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ABSTRACTThe clinical successes of immune checkpoint blockade have invigorated efforts to activate T cell-mediated responses against cancer. Targeting members of the PVR family, consisting of inhibitory receptors TIGIT, CD96, and CD112R, has been an active area of clinical investigation. In this study, the binding interactions and molecular assemblies of the PVR family receptors and ligands have been assessed in vitro. Furthermore, the anti-TIGIT monoclonal antibody BMS-986207 crystal structure in complex with TIGIT was determined and shows that the antibody binds an epitope that is commonly targeted by the CD155 ligand as well as other clinical anti-TIGIT antibodies. In contrast to previously proposed models, where TIGIT outcompetes costimulatory receptor CD226 for binding to CD155 due to much higher affinity (nanomolar range), our data rather suggest that PVR family members all engage in interactions with relatively weak affinity (micromolar range), including TIGIT and CD155 interactions. Thus, TIGIT and other PVR inhibitory receptors likely elicit immune suppression via increased surface expression rather than inherent differences in affinity. This work provides an improved foundational understanding of the PVR family network and mechanistic insight into therapeutic antibody intervention.

Although therapeutically efficacious, ipilimumab can exhibit dose-limiting toxicity that prevents maximal efficacious clinical outcomes and can lead to discontinuation of treatment. We hypothesized that an acidic pH-selective ipilimumab (pH Ipi), which preferentially and reversibly targets the acidic tumor microenvironment over the neutral periphery, may have a more favorable therapeutic index. While ipilimumab has pH-independent CTLA-4 affinity, pH Ipi variants have been engineered to have up to 50-fold enhanced affinity to CTLA-4 at pH 6.0 compared to pH 7.4. In hCTLA-4 knock-in mice, these variants have maintained anti-tumor activity and reduced peripheral activation, a surrogate marker for toxicity. pH-sensitive therapeutic antibodies may be a differentiating paradigm and a novel modality for enhanced tumor targeting and improved safety profiles.

Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) and GITR ligand (GITRL) regulate immune cell activities, including anti-tumor immune responses. Structures and visualization of human and mouse GITR–GITRL complexes offer insight into the architecture of higher-order membrane assemblies, and their signaling.

T cell immunoglobulin mucin domain-containing protein 3 (Tim-3) negatively regulates innate and adaptive immunity in cancer. To identify the mechanisms of Tim-3 in cancer immunity, we evaluated the effects of Tim-3 blockade in human and mouse melanoma. Here, we show that human programmed cell death 1–positive (PD-1+) Tim-3+CD8+ tumor-infiltrating lymphocytes (TILs) upregulate phosphatidylserine (PS), a receptor for Tim-3, and acquire cell surface myeloid markers from antigen-presenting cells (APCs) through transfer of membrane fragments called trogocytosis. Tim-3 blockade acted on Tim-3+ APCs in a PS-dependent fashion to disrupt the trogocytosis of activated tumor antigen–specific CD8+ T cells and PD-1+Tim-3+ CD8+ TILs isolated from patients with melanoma. Tim-3 and PD-1 blockades cooperated to disrupt trogocytosis of CD8+ TILs in 2 melanoma mouse models, decreasing tumor burden and prolonging survival. Deleting Tim-3 in dendritic cells but not in CD8+ T cells impeded the trogocytosis of CD8+ TILs in vivo. Trogocytosed CD8+ T cells presented tumor peptide–major histocompatibility complexes and became the target of fratricide T cell killing, which was reversed by Tim-3 blockade. Our findings have uncovered a mechanism Tim-3 uses to limit antitumor immunity.

The molecular interactions of mouse CD96 to CD155 ligand and to two surrogate antibodies have been investigated. Biophysical and structural studies demonstrate that CD96 forms a homodimer but assembles as 1:1 heterodimeric complexes with CD155 or with one of the surrogate antibodies, which compete for the same binding interface. In comparison, the other surrogate antibody binds across the mouse CD96 dimer and recognizes a quaternary epitope spanning both protomers to block exposure of the ligand-binding site. This study reveals different blocking mechanisms and modalities of these two antibodies and may provide insight into the functional effects of antibodies against CD96.

The development of antibody therapeutics relies on animal models that accurately recapitulate disease biology. Syngeneic mouse models are increasingly used with new molecules to capture the biology of complex cancers and disease states, and to provide insight into the role of the immune system. The establishment of syngeneic mouse models requires the ability to generate surrogate mouse counterparts to antibodies designed for humans. In the field of bispecific antibodies, there remains a dearth of technologies available to generate native IgG-like mouse bispecific antibodies. Thus, we engineered a simple co-expression system for one-step purification of intact mouse IgG1 and IgG2a bispecific antibodies from any antibody pair. We demonstrated proof of concept with CD3/CD20 bispecific antibodies, which highlighted both the quality and efficacy of materials generated by this technology.

Elotuzumab is a humanized therapeutic monoclonal antibody directed to the surface glycoprotein SLAMF7 (CS1, CRACC, CD319), which is highly expressed on multiple myeloma (MM) tumor cells. Improved clinical outcomes have been observed following treatment of MM patients with elotuzumab in combination with lenalidomide or bortezomib. Previous work showed that elotuzumab stimulates NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC), via Fc-domain engagement with FcγRIIIa (CD16). SLAMF7 is also expressed on NK cells, where it can transmit stimulatory signals. We tested whether elotuzumab can directly activate NK cells via ligation with SLAMF7 on NK cells in addition to targeting ADCC through CD16. We show that elotuzumab strongly promoted degranulation and activation of NK cells in a CD16-dependent manner, and a non-fucosylated form of elotuzumab with higher affinity to CD16 exhibited enhanced potency. Using F(ab')2 or Fc-mutant forms of the antibody, the direct binding of elotuzumab to SLAMF7 alone could not stimulate measurable CD69 expression or degranulation of NK cells. However, the addition of soluble elotuzumab could costimulate calcium signaling responses triggered by multimeric engagement of NKp46 and NKG2D in a CD16-independent manner. Thus, while elotuzumab primarily stimulates NK cells through CD16, it can also transduce effective “trans”-costimulatory signals upon direct engagement with SLAMF7, since these responses did not require direct co-engagement with the activating receptors. Trans-costimulation by elotuzumab has potential to reduce activation thresholds of other NK cell receptors engaging with their ligands on myeloma target cell surfaces, thereby potentially further increasing NK cell responsiveness in patients.

Monoclonal antibodies (mAbs) are a growing segment of therapeutics, yet their in vitro characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigen's endogenous concentration, by combining the kinetic exclusion assay and Biacore's calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9), progranulin (PGRN), and fatty acid binding protein (FABP4). We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling.

Therapeutic antibodies are often engineered or selected to have high on-target binding affinities that can be challenging to determine precisely by most biophysical methods. Here, we explore the dynamic range of the kinetic exclusion assay (KinExA) by exploiting the interactions of an anti-DKK antibody with a panel of DKK antigens as a model system. By tailoring the KinExA to each studied antigen, we obtained apparent equilibrium dissociation constants (K(D) values) spanning six orders of magnitude, from approximately 100 fM to 100 nM. Using a previously calibrated antibody concentration and working in a suitable concentration range, we show that a single experiment can yield accurate and precise values for both the apparent K(D) and the apparent active concentration of the antigen, thereby increasing the information content of an assay and decreasing sample consumption. Orthogonal measurements obtained on Biacore and Octet label-free biosensor platforms further validated our KinExA-derived affinity and active concentration determinations. We obtained excellent agreement in the apparent affinities obtained across platforms and within the KinExA method irrespective of the assay orientation employed or the purity of the recombinant or native antigens.

Abstract Uropygial gland secretions of birds consist of host and bacteria derived compounds and play a major sanitary and feather-protective role. Here we report on our microbiome studies of the New Guinean toxic bird Pachycephala schlegelii and the isolation of a member of the Amycolatopsis genus from the uropygial gland secretions. Bioactivity studies in combination with co-cultures, MALDI imaging and HR-MS/MS-based network analyses unveil the basis of its activity against keratinolytic bacteria and fungal skin pathogens. We trace the protective antimicrobial activity of Amycolatopsis sp. PS_44_ISF1 to the production of rifamycin congeners, ciromicin A and of two yet unreported compound families. We perform NMR and HR-MS/MS studies to determine the relative structures of six members belonging to a yet unreported lipopeptide family of pachycephalamides and of one representative of the demiguisins, a new hexapeptide family. We then use a combination of phylogenomic, transcriptomic and knock-out studies to identify the underlying biosynthetic gene clusters responsible for the production of pachycephalamides and demiguisins. Our metabolomics data allow us to map molecular ion features of the identified metabolites in extracts of P. schlegelii feathers, verifying their presence in the ecological setting where they exert their presumed active role for hosts. Our study shows that members of the Actinomycetota may play a role in avian feather protection.

Abstract The use of compounds produced by hosts or symbionts for defence against antagonists has been identified in many organisms, including in fungus-farming termites (Macrotermitinae). The obligate mutualistic fungus Termitomyces plays a pivotal role in plant biomass decomposition and as the primary food source for these termites. Despite the isolation of various specialized metabolites from different Termitomyces species, our grasp of their natural product repertoire remains incomplete. To address this knowledge gap, we conducted a comprehensive analysis of 39 Termitomyces genomes, representing 21 species associated with members of five termite host genera. We identified 754 biosynthetic gene clusters (BGCs) coding for specialized metabolites and categorized 660 BGCs into 61 biosynthetic gene cluster families (GCFs) spanning five compound classes. Seven GCFs were shared by all 21 Termitomyces species and 21 GCFs were present in all genomes of subsets of species. Evolutionary constraint analyses on the 25 most abundant GCFs revealed distinctive evolutionary histories, signifying that millions of years of termite-fungus symbiosis have influenced diverse biosynthetic pathways. This study unveils a wealth of non-random and largely undiscovered chemical potential within Termitomyces and contributes to our understanding of the intricate evolutionary trajectories of biosynthetic gene clusters in the context of long-standing symbiosis.

Abstract The human microbiome emerges as a promising reservoir for diagnostic markers and therapeutics. Since host-associated microbiomes at various body sites differ and diseases do not occur in isolation, a comprehensive analysis strategy highlighting the full potential of microbiomes should include diverse specimen types and various diseases. To ensure robust data quality and comparability across specimen types and diseases, we employ standardized protocols to generate sequencing data from 1931 prospectively collected specimens, including from saliva, plaque, skin, throat, eye, and stool, with an average sequencing depth of 5.3 gigabases. Collected from 515 patients, these samples yield an average of 3.7 metagenomes per patient. Our results suggest significant microbial variations across diseases and specimen types, including unexpected anatomical sites. We identify 583 unexplored species-level genome bins (SGBs) of which 189 are significantly disease-associated. Of note, the existence of microbial resistance genes in one specimen was indicative of the same resistance genes in other specimens of the same patient. Annotated and previously undescribed SGBs collectively harbor 28,315 potential biosynthetic gene clusters (BGCs), with 1050 significant correlations to diseases. Our combinatorial approach identifies distinct SGBs and BGCs, emphasizing the value of pan-body pan-disease microbiomics as a source for diagnostic and therapeutic strategies.

Abstract Fungi constitute the Earth’s second most diverse kingdom, however only a small percentage of these have been thoroughly examined and categorized for their secondary metabolites, which still limits our understanding of the ecological chemical and pharmacological potential of fungi. In this study, we explored members of the co-evolved termite-associated fungal genus Xylaria and identified a family of highly oxygenated polyketide-terpene hybrid natural products using an MS/MS molecular networking-based dereplication approach. Overall, we isolated six no yet reported xylasporin derivatives, of which xylasporin A (1) features a rare cyclic-carbonate moiety. Extensive comparative spectrometric (HRMS2) and spectroscopic (1D and 2D NMR) studies allowed to determine the relative configuration across the xylasporin family, which was supported by chemical shift calculations of more than 50 stereoisomers and DP4+ probability analyses. The absolute configuration of xylasporin A (1) was also proposed based on TDDFT-ECD calculations. Additionally, we were able to revise the relative and absolute configurations of co-secreted xylacremolide B produced by single x-ray crystallography. Comparative genomic and transcriptomic analysis allowed us to deduce the putative biosynthetic assembly line of xylasporins in the producer strain X802, and could guide future engineering efforts of the biosynthetic pathway.

Abstract Background Although Basidiomycota produce pharmaceutically and ecologically relevant natural products, knowledge of how they coordinate their primary and secondary metabolism is virtually non-existent. Upon transition from vegetative mycelium to carpophore formation, mushrooms of the genus Psilocybe use l-tryptophan to supply the biosynthesis of the psychedelic tryptamine alkaloid psilocybin with the scaffold, leading to a strongly increased demand for this particular amino acid as this alkaloid may account for up to 2% of the dry mass. Using Psilocybe mexicana as our model and relying on genetic, transcriptomic, and biochemical methods, this study investigated if l-tryptophan biosynthesis and degradation in P. mexicana correlate with natural product formation. Results A comparative transcriptomic approach of gene expression in P. mexicana psilocybin non-producing vegetative mycelium versus producing carpophores identified the upregulation of l-tryptophan biosynthesis genes. The shikimate pathway genes trpE1, trpD, and trpB (encoding anthranilate synthase, anthranilate phosphoribosyltransferase, and l-tryptophan synthase, respectively) were upregulated in carpophores. In contrast, genes idoA and iasA, encoding indole-2,3-dioxygenase and indole-3-acetaldehyde synthase, i.e., gateway enzymes for l-tryptophan-consuming pathways, were massively downregulated. Subsequently, IasA was heterologously produced in Escherichia coli and biochemically characterized in vitro. This enzyme represents the first characterized microbial l-tryptophan-preferring acetaldehyde synthase. A comparison of transcriptomic data collected in this study with prior data of Psilocybe cubensis showed species-specific differences in how l-tryptophan metabolism genes are regulated, despite the close taxonomic relationship. Conclusions The upregulated l-tryptophan biosynthesis genes and, oppositely, the concomitant downregulated genes encoding l-tryptophan-consuming enzymes reflect a well-adjusted cellular system to route this amino acid toward psilocybin production. Our study has pilot character beyond the genus Psilocybe and provides, for the first time, insight in the coordination of mushroom primary and secondary metabolism.

Abstract β-Amino acid-containing macrolactams represent a structurally diverse group of bioactive natural products derived from polyketides; however we are currently lacking a comprehensive overview about their abundance across bacterial families and the underlying biosynthetic diversity. In this study, we employed a targeted β-amino acid-specific homology-based multi-query search to identify potential bacterial macrolactam producers. Here we demonstrate that approximately 10% of each of the identified actinobacterial genera harbor a biosynthetic gene cluster (BGC) encoding macrolactam production. Based on our comparative study, we propose that mutations occurring in specific regions of polyketide synthases (PKS) are the primary drivers behind the variation in macrolactam ring sizes. We successfully validated two producers of ciromicin A from the genus Amycolatopsis, revised the composition of the biosynthetic gene cluster region mte of macrotermycins, and confirmed the ciromicin biosynthetic pathway through heterologous expression. Additionally, network-based metabolomic analysis uncovered three previously unreported macrotermycin congeners from Amycolatopsis sp. M39. The combination of targeted mining and network-based analysis serves as a powerful tool for identifying macrolactam producers and our studies will catalyze the future discovery of yet unreported macrolactams.

Abstract Macrotermitinae termites have farmed fungi in the genus Termitomyces as a food source for millions of years. However, the biochemical mechanisms orchestrating this mutualistic relationship are largely unknown. To deduce fungal signals and ecological patterns that relate to the stability of this symbiosis, we explored the volatile organic compound (VOC) repertoire of Termitomyces from Macrotermes natalensis colonies. Results show that mushrooms emit a VOC pattern that differs from mycelium grown in fungal gardens and laboratory cultures. The abundance of sesquiterpenoids from mushrooms allowed targeted isolation of five drimane sesquiterpenes from plate cultivations. The total synthesis of one of these, drimenol, and related drimanes assisted in structural and comparative analysis of volatile organic compounds (VOCs) and antimicrobial activity testing. Enzyme candidates putatively involved in terpene biosynthesis were heterologously expressed and while these were not involved in the biosynthesis of the complete drimane skeleton, they catalyzed the formation of two structurally related monocyclic sesquiterpenes named nectrianolins.

Summary: Background: Rheumatoid arthritis (RA) is a common autoimmune disease with emerging environmental and microbiome risk factors. The western diet is typically deficient in magnesium (Mg), and there is some evidence suggesting that Mg may have anti-inflammatory properties. But the actual role of Mg supplementation in arthritis or in T cell subsets has not been explored. Methods: We investigated the role of a high Mg diet in two different mouse models of RA induced with the KRN serum, and collagen-induced arthritis. We also characterized the phenotypes of splenocytes, gene expression, and an extensive intestinal microbiome analyses including fecal material transplantation (FMT). Findings: The high Mg diet group was significantly protected with reduced arthritis severity and joint damage, and reduced expression of IL-1β, IL-6, and TNFα. The high Mg group also had increased numbers of Foxp3+ Treg cells and IL-10-producing T cells. The high Mg protective effect disappeared in IL-10 knockout mice. FMT from the high Mg diet mice recreated the phenotypes seen in the diet-treated mice, with reduced arthritis severity, increased Foxp3+ Treg, and increased IL-10-producing T cells. Intestinal microbiome analyses using 16S rDNA sequencing revealed diet-specific changes, including reduced levels of RA-associated Prevotella in the high Mg group, while increasing levels of Bacteroides and other bacteria associated with increased production of short-chain fatty acids. Metagenomic analyses implicated additional pathways including L-tryptophan biosynthesis and arginine deiminase. Interpretation: We describe a new role for Mg in suppressing arthritis, in expanding Foxp3+ T reg cells and in the production of IL-10, and show that these effects are mediated by the intestinal microbiome. Our discoveries suggest a novel strategy for modifying the intestinal microbiome to treat RA and other autoimmune and inflammatory diseases. Funding: None.

Aromatic prenylated metabolites have important biological roles and activities in all living organisms. Compared to their importance in all domains of life, we know relatively little about their substrate scopes and metabolic functions. Here, we describe a new UbiA-like prenyltransferase (Ptase) Ubi-297 encoded in a conserved operon of several bacterial taxa, including marine Flavobacteria and the genus Sacchromonospora. In silico analysis of Ubi-297 homologs indicated that members of this Ptase group are composed of several transmembrane α-helices and carry a conserved and distinct aspartic-rich Mg2+-binding domain. We heterologously produced UbiA-like Ptases from the bacterial genera Maribacter, Zobellia, and Algoriphagus in Escherichia coli. Investigation of their substrate scope uncovered the preferential farnesylation of quinoline derivatives, such as 8-hydroxyquinoline-2-carboxylic acid (8-HQA) and quinaldic acid. The results of this study provide new insights into the abundance and diversity of Ptases in marine Flavobacteria and beyond.

Abstract Rheumatoid arthritis (RA)-associated interstitial lung disease (RA-ILD) is the most common pulmonary complication of RA, increasing morbidity and mortality. Anti-citrullinated protein antibodies have been associated with the development and progression of both RA and fibrotic lung disease; however, the role of protein citrullination in RA-ILD remains unclear. Here, we demonstrate that the expression of peptidylarginine deiminase 2 (PAD2), an enzyme that catalyzes protein citrullination, is increased in lung homogenates from subjects with RA-ILD and their lung fibroblasts. Chemical inhibition or genetic knockdown of PAD2 in RA-ILD fibroblasts attenuated their activation, marked by decreased myofibroblast differentiation, gel contraction, and extracellular matrix gene expression. Treatment of RA-ILD fibroblasts with the proteoglycan syndecan-2 (SDC2) yielded similar antifibrotic effects through regulation of PAD2 expression, phosphoinositide 3-kinase/Akt signaling, and Sp1 activation in a CD148-dependent manner. Furthermore, SDC2-transgenic mice exposed to bleomycin-induced lung injury in an inflammatory arthritis model expressed lower levels of PAD2 and were protected from the development of pulmonary fibrosis. Together, our results support a SDC2-sensitive profibrotic role for PAD2 in RA-ILD fibroblasts and identify PAD2 as a promising therapeutic target of RA-ILD.

Targeting the Kv1.3 potassium channel has proven effective in reducing obesity and the severity of animal models of autoimmune disease. Stichodactyla toxin (ShK), isolated from the sea anemone Stichodactyla helianthus, is a potent blocker of Kv1.3. Several of its analogs are some of the most potent and selective blockers of this channel. However, like most biologics, ShK and its analogs require injections for their delivery, and repeated injections reduce patient compliance during the treatment of chronic diseases. We hypothesized that inducing the expression of an ShK analog by hepatocytes would remove the requirement for frequent injections and lead to a sustained level of Kv1.3 blocker in the circulation. To this goal, we tested the ability of Adeno-Associated Virus (AAV)8 vectors to target hepatocytes for expressing the ShK analog, ShK-235 (AAV-ShK-235) in rodents. We designed AAV8 vectors expressing the target transgene, ShK-235, or Enhanced Green fluorescent protein (EGFP). Transduction of mouse livers led to the production of sufficient levels of functional ShK-235 in the serum from AAV-ShK-235 single-injected mice to block Kv1.3 channels. However, AAV-ShK-235 therapy was not effective in reducing high-fat diet-induced obesity in mice. In addition, injection of even high doses of AAV8-ShK-235 to rats resulted in a very low liver transduction efficiency and failed to reduce inflammation in a well-established rat model of delayed-type hypersensitivity. In conclusion, the AAV8-based delivery of ShK-235 was highly effective in inducing the secretion of functional Kv1.3-blocking peptide in mouse, but not rat, hepatocytes yet did not reduce obesity in mice fed a high-fat diet.

ABSTRACT Serine palmitoyltransferase catalyzes the first step of the sphingolipid biosynthesis. Recently, sphingolipid homeostasis has been connected to several human diseases, making serine palmitoyltransferases an interesting therapeutic target. Known and efficient serine palmitoyltransferase-inhibitors are sphingofungins, a group of natural products isolated from fungi. To further characterize newly isolated sphingofungins, we designed an easy to use colorimetric serine palmitoyltransferase activity assay using FadD, which can be performed in 96-well plates. Because sphingofungins exert antifungal activitiy as well, we compared the in vitro assay results with an in vivo growth assay using Saccharomyces cerevisiae. The reported experiments showed differences among the assayed sphingofungins, highlighting an increase of activity based on the saturation levels of the polyketide tail. IMPORTANCE Targeting the cellular sphingolipid metabolism is often discussed as a potential approach to treat associated human diseases such as cancer and Alzheimer's disease. Alternatively, it is also a possible target for the development of antifungal compounds, which are direly needed. A central role is played by the serine palmitoyltransferase, which catalyzes the initial and rate limiting step of sphingolipid de novo synthesis and, as such, the development of inhibitory compounds for this enzyme is of interest. Our work here established an alternative approach for determining the activity of serine palmitoyltransferase adding another tool for the validation of its inhibition. We also determined the effect of different modifications to sphingofungins on their inhibitory activity against serine palmitoyltransferase, revealing important differences on said activity against enzymes of bacterial and fungal origin.

ABSTRACT Macrotermitinae termites have domesticated fungi of the genus Termitomyces as food for their colony, analogously to human farmers growing crops. Termites propagate the fungus by continuously blending foraged and predigested plant material with fungal mycelium and spores (fungus comb) within designated subterranean chambers. To test the hypothesis that the obligate fungal symbiont emits specific volatiles (odor) to orchestrate its life cycle and symbiotic relations, we determined the typical volatile emission of fungus comb biomass and Termitomyces nodules, revealing α-pinene, camphene, and d-limonene as the most abundant terpenes. Genome mining of Termitomyces followed by gene expression studies and phylogenetic analysis of putative enzymes related to secondary metabolite production encoded by the genomes uncovered a conserved and specific biosynthetic repertoire across strains. Finally, we proved by heterologous expression and in vitro enzymatic assays that a highly expressed gene sequence encodes a rare bifunctional mono-/sesquiterpene cyclase able to produce the abundant comb volatiles camphene and d-limonene. IMPORTANCE The symbiosis between macrotermitinae termites and Termitomyces is obligate for both partners and is one of the most important contributors to biomass conversion in the Old World tropic’s ecosystems. To date, research efforts have dominantly focused on acquiring a better understanding of the degradative capabilities of Termitomyces to sustain the obligate nutritional symbiosis, but our knowledge of the small-molecule repertoire of the fungal cultivar mediating interspecies and interkingdom interactions has remained fragmented. Our omics-driven chemical, genomic, and phylogenetic study provides new insights into the volatilome and biosynthetic capabilities of the evolutionarily conserved fungal genus Termitomyces, which allows matching metabolites to genes and enzymes and, thus, opens a new source of unique and rare enzymatic transformations.

Voltage-gated Kv1.3 potassium channels are key regulators of T lymphocyte activation, proliferation and cytokine production, by providing the necessary membrane hyper-polarization for calcium influx following immune stimulation. It is noteworthy that an accumulating body of in vivo and in vitro evidence links these channels to multiple sclerosis pathophysiology. Here we studied the electrophysiological properties and the transcriptional and translational expression of T lymphocyte Kv1.3 channels in multiple sclerosis, by combining patch clamp recordings, reverse transcription polymerase chain reaction and flow cytometry on freshly isolated peripheral blood T lymphocytes from two patient cohorts with multiple sclerosis, as well as from healthy and disease controls. Our data demonstrate that T lymphocytes in MS, manifest a significant up-regulation of Kv1.3 mRNA, Kv1.3 membrane protein and Kv1.3 current density and therefore of functional membrane channel protein, compared to control groups (p < 0.001). Interestingly, patient sub-grouping shows that Kv1.3 channel density is significantly higher in secondary progressive, compared to relapsing-remitting multiple sclerosis (p < 0.001). Taking into account the tight connection between Kv1.3 channel activity and calcium-dependent processes, our data predict and could partly explain the reported alterations of T lymphocyte function in multiple sclerosis, while they highlight Kv1.3 channels as potential therapeutic targets and peripheral biomarkers for the disease.

Trillions of microbes representing all kingdoms of life are resident in, and on, humans holding essential roles for the host development and physiology. The last decade over a dozen online tools and servers, accessible via public domain, have been developed for the analysis of bacterial sequences; however, the analysis of fungi is still in its infancy. Here, we present a web server dedicated to the comprehensive analysis of the human mycobiome for (i) translating raw sequencing reads to data tables and high-standard figures, (ii) integrating statistical analysis and machine learning with a manually curated relational database and (iii) comparing the user’s uploaded datasets with publicly available from the Sequence Read Archive. Using 1,266 publicly available Internal transcribed spacers (ITS) samples, we demonstrated the utility of DAnIEL web server on large scale datasets and show the differences in fungal communities between human skin and soil sites.

Summary: Insights into the genomic consequences of symbiosis for basidiomycete fungi associated with social insects remain sparse. Capitalizing on viability of spores from centuries-old herbarium specimens of free-living, facultative, and specialist termite-associated Podaxis fungi, we obtained genomes of 10 specimens, including two type species described by Linnaeus >240 years ago. We document that the transition to termite association was accompanied by significant reductions in genome size and gene content, accelerated evolution in protein-coding genes, and reduced functional capacities for oxidative stress responses and lignin degradation. Functional testing confirmed that termite specialists perform worse under oxidative stress, while all lineages retained some capacity to cleave lignin. Mitochondrial genomes of termite associates were significantly larger; possibly driven by smaller population sizes or reduced competition, supported by apparent loss of certain biosynthetic gene clusters. Our findings point to relaxed selection that mirrors genome traits observed among obligate endosymbiotic bacteria of many insects.

Bacterial biofilms profoundly influence the recruitment and settlement of marine invertebrates, critical steps for diverse marine processes such as the formation of coral reefs, the maintenance of marine fisheries, and the fouling of submerged surfaces. However, the complex composition of biofilms often makes the characterization of individual signals and regulatory mechanisms challenging.

Fungus-growing termites have optimized the decomposition of recalcitrant plant biomass to access valuable nutrients by engaging in a tripartite symbiosis with complementary contributions from a fungal mutualist and a codiversified gut microbiome. This complex symbiotic interplay makes them one of the most successful and important decomposers for carbon cycling in Old World ecosystems.

Actinobacteria

Antibiotics are commonly used in the Intensive Care Unit (ICU); however, several studies showed that the impact of antibiotics to prevent infection, multi-organ failure, and death in the ICU is less clear than their benefit on course of infection in the absence of organ dysfunction. We characterized here the compositional and metabolic changes of the gut microbiome induced by critical illness and antibiotics in a cohort of 75 individuals in conjunction with 2,180 gut microbiome samples representing 16 different diseases. We revealed an “infection-vulnerable” gut microbiome environment present only in critically ill treated with antibiotics (ICU+). Feeding of Caenorhabditis elegans with Bifidobacterium animalis and Lactobacillus crispatus, species that expanded in ICU+ patients, revealed a significant negative impact of these microbes on host viability and developmental homeostasis. These results suggest that antibiotic administration can dramatically impact essential functional activities in the gut related to immune responses more than critical illness itself, which might explain in part untoward effects of antibiotics in the critically ill.

Abstract Background Fibroblast-like synoviocytes (FLS) and CCR7− effector memory T (TEM) cells are two of the major cell types implicated in the progression of rheumatoid arthritis (RA). In particular, FLS become highly invasive, whereas TEM cells proliferate and secrete proinflammatory cytokines, during RA. FLS and T cells may also interact and influence each other’s phenotypes. Inhibition of the pathogenic phenotypes of both FLS and TEM cells can be accomplished by selectively blocking the predominant potassium channels that they upregulate during RA: KCa1.1 (BK, Slo1, MaxiK, KCNMA1) upregulated by FLS and Kv1.3 (KCNA3) upregulated by activated TEM cells. In this study, we investigated the roles of KCa1.1 and Kv1.3 in regulating the interactions between FLS and TEM cells and determined if combination therapies of KCa1.1- and Kv1.3-selective blockers are more efficacious than monotherapies in ameliorating disease in rat models of RA. Methods We used in vitro functional assays to assess the effects of selective KCa1.1 and Kv1.3 channel inhibitors on the interactions of FLS isolated from rats with collagen-induced arthritis (CIA) with syngeneic TEM cells. We also used flow cytometric analyses to determine the effects of KCa1.1 blockers on the expression of proteins used for antigen presentation on CIA-FLS. Finally, we used the CIA and pristane-induced arthritis models to determine the efficacy of combinatorial therapies of KCa1.1 and Kv1.3 blockers in reducing disease severity compared with monotherapies. Results We show that the interactions of FLS from rats with CIA and of rat TEM cells are regulated by KCa1.1 and Kv1.3. Inhibiting KCa1.1 on FLS reduces the ability of FLS to stimulate TEM cell proliferation and migration, and inhibiting Kv1.3 on TEM cells reduces TEM cells’ ability to enhance FLS expression of KCa1.1 and major histocompatibility complex class II protein, as well as stimulates their invasion. Furthermore, we show that combination therapies of selective KCa1.1 and Kv1.3 blockers are more efficacious than monotherapies at reducing signs of disease in two rat models of RA. Conclusions Our results demonstrate the importance of KCa1.1 and Kv1.3 in regulating FLS and TEM cells during RA, as well as the value of combined therapies targeting both of these cell types to treat RA.

Fungus-farming termites host gut microbial communities that contribute to the pre-digestion of plant biomass for manuring the fungal mutualist, and potentially to the production of defensive compounds that suppress antagonists. Termite colonies are characterized by complex division of labor and differences in diet between termite size (minor and major) and morphological (worker and soldier) castes, and this extends to the composition of their gut microbial communities. We hypothesized that gut metabolomes should mirror these differences and tested this through untargeted LC-MS/MS analyses of three South African species of fungus-farming termites. We found distinct metabolomes between species and across castes, especially between soldiers and workers. Primary metabolites dominate the metabolomes and the high number of overlapping features with the mutualistic fungus and plant material show distinct impacts of diet and the environment. The identification of a few bioactive compounds of likely microbial origin underlines the potential for compound discovery among the many unannotated features. Our untargeted approach provides a first glimpse into the complex gut metabolomes and our dereplication suggests the presence of bioactive compounds with potential defensive roles to be targeted in future studies.

Abstract The peptide HsTX1[R14A] is a potent and selective blocker of the voltage-gated potassium channel Kv1.3, which is a highly promising target for the treatment of autoimmune diseases and other conditions. In order to assess the biodistribution of this peptide, it was conjugated with NOTA and radiolabelled with copper-64. [64Cu]Cu-NOTA-HsTX1[R14A] was synthesised in high radiochemical purity and yield. The radiotracer was evaluated in vitro and in vivo. The biodistribution and PET studies after intravenous and subcutaneous injections showed similar patterns and kinetics. The hydrophilic peptide was rapidly distributed, showed low accumulation in most of the organs and tissues, and demonstrated high molecular stability in vitro and in vivo. The most prominent accumulation occurred in the epiphyseal plates of trabecular bones. The high stability and bioavailability, low normal-tissue uptake of [64Cu]Cu-NOTA-HsTX1[R14A], and accumulation in regions of up-regulated Kv channels both in vitro and in vivo demonstrate that HsTX1[R14A] represents a valuable lead for conditions treatable by blockade of the voltage-gated potassium channel Kv1.3. The pharmacokinetics shows that both intravenous and subcutaneous applications are viable routes for the delivery of this potent peptide.