proteins are actually expressed. The comparison with published sequences of other equids revealed that the deletion has probably occurred before the ancestor of present-day asses and zebras diverged from the horse lineage.

The emblematic hydrothermal worm Alvinella pompejana is one of the most thermo tolerant animal known on Earth. It relies on a symbiotic association offering a unique opportunity to discover biochemical adaptations that allow animals to thrive in such a hostile habitat. Here, by studying the Pompeii worm, we report on the discovery of the first antibiotic peptide from a deep-sea organism, namely alvinellacin. After purification and peptide sequencing, both the gene and the peptide tertiary structures were elucidated. As epibionts are not cultivated so far and because of lethal decompression effects upon Alvinella sampling, we developed shipboard biological assays to demonstrate that in addition to act in the first line of defense against microbial invasion, alvinellacin shapes and controls the worm's epibiotic microflora. Our results provide insights into the nature of an abyssal antimicrobial peptide (AMP) and into the manner in which an extremophile eukaryote uses it to interact with the particular microbial community of the hydrothermal vent ecosystem. Unlike earlier studies done on hydrothermal vents that all focused on the microbial side of the symbiosis, our work gives a view of this interaction from the host side.