Importance of quorum sensing crosstalk in the brown alga Saccharina latissima epimicrobiome.

Brown macroalgae are colonized by diverse microorganisms influencing the physiology of their host. However, cell-cell interactions within the surface microbiome (epimicrobiome) are largely unexplored, despite the significance of specific chemical mediators in maintaining host-microbiome homeostasis. In this study, by combining liquid chromatography coupled to mass spectrometry (LC-MS) analysis and bioassays, we demonstrated that the widely diverse fungal epimicrobiota of the brown alga Saccharina latissima can affect quorum sensing (QS), a type of cell-cell interaction, as well as bacterial biofilm formation. We also showed the ability of the bacterial epimicrobiota to form and inhibit biofilm growth, as well as to activate or inhibit QS pathways. Overall, we demonstrate that QS and anti-QS compounds produced by the epimicrobiota are key metabolites in these brown algal epimicrobiota communities and highlight the importance of exploring this epimicrobiome for the discovery of new bioactive compounds, including potentially anti-QS molecules with antifouling properties.

Empirical evidence for metabolic drift in plant and algal lipid biosynthesis pathways.

Metabolic pathway drift has been formulated as a general principle to help in the interpretation of comparative analyses between biosynthesis pathways. Indeed, such analyses often indicate substantial differences, even in widespread pathways that are sometimes believed to be conserved. Here, our purpose is to check how much this interpretation fits to empirical data gathered in the field of plant and algal biosynthesis pathways. After examining several examples representative of the diversity of lipid biosynthesis pathways, we explain why it is important to compare closely related species to gain a better understanding of this phenomenon. Furthermore, this comparative approach brings us to the question of how much biotic interactions are responsible for shaping this metabolic plasticity. We end up introducing some model systems that may be promising for further exploration of this question.

Exploring uranium bioaccumulation in the brown alga Ascophyllum nodosum: insights from multi-scale spectroscopy and imaging.

Legacy radioactive waste can be defined as the radioactive waste produced during the infancy of the civil nuclear industry's development in the mid-20th Century, a time when, unfortunately, waste storage and treatment were not well planned. The marine environment is one of the environmental compartments worth studying in this regard because of legacy waste in specific locations of the seabed. Comprising nearly 70% of the earth's service, the oceans are the largest and indeed the final destination for contaminated fresh waters. For this reason, long-term studies of the accumulation biochemical mechanisms of metallic radionuclides in the marine ecosystem are required. In this context the brown algal compartment may be ecologically relevant because of forming large and dense algal beds in coastal areas and potential important biomass for contamination. This report presents the first step in the investigation of uranium (U, an element used in the nuclear cycle) bioaccumulation in the brown alga Ascophyllum nodosum using a multi-scale spectroscopic and imaging approach. Contamination of A. nodosum specimens in closed aquaria at 13 °C was performed with a defined quantity of U(VI) (10-5 M). The living algal uptake was quantified by ICP-MS and a localization study in the various algal compartments was carried out by combining electronic microscopy imaging (SEM), X-ray Absorption spectroscopy (XAS) and micro X-ray Florescence (µ-XRF). Data indicate that the brown alga is able to concentrate U(VI) by an active bioaccumulation mechanism, reaching an equilibrium state after 200 h of daily contamination. A comparison between living organisms and dry biomass confirms a stress-response process in the former, with an average bioaccumulation factor (BAF) of 10 ± 2 for living specimens (90% lower compared to dry biomass, 142 ± 5). Also, these results open new perspectives for a potential use of A. nodosum dry biomass as uranium biosorbent. The different partial BAFs (bioaccumulation factors) range from 3 (for thallus) to 49 (for receptacles) leading to a compartmentalization of uranium within the seaweed. This reveals a higher accumulation capacity in the receptacles, the algal reproductive parts. SEM images highlight the different tissue distributions among the compartments with a superficial absorption in the thallus and lateral branches and several hotspots in the oospheres of the female individuals. A preliminary speciation XAS analysis identified a distinct U speciation in the gametes-containing receptacles as a pseudo-autunite phosphate phase. Similarly, XAS measurements on the lateral branches (XANES) were not conclusive with regards to the occurrence of an alginate-U complex in these tissues. Nonetheless, the hypothesis that alginate may play a role in the speciation of U in the algal thallus tissues is still under consideration.

Corrigendum: Identification of novel anti-amoebic pharmacophores from kinase inhibitor chemotypes.

[This corrects the article DOI: 10.3389/fmicb.2023.1149145.].

Aldehyde perception induces specific molecular responses in Laminaria digitata and affects algal consumption by a specialist grazer.

In the marine environment, distance signaling based on water-borne cues occurs during interactions between macroalgae and herbivores. In the brown alga Laminaria digitata from North-Atlantic Brittany, oligoalginates elicitation or grazing was shown to induce chemical and transcriptomic regulations, as well as emission of a wide range of volatile aldehydes, but their biological roles as potential defense or warning signals in response to herbivores remain unknown. In this context, bioassays using the limpet Patella pellucida and L. digitata were carried out for determining the effects of algal transient incubation with 4-hydroxyhexenal (4-HHE), 4-hydroxynonenal (4-HNE) and dodecadienal on algal consumption by grazers. Simultaneously, we have developed metabolomic and transcriptomic approaches to study algal molecular responses after treatments of L. digitata with these chemical compounds. The results indicated that, unlike the treatment of the plantlets with 4-HNE or dodecadienal, treatment with 4-HHE decreases algal consumption by herbivores at 100 ng.ml-1 . Moreover, we showed that algal metabolome was significantly modified according to the type of aldehydes, and more specifically the metabolite pathways linked to fatty acid degradation. RNAseq analysis further showed that 4-HHE at 100 ng.ml-1 can activate the regulation of genes related to oxylipin signaling pathways and specific responses, compared to oligoalginates elicitation. As kelp beds constitute complex ecosystems consisting of habitat and food source for marine herbivores, the algal perception of specific aldehydes leading to targeted molecular regulations could have an important biological role on kelps/grazers interactions.

Inferring and comparing metabolism across heterogeneous sets of annotated genomes using AuCoMe.

Comparative analysis of genome-scale metabolic networks (GSMNs) may yield important information on the biology, evolution, and adaptation of species. However, it is impeded by the high heterogeneity of the quality and completeness of structural and functional genome annotations, which may bias the results of such comparisons. To address this issue, we developed AuCoMe, a pipeline to automatically reconstruct homogeneous GSMNs from a heterogeneous set of annotated genomes without discarding available manual annotations. We tested AuCoMe with three data sets, one bacterial, one fungal, and one algal, and showed that it successfully reduces technical biases while capturing the metabolic specificities of each organism. Our results also point out shared and divergent metabolic traits among evolutionarily distant algae, underlining the potential of AuCoMe to accelerate the broad exploration of metabolic evolution across the tree of life.

Identification of novel anti-amoebic pharmacophores from kinase inhibitor chemotypes.

Acanthamoeba species, Naegleria fowleri, and Balamuthia mandrillaris are opportunistic pathogens that cause a range of brain, skin, eye, and disseminated diseases in humans and animals. These pathogenic free-living amoebae (pFLA) are commonly misdiagnosed and have sub-optimal treatment regimens which contribute to the extremely high mortality rates (>90%) when they infect the central nervous system. To address the unmet medical need for effective therapeutics, we screened kinase inhibitor chemotypes against three pFLA using phenotypic drug assays involving CellTiter-Glo 2.0. Herein, we report the activity of the compounds against the trophozoite stage of each of the three amoebae, ranging from nanomolar to low micromolar potency. The most potent compounds that were identified from this screening effort were: 2d (A. castellanii EC50: 0.92 ± 0.3 µM; and N. fowleri EC50: 0.43 ± 0.13 µM), 1c and 2b (N. fowleri EC50s: <0.63 µM, and 0.3 ± 0.21 µM), and 4b and 7b (B. mandrillaris EC50s: 1.0 ± 0.12 µM, and 1.4 ± 0.17 µM, respectively). With several of these pharmacophores already possessing blood-brain barrier (BBB) permeability properties, or are predicted to penetrate the BBB, these hits present novel starting points for optimization as future treatments for pFLA-caused diseases.

Chemical Optimization of CBL0137 for Human African Trypanosomiasis Lead Drug Discovery.

The carbazole CBL0137 (1) is a lead for drug development against human African trypanosomiasis (HAT), a disease caused by Trypanosoma brucei. To advance 1 as a candidate drug, we synthesized new analogs that were evaluated for the physicochemical properties, antitrypanosome potency, selectivity against human cells, metabolism in microsomes or hepatocytes, and efflux ratios. Structure-activity/property analyses of analogs revealed eight new compounds with higher or equivalent selectivity indices (5j, 5t, 5v, 5w, 5y, 8d, 13i, and 22e). Based on the overall compound profiles, compounds 5v and 5w were selected for assessment in a mouse model of HAT; while 5v demonstrated a lead-like profile for HAT drug development, 5w showed a lack of efficacy. Lessons from these studies will inform further optimization of carbazoles for HAT and other indications.

Iodine uptake in brown seaweed exposed to radioactive liquid discharges from the reprocessing plant of ORANO La Hague.

Iodine-129 is present in controlled liquid radioactive waste routinely released in seawater by the ORANO nuclear fuel reprocessing plant in La Hague (Normandy, France). Brown algae are known for their exceptional ability to concentrate iodine from seawater. They also potentially emit volatile iodine compounds in response to various stresses, such as during emersion at low tide. For these reasons, brown seaweed is routinely collected for radioactivity monitoring in the marine environment (Fucus serratus and Laminaria digitata). Despite the high concentration ratio, the exact mechanism of iodine uptake is still unclear. Chemical imaging by laser desorption/ionization mass spectrometry provided evidence that iodine is stored by kelps as I-. In this study we investigate in vivo iodine uptake in kelps (L. digitata) with an emphasis on seawater iodine chemical speciation. Our results showed that kelp plantlets were able to take up iodine in the forms of both IO3- and I-. We also observed transient net efflux of I- back to seawater but no IO3- efflux. Since the seaweed stores I- but takes up both IO3- and I-, IO3- was likely to be converted into I- at some point in the plantlet. One major outcome of our experiments was the direct observation of the kelp-based biogenic conversion of seawater IO3- into I-. On the basis of both IO3- and I- uptakes by the seaweed, we propose new steps in the possible iodine concentration mechanism used by brown algae.

X-ray structure and mechanism of ZgHAD, a l-2-haloacid dehalogenase from the marine Flavobacterium Zobellia galactanivorans.

Haloacid dehalogenases are potentially involved in bioremediation of contaminated environments and few have been biochemically characterized from marine organisms. The l-2-haloacid dehalogenase (l-2-HAD) from the marine Bacteroidetes Zobellia galactanivorans DsijT (ZgHAD) has been shown to catalyze the dehalogenation of C2 and C3 short-chain l-2-haloalkanoic acids. To better understand its catalytic properties, its enzymatic stability, active site, and 3D structure were analyzed. ZgHAD demonstrates high stability to solvents and a conserved catalytic activity when heated up to 60°C, its melting temperature being at 65°C. The X-ray structure of the recombinant enzyme was solved by molecular replacement. The enzyme folds as a homodimer and its active site is very similar to DehRhb, the other known l-2-HAD from a marine Rhodobacteraceae. Marked differences are present in the putative substrate entrance sites of the two enzymes. The H179 amino acid potentially involved in the activation of a catalytic water molecule was confirmed as catalytic amino acid through the production of two inactive site-directed mutants. The crystal packing of 13 dimers in the asymmetric unit of an active-site mutant, ZgHAD-H179N, reveals domain movements of the monomeric subunits relative to each other. The involvement of a catalytic His/Glu dyad and substrate binding amino acids was further confirmed by computational docking. All together our results give new insights into the catalytic mechanism of the group of marine l-2-HAD.

JDQ443, a Structurally Novel, Pyrazole-Based, Covalent Inhibitor of KRASG12C for the Treatment of Solid Tumors.

Rapid emergence of tumor resistance via RAS pathway reactivation has been reported from clinical studies of covalent KRASG12C inhibitors. Thus, inhibitors with broad potential for combination treatment and distinct binding modes to overcome resistance mutations may prove beneficial. JDQ443 is an investigational covalent KRASG12C inhibitor derived from structure-based drug design followed by extensive optimization of two dissimilar prototypes. JDQ443 is a stable atropisomer containing a unique 5-methylpyrazole core and a spiro-azetidine linker designed to position the electrophilic acrylamide for optimal engagement with KRASG12C C12. A substituted indazole at pyrazole position 3 results in novel interactions with the binding pocket that do not involve residue H95. JDQ443 showed PK/PD activity in vivo and dose-dependent antitumor activity in mouse xenograft models. JDQ443 is now in clinical development, with encouraging early phase data reported from an ongoing Phase Ib/II clinical trial (NCT04699188).

Pathogen exposure leads to a transcriptional downregulation of core cellular functions that may dampen the immune response in a macroalga.

Diseases in marine eukaryotic organisms caused by opportunistic pathogens represent a serious threat to our oceans with potential downstream consequences for ecosystem functioning. Disease outbreaks affecting macroalgae are of particular concern due to their critical role as habitat-forming organisms. However, there is limited understanding of the molecular strategies used by macroalgae to respond to opportunistic pathogens. In this study, we used mRNA-sequencing analysis to investigate the early antipathogen response of the model macroalga Delisea pulchra (Rhodophyta) under the environmental conditions that are known to promote the onset of disease. Using de novo assembly methods, 27,586 unique transcripts belonging to D. pulchra were identified that were mostly affiliated with stress response and signal transduction processes. Differential gene expression analysis between a treatment with the known opportunistic pathogen, Aquimarina sp. AD1 (Bacteroidota), and a closely related benign strain (Aquimarina sp. AD10) revealed a downregulation of genes coding for predicted protein metabolism, stress response, energy generation and photosynthesis functions. The rapid repression of genes coding for core cellular processes is likely to interfere with the macroalgal antipathogen response, later leading to infection, tissue damage and bleaching symptoms. Overall, this study provides valuable insight into the genetic features of D. pulchra, highlighting potential antipathogen response mechanisms of macroalgae and contributing to an improved understanding of host-pathogen interactions in a changing environment.

Discovery, Preclinical Characterization, and Early Clinical Activity of JDQ443, a Structurally Novel, Potent, and Selective Covalent Oral Inhibitor of KRASG12C.

Covalent inhibitors of KRASG12C have shown antitumor activity against advanced/metastatic KRASG12C-mutated cancers, though resistance emerges and additional strategies are needed to improve outcomes. JDQ443 is a structurally unique covalent inhibitor of GDP-bound KRASG12C that forms novel interactions with the switch II pocket. JDQ443 potently inhibits KRASG12C-driven cellular signaling and demonstrates selective antiproliferative activity in KRASG12C-mutated cell lines, including those with G12C/H95 double mutations. In vivo, JDQ443 induces AUC exposure-driven antitumor efficacy in KRASG12C-mutated cell-derived (CDX) and patient-derived (PDX) tumor xenografts. In PDX models, single-agent JDQ443 activity is enhanced by combination with inhibitors of SHP2, MEK, or CDK4/6. Notably, the benefit of JDQ443 plus the SHP2 inhibitor TNO155 is maintained at reduced doses of either agent in CDX models, consistent with mechanistic synergy. JDQ443 is in clinical development as monotherapy and in combination with TNO155, with both strategies showing antitumor activity in patients with KRASG12C-mutated tumors. SIGNIFICANCE: JDQ443 is a structurally novel covalent KRASG12C inhibitor with a unique binding mode that demonstrates potent and selective antitumor activity in cell lines and in vivo models. In preclinical models and patients with KRASG12C-mutated malignancies, JDQ443 shows potent antitumor activity as monotherapy and in combination with the SHP2 inhibitor TNO155. This article is highlighted in the In This Issue feature, p. 1397.

Functional Characterization of a L-2-Haloacid Dehalogenase From Zobellia galactanivorans DsijT Suggests a Role in Haloacetic Acid Catabolism and a Wide Distribution in Marine Environments.

L-2-halocid dehalogenases (L-2-HADs) have been mainly characterized from terrestrial polluted environments. By contrast, knowledge is still scarce about their role in detoxification of predominant halocarbons in marine environments. Here, phylogenetic analyses showed a wide diversity of homologous L-2-HADs, especially among those belonging to marine bacteria. Previously characterized terrestrial L-2-HADs were part of a monophyletic group (named group A) including proteins of terrestrial and marine origin. Another branch (named group B) contained mostly marine L-2-HADs, with two distinct clades of Bacteroidetes homologs, closely linked to Proteobacteria ones. This study further focused on the characterization of the only L-2-HAD from the flavobacterium Zobellia galactanivorans DsijT (ZgHAD), belonging to one of these Group B clades. The recombinant ZgHAD was shown to dehalogenate bromo- and iodoacetic acids, and gene knockout in Z. galactanivorans revealed a direct role of ZgHAD in tolerance against both haloacetic acids. Analyses of metagenomic and metatranscriptomic datasets confirmed that L-2-HADs from group A were well-represented in terrestrial and marine bacteria, whereas ZgHAD homologs (group B L-2-HADs) were mainly present in marine bacteria, and particularly in host-associated species. Our results suggest that ZgHAD homologs could be key enzymes for marine Bacteroidetes, by conferring selective advantage for the recycling of toxic halogen compounds produced in particular marine habitats, and especially during interactions with macroalgae.

Synergistic effects of temperature and light affect the relationship between Taonia atomaria and its epibacterial community: a controlled conditions study.

In the context of global warming, this study aimed to assess the effect of temperature and irradiance on the macroalgal Taonia atomaria holobiont dynamics. We developed an experimental set-up using aquaria supplied by natural seawater with three temperatures combined with three irradiances. The holobiont response was monitored over 14 days using a multi-omics approach coupling algal surface metabolomics and metabarcoding. Both temperature and irradiance appeared to shape the microbiota and the surface metabolome, but with a distinct temporality. Epibacterial community first changed according to temperature, and later in relation to irradiance, while the opposite occurred for the surface metabolome. An increased temperature revealed a decreasing richness of the epiphytic community together with an increase of several bacterial taxa. Irradiance changes appeared to quickly impact surface metabolites production linked with the algal host photosynthesis (e.g. mannitol, fucoxanthin, dimethylsulfoniopropionate), which was hypothesized to explain modifications of the structure of the epiphytic community. Algal host may also directly adapt its surface metabolome to changing temperature with time (e.g. lipids content) and also in response to changing microbiota (e.g. chemical defences). Finally, this study brought new insights highlighting complex direct and indirect responses of seaweeds and their associated microbiota under changing environments.

Semi-Quantitative Targeted Gas Chromatography-Mass Spectrometry Profiling Supports a Late Side-Chain Reductase Cycloartenol-to-Cholesterol Biosynthesis Pathway in Brown Algae.

Sterols are biologically important molecules that serve as membrane fluidity regulators and precursors of signaling molecules, either endogenous or involved in biotic interactions. There is currently no model of their biosynthesis pathways in brown algae. Here, we benefit from the availability of genome data and gas chromatography-mass spectrometry (GC-MS) sterol profiling using a database of internal standards to build such a model. We expand the set of identified sterols in 11 species of red, brown, and green macroalgae and integrate these new data with genomic data. Our analyses suggest that some metabolic reactions may be conserved despite the loss of canonical eukaryotic enzymes, like the sterol side-chain reductase (SSR). Our findings are consistent with the principle of metabolic pathway drift through enzymatic replacement and show that cholesterol synthesis from cycloartenol may be a widespread but variable pathway among chlorophyllian eukaryotes. Among the factors contributing to this variability, one could be the recruitment of cholesterol biosynthetic intermediates to make signaling molecules, such as the mozukulins. These compounds were found in some brown algae belonging to Ectocarpales, and we here provide a first mozukulin biosynthetic model. Our results demonstrate that integrative approaches can already be used to infer experimentally testable models, which will be useful to further investigate the biological roles of those newly identified algal pathways.

A community perspective on the concept of marine holobionts: current status, challenges, and future directions.

Host-microbe interactions play crucial roles in marine ecosystems. However, we still have very little understanding of the mechanisms that govern these relationships, the evolutionary processes that shape them, and their ecological consequences. The holobiont concept is a renewed paradigm in biology that can help to describe and understand these complex systems. It posits that a host and its associated microbiota with which it interacts, form a holobiont, and have to be studied together as a coherent biological and functional unit to understand its biology, ecology, and evolution. Here we discuss critical concepts and opportunities in marine holobiont research and identify key challenges in the field. We highlight the potential economic, sociological, and environmental impacts of the holobiont concept in marine biological, evolutionary, and environmental sciences. Given the connectivity and the unexplored biodiversity specific to marine ecosystems, a deeper understanding of such complex systems requires further technological and conceptual advances, e.g., the development of controlled experimental model systems for holobionts from all major lineages and the modeling of (info)chemical-mediated interactions between organisms. Here we propose that one significant challenge is to bridge cross-disciplinary research on tractable model systems in order to address key ecological and evolutionary questions. This first step is crucial to decipher the main drivers of the dynamics and evolution of holobionts and to account for the holobiont concept in applied areas, such as the conservation, management, and exploitation of marine ecosystems and resources, where practical solutions to predict and mitigate the impact of human activities are more important than ever.

Mass spectrometry - based imaging techniques for iodine-127 and iodine-129 detection and localization in the brown alga Laminaria digitata.

129I is one of the main radioisotopes of iodine derived from the nuclear fuel cycle that can be found sustainably in the environment due to its long half-life. In coastal marine environment, brown macroalgae, such laminariales (or kelps), are known to naturally feature highest rates of iodine accumulation, and to be an important source of biogenic volatile iodinated compounds released to the atmosphere. These seaweeds are therefore likely to be significantly marked by but also potential vectors of radioactive iodine. In order to better understand the chemical and isotopic speciation of iodine in brown algal tissues, we combined mass spectrometry-based imaging approaches in natural samples of Laminaria digitata young sporophytes, collected at two different locations along the south coast of the English Channel (Roscoff and Goury). Laser desorption ionization (LDI) and desorption electrospray-ionization techniques (DESI), coupled with mass spectrometry, confirmed the predominance of inorganic I- species on the surface of fresh algae, and a peripheral iodine localization when applied on micro-sections. Moreover, radioactive isotope 129I was not detected on plantlet surface or in stipe sections of algal samples collected near Roscoff but was detected in L. digitata samples collected at Goury, near La Hague, where controlled liquid radioactive discharges from the ORANO La Hague reprocessing plant occur. At the subcellular scale, cryo-fixed micro-sections of algal blade samples from both sites were further analyzed by secondary ion mass spectrometry (nano-SIMS), leading to similar results. Even if the signal detected for 129I was much weaker than for 127I in samples from Goury, the chemical imaging revealed some differences in extracellular distribution between radioactive and stable iodine isotopes. Altogether LDI and nano-SIMS are complementary and powerful techniques for the detection and localization of iodine isotopes in algal samples, and for a better understanding of radioactive and stable iodine uptake mechanisms in the marine environment.

Accumulation of detached kelp biomass in a subtidal temperate coastal ecosystem induces succession of epiphytic and sediment bacterial communities.

Kelps are dominant primary producers in temperate coastal ecosystems. Large amounts of kelp biomass can be exported to the seafloor during the algal growth cycle or following storms, creating new ecological niches for the associated microbiota. Here, we investigated the bacterial community associated with the kelp Laminaria hyperborea during its accumulation and degradation on the seafloor. Kelp tissue, seawater and sediment were sampled during a 6-month in situ experiment simulating kelp detritus accumulation. Evaluation of the epiphytic bacterial community abundance, structure, taxonomic composition and predicted functional profiles evidenced a biphasic succession. Initially, dominant genera (Hellea, Litorimonas, Granulosicoccus) showed a rapid and drastic decrease in sequence abundance, probably outcompeted by algal polysaccharide-degraders such as Bacteroidia members which responded within 4 weeks. Acidimicrobiia, especially members of the Sva0996 marine group, colonized the degrading kelp biomass after 11 weeks. These secondary colonizers could act as opportunistic scavenger bacteria assimilating substrates exposed by early degraders. In parallel, kelp accumulation modified bacterial communities in the underlying sediment, notably favouring anaerobic taxa potentially involved in the sulfur and nitrogen cycles. Overall, this study provides insights into the bacterial degradation of algal biomass in situ, an important link in coastal trophic chains.

Correction to "2-Formylpyridyl Ureas as Highly Selective Reversible-Covalent Inhibitors of Fibroblast Growth Factor Receptor 4".

[This corrects the article DOI: 10.1021/acsmedchemlett.7b00485.].