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Abstract Background Staphylococcus aureus is the predominant pathogen isolated in diabetic foot infections. Recently, the skin commensal bacterium, Helcococcus kunzii, was found to modulate the virulence of this pathogen in an in vivo model. This study aims to elucidate the molecular mechanisms underlying the interaction between these two bacterial species, using a proteomic approach. Results Our results reveal that H. kunzii can coexist and proliferate alongside S. aureus in a Chronic Wound Media (CWM), thereby mimicking an in vitro chronic wound environment. We noted that the secreted proteome of H. kunzii induced a transcriptional effect on S. aureus virulence, resulting in a decrease in the expression level of agrA, a gene involved in quorum sensing. The observed effect could be ascribed to specific proteins secreted by H. kunzii including polysaccharide deacetylase, peptidoglycan DD-metalloendopeptidase, glyceraldehyde-3-phosphate dehydrogenase, trypsin-like peptidase, and an extracellular solute-binding protein. These proteins potentially interact with the agr system, influencing S. aureus virulence. Additionally, the virulence of S. aureus was notably affected by modifications in iron-related pathways and components of cell wall architecture in the presence of H. kunzii. Furthermore, the overall metabolism of S. aureus was reduced when cocultured with H. kunzii. Conclusion Future research will focus on elucidating the role of these excreted factors in modulating virulence.

Abstract Background By analyzing the proteins which are the workhorses of biological systems, metaproteomics allows us to list the taxa present in any microbiota, monitor their relative biomass, and characterize the functioning of complex biological systems. Results Here, we present a new strategy for rapidly determining the microbial community structure of a given sample and designing a customized protein sequence database to optimally exploit extensive tandem mass spectrometry data. This approach leverages the capabilities of the first generation of Quadrupole Orbitrap mass spectrometer incorporating an asymmetric track lossless (Astral) analyzer, offering rapid MS/MS scan speed and sensitivity. We took advantage of data-dependent acquisition and data-independent acquisition strategies using a peptide extract from a human fecal sample spiked with precise amounts of peptides from two reference bacteria. Conclusions Our approach, which combines both acquisition methods, proves to be time-efficient while processing extensive generic databases and massive datasets, achieving a coverage of more than 122,000 unique peptides and 38,000 protein groups within a 30-min DIA run. This marks a significant departure from current state-of-the-art metaproteomics methodologies, resulting in broader coverage of the metabolic pathways governing the biological system. In combination, our strategy and the Astral mass analyzer represent a quantum leap in the functional analysis of microbiomes. Video Abstract

Hyperglycemia increases the heart sensitivity to ischemia-reperfusion (IR), but the underlying cellular mechanisms remain unclear. Mitochondrial dynamics (the processes that govern mitochondrial morphology and their interactions with other organelles, such as the reticulum), has emerged as a key factor in the heart vulnerability to IR. However, it is unknown whether mitochondrial dynamics contributes to hyperglycemia deleterious effect during IR. We hypothesized that (i) the higher heart vulnerability to IR in hyperglycemic conditions could be explained by hyperglycemia effect on the complex interplay between mitochondrial dynamics, Ca2+ homeostasis, and reactive oxygen species (ROS) production; and (ii) the activation of DRP1, a key regulator of mitochondrial dynamics, could play a central role. Using transmission electron microscopy and proteomic analysis, we showed that the interactions between sarcoplasmic reticulum and mitochondria and mitochondrial fission were increased during IR in isolated rat hearts perfused with a hyperglycemic buffer compared with hearts perfused with a normoglycemic buffer. In isolated mitochondria and cardiomyocytes, hyperglycemia increased mitochondrial ROS production and Ca2+ uptake. This was associated with higher RyR2 instability. These results could contribute to explain the early mPTP activation in mitochondria from isolated hearts perfused with a hyperglycemic buffer and in hearts from streptozotocin-treated rats (to increase the blood glucose). DRP1 inhibition by Mdivi-1 during the hyperglycemic phase and before IR induction, normalized Ca2+ homeostasis, ROS production, mPTP activation, and reduced the heart sensitivity to IR in streptozotocin-treated rats. In conclusion, hyperglycemia-dependent DRP1 activation results in higher reticulum-mitochondria calcium exchange that contribute to the higher heart vulnerability to IR.

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.

Abstract Proteogenomic methodologies have enabled the identification of protein sequences in wild species without annotated genomes, shedding light on molecular mechanisms affected by pollution. However, proteomic resources for sentinel species are limited, and organ-level investigations are necessary to expand our understanding of their molecular biology. This study presents proteomic resources obtained from proteogenomic analyses of key organs (hepatopancreas, gills, hemolymph) from three established aquatic sentinel invertebrate species of interest in ecotoxicological/ecological research and environmental monitoring: Gammarus fossarum, Dreissena polymorpha, and Palaemon serratus. Proteogenomic analyses identified thousands of proteins for each species, with over 90% of them being annotated to putative function. Functional analysis validated the relevance of the proteomic atlases by revealing similarities in functional annotation of catalogues of proteins across analogous organs in the three species, while deep contrasts between functional profiles are delimited across different organs in the same organism. These organ-level proteomic atlases are crucial for future research on these sentinel animals, aiding in the evaluation of aquatic environmental risks and providing a valuable resource for ecotoxicological studies.

Coriolopsis trogii is a basidiomycete fungus which utilizes a large array of lignin-modifying enzymes to colonize and decompose dead wood. Its extracellular enzymatic arsenal includes laccases, i.e., polyphenol oxidases of relevant interest for different industrial applications thanks to their ability to oxidize a diverse range of natural and synthetic compounds. In this work, the production of laccases in C. trogii MUT3379 was explored and improved. From an initial production of ca. 10,000 U L−1, the fermentation process was gradually optimized, reaching a final yield of ca. 200,000 U L−1. An SDS-PAGE analysis of the secretome highlighted the presence of a main protein of ca. 60 kDa showing laccase activity, which was designated as Lac3379-1 once its primary sequence was established by tandem mass spectrometry. The characterization of Lac3379-1 revealed a remarkable enzymatic stability in the presence of surfactants and solvents and a diversified activity on a broad range of substrates, positioning it as an interesting tool for diverse biotechnological applications. The high-yield and robust production process indicates C. trogii MUT3379 as a promising cell factory for laccases, offering new perspectives for industrial applications of lignin-modifying enzymes.

Abstract Background Individual functional modifications shape the ability of wildlife populations to cope with anthropogenic environmental changes. But instead of adaptive response, human-altered environments can generate a succession of deleterious functional changes leading to the extinction of the population. To study how persistent anthropogenic changes impacted local species’ population status, we characterised population structure, genetic diversity and individual response of gene expression in the tree frog Hyla orientalis along a gradient of radioactive contamination around the Chernobyl nuclear power plant. Results We detected lower effective population size in populations most exposed to ionizing radiation in the Chernobyl Exclusion Zone that is not compensated by migrations from surrounding areas. We also highlighted a decreased body condition of frogs living in the most contaminated area, a distinctive transcriptomics signature and stop-gained mutations in genes involved in energy metabolism. While the association with dose will remain correlational until further experiments, a body of evidence suggests the direct or indirect involvement of radiation exposure in these changes. Conclusions Despite ongoing migration and lower total dose rates absorbed than at the time of the accident, our results demonstrate that Hyla orientalis specimens living in the Chernobyl Exclusion Zone are still undergoing deleterious changes, emphasizing the long-term impacts of the nuclear disaster.

Abstract Lytic polysaccharide monooxygenases (LPMOs) are taxonomically widespread copper-enzymes boosting biopolymers conversion (e.g. cellulose, chitin) in Nature. White-rot Polyporales, which are major fungal wood decayers, may possess up to 60 LPMO-encoding genes belonging to the auxiliary activities family 9 (AA9). Yet, the functional relevance of such multiplicity remains to be uncovered. Previous comparative transcriptomic studies of six Polyporales fungi grown on cellulosic substrates had shown the overexpression of numerous AA9-encoding genes, including some holding a C-terminal domain of unknown function (“X282”). Here, after carrying out structural predictions and phylogenetic analyses, we selected and characterized six AA9-X282s with different C-term modularities and atypical features hitherto unreported. Unexpectedly, after screening a large array of conditions, these AA9-X282s showed only weak binding properties to cellulose, and low to no cellulolytic oxidative activity. Strikingly, proteomic analysis revealed the presence of multiple phosphorylated residues at the surface of these AA9-X282s, including a conserved residue next to the copper site. Further analyses focusing on a 9 residues glycine-rich C-term extension suggested that it could hold phosphate-binding properties. Our results question the involvement of these AA9 proteins in the degradation of plant cell wall and open new avenues as to the divergence of function of some AA9 members.

Abstract Background Osteoarthritis is an age-related disease that currently faces a lack of symptomatic treatment. Inflammation, which is mainly sustained by pro-inflammatory cytokines such as IL-1b, TNF, and IL-6, plays an important role in osteoarthritis progression. In this context, pro-inflammatory cytokines are widely used to mimic the inflammatory component of osteoarthritis in vitro. However, the therapeutic failures of clinical trials evaluating anti-cytokines drugs highlight the lack of overall understanding of the effects of these cytokines on chondrocytes. Methods Here, we generated a comprehensive transcriptomic and proteomic dataset of osteoarthritic chondrocytes treated with these cytokines to describe their pro-inflammatory signature and compare it to the transcriptome of non-osteoarthritic chondrocytes. Then, the dysregulations highlighted at the molecular level were functionally confirmed by real-time cellular metabolic assays. Results We identified dysregulation of metabolic-related genes in osteoarthritic chondrocytes but not in non-osteoarthritic chondrocytes. A metabolic shift, toward increased glycolysis at the expense of mitochondrial respiration, was specifically confirmed in osteoarthritic chondrocytes treated with IL-1b or TNF. Conclusion These data show a strong and specific association between inflammation and metabolism in osteoarthritic chondrocytes, which was not found in non-osteoarthritic chondrocytes. This indicates that the link between inflammation and metabolic dysregulation may be exacerbated during chondrocyte damage in osteoarthritis. Video Abstract

Objectives: At the time when the COVID-19 pandemic was responsible for more than six million deaths worldwide, the antiquity of coronaviruses remains undefined. We investigated individuals buried during the 16th century in France for the direct and paleoserological diagnosis of the coronavirus. Methods: The 2011-2012 excavation of Abbey Saint-Pierre in Baume-Les-Messieurs, France uncovered 12 skeletons of individuals from the 13th to the 18th century. The total proteins extracted from dental pulps were subjected to microbial paleoserology, targeting SARS-CoV-2, human-associated coronavirus (HCoV)-229E, and OC43 antigens and for coronavirus peptide research using metaproteomics, in parallel to negative controls. Results: Three peptide sequences totaling 36 amino acids indicative of a coronavirus were retrieved from the dental pulp remains collected from two individuals buried circa 16th century, in whom paleoserology confirmed a specific immunological response against modern-day SARS-CoV-2 and HCoV-229E. Conclusion: We provide serological and proteomic evidence for a betacoronavirus with no modern correspondent, infecting populations in the 16th century, extending the antiquity of coronaviruses by more than three centuries. Historical, archaeozoological, and paleoproteomic data suggested close contacts between these two individuals and domestic swine, cattle, and poultry, suggesting an ancient zoonotic coronavirus. Coronaviruses have been undesirable companions of populations long before the ongoing coronavirus disease 2019 outbreak emerged.

Abstract Lignocellulosic residues are amongst the most abundant waste products on Earth. Therefore, there is an increasing interest in the utilization of these residues for bioethanol production and for biorefineries to produce compounds of industrial interest. Enzymes that breakdown cellulose and hemicellulose into oligomers and monosaccharides are required in these processes and cellulolytic enzymes with optimum activity at a low pH area are desirable for industrial processes. Here, we explore the fungal biodiversity of Rıo Tinto, the largest acidic ecosystem on Earth, as far as the secretion of cellulolytic enzymes is concerned. Using colorimetric and industrial substrates, we show that a high proportion of the fungi present in this extremophilic environment secrete a wide range of enzymes that are able to hydrolyze cellulose and hemicellulose at acidic pH (4.5–5). Shotgun proteomic analysis of the secretomes of some of these fungi has identified different cellulases and hemicellulolytic enzymes as well as a number of auxiliary enzymes. Supplementation of pre‐industrial cocktails from Myceliophtora with Rio Tinto secretomes increased the amount of monosaccharides released from corn stover or sugar cane straw. We conclude that the Rio Tinto fungi display a good variety of hydrolytic enzymes with high industrial potential.

Despite various plans to rationalize antibiotic use, antibiotic resistance in environmental bacteria is increasing due to the accumulation of antibiotic residues in the environment. This study aimed to test the ability of basidiomycete fungal strains to biotransform the antibiotic levofloxacin, a widely-used third-generation broad-spectrum fluoroquinolone, and to propose enzyme targets potentially involved in this biotransformation. The biotransformation process was performed using fungal strains. Levofloxacin biotransformation reached 100% after 9 days of culture with Porostereum spadiceum BS34. Using genomics and proteomics analyses coupled with activity tests, we showed that P. spadiceum produces several heme-peroxidases together with H2O2-producing enzymes that could be involved in the antibiotic biotransformation process. Using UV and high-resolution mass spectrometry, we were able to detect five levofloxacin degradation products. Their putative identity based on their MS2 fragmentation patterns led to the conclusion that the piperazine moiety was the main target of oxidative modification of levofloxacin by P. spadiceum, leading to a decrease in antibiotic activity.

ABSTRACTThe Artificial Gravity Bed Rest – European Space Agency (AGBRESA) study was the first joint bed rest study by ESA, DLR, and NASA that examined the effect of simulated weightlessness on the human body and assessed the potential benefits of artificial gravity as a countermeasure in an analog of long-duration spaceflight. In this study, we investigated the impact of simulated microgravity on the gut microbiome of 12 participants during a 60-day head-down tilt bed rest at the :envihab facilities. Over 60 days of simulated microgravity resulted in a mild change in the gut microbiome, with distinct microbial patterns and pathway expression in the feces of the countermeasure group compared to the microgravity simulation-only group. Additionally, we found that the countermeasure protocols selectively increased the abundance of beneficial short-chain fatty acids in the gut, such as acetate, butyrate, and propionate. Some physiological signatures also included the modulation of taxa reported to be either beneficial or opportunistic, indicating a mild adaptation in the microbiome network balance. Our results suggest that monitoring the gut microbial catalog along with pathway clustering and metabolite profiling is an informative synergistic strategy to determine health disturbances and the outcome of countermeasure protocols for future space missions.

This study aims at identifying molecular biomarkers differentiating responders and non-responders to treatment with Tumor Necrosis Factor inhibitors (TNFi) among patients with axial spondyloarthritis (axSpA). Whole blood mRNA and plasma proteins were measured in a cohort of biologic-naïve axSpA patients (n = 35), pre and post (14 weeks) TNFi treatment with adalimumab. Differential expression analysis was used to identify the most enriched pathways and in predictive models to distinguish responses to TNFi. A treatment-associated signature suggests a reduction in inflammatory activity. We found transcripts and proteins robustly differentially expressed between baseline and week 14 in responders. C-reactive protein (CRP) and Haptoglobin (HP) proteins showed strong and early decrease in the plasma of axSpA patients, while a cluster of apolipoproteins (APOD, APOA2, APOA1) showed increased expression at week 14. Responders to TNFi treatment present higher levels of markers of innate immunity at baseline, and lower levels of adaptive immunity markers, particularly B-cells. A logistic regression model incorporating ASDAS-CRP, gender, and AFF3, the top differentially expressed gene at baseline, enabled an accurate prediction of response to adalimumab in our cohort (AUC = 0.97). In conclusion, innate and adaptive immune cell type composition at baseline may be a major contributor to response to adalimumab in axSpA patients. A model including clinical and gene expression variables should also be considered.

Creatine transporter deficiency (CTD) is an X-linked disease caused by mutations in the SLC6A8 gene. The impaired creatine uptake in the brain results in intellectual disability, behavioral disorders, language delay, and seizures. In this work, we generated human brain organoids from induced pluripotent stem cells of healthy subjects and CTD patients. Brain organoids from CTD donors had reduced creatine uptake compared with those from healthy donors. The expression of neural progenitor cell markers SOX2 and PAX6 was reduced in CTD-derived organoids, while GSK3β, a key regulator of neurogenesis, was up-regulated. Shotgun proteomics combined with integrative bioinformatic and statistical analysis identified changes in the abundance of proteins associated with intellectual disability, epilepsy, and autism. Re-establishment of the expression of a functional SLC6A8 in CTD-derived organoids restored creatine uptake and normalized the expression of SOX2, GSK3β, and other key proteins associated with clinical features of CTD patients. Our brain organoid model opens new avenues for further characterizing the CTD pathophysiology and supports the concept that reinstating creatine levels in patients with CTD could result in therapeutic efficacy.

ABSTRACT The xanthine oxidoreductase (XOR) family are metal-containing enzymes that use the molybdenum cofactor (Moco), 2Fe-2S clusters, and flavin adenine dinucleotide (FAD) for their catalytic activity. This large molybdoenzyme family includes xanthine, aldehyde, and CO dehydrogenases. XORs are widely distributed from bacteria to humans due to their key roles in the catabolism of purines, aldehydes, drugs, and xenobiotics, as well as interconversions between CO and CO2. Assessing the effect of excess metals on the Rubrivivax gelatinosus bacterium, we found that exposure to copper (Cu) or cadmium (Cd) caused a dramatic decrease in the activity of a high-molecular-weight soluble complex exhibiting nitroblue tetrazolium reductase activity. Mass spectrometry and genetic analyses showed that the complex corresponds to a putative CO dehydrogenase (pCOD). Using mutants that accumulate either Cu+ or Cd2+ in the cytoplasm, we show that Cu+ or Cd2+ is a potent inhibitor of XORs (pCOD and the xanthine dehydrogenase [XDH]) in vivo. This is the first in vivo demonstration that Cu+ affects Moco-containing enzymes. The specific inhibitory effect of these compounds on the XOR activity is further supported in vitro by direct addition of competing metals to protein extracts. Moreover, emphasis is given on the inhibitory effect of Cu on bovine XOR, showing that the XOR family could be a common target of Cu. Given the conservation of XOR structure and function across the tree of life, we anticipate that our findings could be transferable to other XORs and organisms. IMPORTANCE The high toxicity of Cu, Cd, Pb, As, and other metals arises from their ability to cross membranes and target metalloenzymes in the cytoplasm. Identifying these targets provides insights into the toxicity mechanisms. The vulnerability of metalloenzymes arises from the accessibility of their cofactors to ions. Accordingly, many enzymes whose cofactors are solvent exposed are likely to be targets of competing metals. Here, we describe for the first time, with in vivo and in vitro experiments, a direct effect of excess Cu on the xanthine oxidoreductase family (XOR/XDH/pCOD). We show that toxic metal affects these Moco enzymes, and we suggest that access to the Moco center by Cu ions could explain the Cu inhibition of XORs in living organisms. Human XOR activity is associated with hyperuricemia, xanthinuria, gout arthritis, and other diseases. Our findings in vivo highlight XOR as a Cu target and thus support the potential use of Cu in metal-based therapeutics against these diseases.

IntroductionBurkholderia thailandensis is a study model for Burkholderia pseudomallei, a highly virulent pathogen, known to be the causative agent of melioidosis and a potential bioterrorism agent. These two bacteria use an (acyl-homoserine lactone) AHL-mediated quorum sensing (QS) system to regulate different behaviors including biofilm formation, secondary metabolite productions, and motility.MethodsUsing an enzyme-based quorum quenching (QQ) strategy, with the lactonase SsoPox having the best activity on B. thailandensis AHLs, we evaluated the importance of QS in B. thailandensis by combining proteomic and phenotypic analyses.ResultsWe demonstrated that QS disruption largely affects overall bacterial behavior including motility, proteolytic activity, and antimicrobial molecule production. We further showed that QQ treatment drastically decreases B. thailandensis bactericidal activity against two bacteria (Chromobacterium violaceum and Staphylococcus aureus), while a spectacular increase in antifungal activity was observed against fungi and yeast (Aspergillus niger, Fusarium graminearum and Saccharomyces cerevisiae).DiscussionThis study provides evidence that QS is of prime interest when it comes to understanding the virulence of Burkholderia species and developing alternative treatments.

Sampling small amounts of biofilm from harsh environments such as the biofilm present on the walls of a radioactive material storage pool offers few analytical options if taxonomic characterization and estimation of the different biomass contributions are the objectives. Although 16S/18S rRNA amplification on extracted DNA and sequencing is the most widely applied method, its reliability in terms of quantitation has been questioned as yields can be species-dependent. Here, we propose a tandem-mass spectrometry proteotyping approach consisting of acquiring peptide data and interpreting then against a generalist database without any a priori. The peptide sequence information is transformed into useful taxonomical information that allows to obtain the different biomass contributions at different taxonomical ranks. This new methodology is applied for the first time to analyze the composition of biofilms from minute quantities of material collected from a pool used to store radioactive sources in a nuclear facility. For these biofilms, we report the identification of three genera, namely Sphingomonas, Caulobacter, and Acidovorax, and their functional characterization by metaproteomics which shows that these organisms are metabolic active. Differential expression of Gene Ontology GOslim terms between the two main microorganisms highlights their metabolic specialization.

Creatine transporter deficiency (CTD), a leading cause of intellectual disability is a result of the mutation in the gene encoding the creatine transporter SLC6A8, which prevents creatine uptake into the brain, causing mental retardation, expressive speech and language delay, autistic-like behavior and epilepsy. Preclinical in vitro and in vivo data indicate that dodecyl creatine ester (DCE) which increases the creatine brain content, might be a therapeutic option for CTD patients. To gain a better understanding of the pathophysiology and DCE treatment efficacy in CTD, this study focuses on the identification of biomarkers related to cognitive improvement in a Slc6a8 knockout mouse model (Slc6a8−/y) engineered to mimic the clinical features of CTD patients which have low brain creatine content. Shotgun proteomics analysis of 4,035 proteins in four different brain regions; the cerebellum, cortex, hippocampus (associated with cognitive functions) and brain stem, and muscle as a control, was performed in 24 mice. Comparison of the protein abundance in the four brain regions between DCE-treated intranasally Slc6a8−/y mice and wild type and DCE-treated Slc6a8−/y and vehicle group identified 14 biomarkers, shedding light on the mechanism of action of DCE. Integrative bioinformatics and statistical modeling identified key proteins in CTD, including KIF1A and PLCB1. The abundance of these proteins in the four brain regions was significantly correlated with both the object recognition and the Y-maze tests. Our findings suggest a major role for PLCB1, KIF1A, and associated molecules in the pathogenesis of CTD.

The authors present CAMPI, a large-scale multi-lab comparison of diverse metaproteomics workflows. CAMPI provides insights into the robustness of current methods, suggests further improvements to the field, and may pave the way for future community-driven metaproteomics projects.

Abstract Through connecting genomic and metabolic information, metaproteomics is an essential approach for understanding how microbiomes function in space and time. The international metaproteomics community is delighted to announce the launch of the Metaproteomics Initiative (www.metaproteomics.org), the goal of which is to promote dissemination of metaproteomics fundamentals, advancements, and applications through collaborative networking in microbiome research. The Initiative aims to be the central information hub and open meeting place where newcomers and experts interact to communicate, standardize, and accelerate experimental and bioinformatic methodologies in this field. We invite the entire microbiome community to join and discuss potential synergies at the interfaces with other disciplines, and to collectively promote innovative approaches to gain deeper insights into microbiome functions and dynamics. Video Abstract.

Abstract Background Bacillus cereus is a notorious foodborne pathogen, which can grow under anoxic conditions. Anoxic growth is supported by endogenous redox metabolism, for which the thiol redox proteome serves as an interface. Here, we studied the cysteine (Cys) proteome dynamics of B. cereus ATCC 14579 cells grown under fermentative anoxic conditions. We used a quantitative thiol trapping method combined with proteomics profiling. Results In total, we identified 153 reactive Cys residues in 117 proteins participating in various cellular processes and metabolic pathways, including translation, carbohydrate metabolism, and stress response. Of these reactive Cys, 72 were detected as reduced Cys. The B. cereus Cys proteome evolved during growth both in terms of the number of reduced Cys and the Cys-containing proteins identified, reflecting its growth-phase-dependence. Interestingly, the reduced status of the B. cereus thiol proteome increased during growth, concomitantly to the decrease of extracellular oxidoreduction potential. Conclusions Taken together, our data show that the B. cereus Cys proteome during unstressed fermentative anaerobic growth is a dynamic entity and provide an important foundation for future redox proteomic studies in B. cereus and other organisms.

Abstract Background Soil and sediment microorganisms are highly phylogenetically diverse but are currently largely under-represented in public molecular databases. Their functional characterization by means of metaproteomics is usually performed using metagenomic sequences acquired for the same sample. However, such hugely diverse metagenomic datasets are difficult to assemble; in parallel, theoretical proteomes from isolates available in generic databases are of high quality. Both these factors advocate for the use of theoretical proteomes in metaproteomics interpretation pipelines. Here, we examined a number of database construction strategies with a view to increasing the outputs of metaproteomics studies performed on soil samples. Results The number of peptide-spectrum matches was found to be of comparable magnitude when using public or sample-specific metagenomics-derived databases. However, numbers were significantly increased when a combination of both types of information was used in a two-step cascaded search. Our data also indicate that the functional annotation of the metaproteomics dataset can be maximized by using a combination of both types of databases. Conclusions A two-step strategy combining sample-specific metagenome database and public databases such as the non-redundant NCBI database and a massive soil gene catalog allows maximizing the metaproteomic interpretation both in terms of ratio of assigned spectra and retrieval of function-derived information. Video abstract

ABSTRACT Global warming endangers our world, with probably a drastic drop in food production as one of the first vital consequences for humanity. To maintain or improve quality and sustainable yields, the burning imperative for agriculture is to rapidly integrate the essential component for plant development and soil regeneration, namely, the soil microbiome. Although enormous progress has been made in identifying the components of this microbiome, the way in which they interact with each other and with plants remains poorly understood. Lidbury I, Raguideau S, Borsetto C, Murphy A, et al. (mSystems 7:e00025-22, 2022, https://doi.org/10.1128/mSystems.00025-22) illustrate how metaproteomics helps define key interactions between plants and microorganisms at the rhizospheric interface. The many extracellular proteins identified and quantified by this methodology uniquely explain the observed phenotype. This study shows that the adoption of metaproteomics is no longer an option that microbiologists should consider but a must!

Microorganisms proteotyping by tandem mass spectrometry has been recently shown as a powerful methodology to identify the wide-range taxonomy and biomass of microbiota. Sputum is the recommended specimen for routine microbiological monitoring of Cystic Fibrosis (CF) patients but has been rarely submitted to tandem mass spectrometry-based proteotyping. In this study, we compared the microbial components of spontaneous and induced sputum samples from three cystic fibrosis patients. Although the presence of microbial proteins is much lower than host proteins, we report that the microbiota’s components present in the samples can be identified, as well as host biomarkers and functional insights into the microbiota. No significant difference was found in microorganism abundance between paired spontaneous and induced sputum samples. Microbial proteins linked to resistance, iron uptake, and biofilm-forming ability were observed in sputa independently of the sampling method. This unbiased and enlarged view of the CF microbiome could be highly complementary to culture and relevant for the clinical management of CF patients by improving knowledge about the host-pathogen dynamics and CF pathophysiology.

Abstract Metal sulfides are a common group of extracellular bacterial biominerals. However, only a few cases of intracellular biomineralization are reported in this group, mostly limited to greigite (Fe3S4) in magnetotactic bacteria. Here, a previously unknown periplasmic biomineralization of copper sulfide produced by the magnetotactic bacterium Desulfamplus magnetovallimortis strain BW‐1, a species known to mineralize greigite (Fe3S4) and magnetite (Fe3O4) in the cytoplasm is reported. BW‐1 produces hundreds of spherical nanoparticles, composed of 1–2 nm substructures of a poorly crystalline hexagonal copper sulfide structure that remains in a thermodynamically unstable state. The particles appear to be surrounded by an organic matrix as found from staining and electron microscopy inspection. Differential proteomics suggests that periplasmic proteins, such as a DegP‐like protein and a heavy metal‐binding protein, could be involved in this biomineralization process. The unexpected periplasmic formation of copper sulfide nanoparticles in BW‐1 reveals previously unknown possibilities for intracellular biomineralization that involves intriguing biological control and holds promise for biological metal recovery in times of copper shortage.

Staphylococcus epidermidis is one of the most common bacteria of the human skin microbiota. Despite its role as a commensal, S. epidermidis has emerged as an opportunistic pathogen, associated with 80% of medical devices related infections. Moreover, these bacteria are extremely difficult to treat due to their ability to form biofilms and accumulate resistance to almost all classes of antimicrobials. Thus new preventive and therapeutic strategies are urgently needed. However, the molecular mechanisms associated with S. epidermidis colonisation and disease are still poorly understood. A deeper understanding of the metabolic and cellular processes associated with response to environmental factors characteristic of SE ecological niches in health and disease might provide new clues on colonisation and disease processes. Here we studied the impact of pH conditions, mimicking the skin pH (5.5) and blood pH (7.4), in a S. epidermidis commensal strain by means of next-generation proteomics and 1H NMR-based metabolomics. Moreover, we evaluated the metabolic changes occurring during a sudden pH change, simulating the skin barrier break produced by a catheter. We found that exposure of S. epidermidis to skin pH induced oxidative phosphorylation and biosynthesis of peptidoglycan, lipoteichoic acids and betaine. In contrast, at blood pH, the bacterial assimilation of monosaccharides and its oxidation by glycolysis and fermentation was promoted. Additionally, several proteins related to virulence and immune evasion, namely extracellular proteases and membrane iron transporters were more abundant at blood pH. In the situation of an abrupt skin-to-blood pH shift we observed the decrease in the osmolyte betaine and changes in the levels of several metabolites and proteins involved in cellular redoxl homeostasis. Our results suggest that at the skin pH S. epidermidis cells are metabolically more active and adhesion is promoted, while at blood pH, metabolism is tuned down and cells have a more virulent profile. pH increase during commensal-to-pathogen conversion appears to be a critical environmental signal to the remodelling of the S. epidermidis metabolism toward a more pathogenic state. Targeting S. epidermidis proteins induced by pH 7.4 and promoting the acidification of the medical device surface or surrounding environment might be new strategies to treat and prevent S. epidermidis infections.

Microbial life can thrive in the most inhospitable places, such as nuclear facilities with high levels of ionizing radiation. Using direct meta-analyses, we have previously highlighted the presence of bacteria belonging to twenty-five different genera in the highly radioactive water of the cooling pool of an operating nuclear reactor core. In the present study, we further characterize this specific environment by isolating and identifying some of these microorganisms and assessing their radiotolerance and their ability to decontaminate uranium. This metal is one of the major radioactive contaminants of anthropogenic origin in the environment due to the nuclear and mining industries and agricultural practices. The microorganisms isolated when sampling was performed during the reactor operation consisted mainly of Actinobacteria and Firmicutes, whereas Proteobacteria were dominant when sampling was performed during the reactor shutdown. We investigated their tolerance to gamma radiation under different conditions. Most of the bacterial strains studied were able to survive 200 Gy irradiation. Some were even able to withstand 1 kGy, with four of them showing more than 10% survival at this dose. We also assessed their uranium uptake capacity. Seven strains were able to remove almost all the uranium from a 5 µM solution. Four strains displayed high efficiency in decontaminating a 50 µM uranium solution, demonstrating promising potential for use in bioremediation processes in environments contaminated by radionuclides.

A phyloprofile of Frankia genomes was carried out to identify those genes present in symbiotic strains of clusters 1, 1c, 2 and 3 and absent in non-infective strains of cluster 4. At a threshold of 50% AA identity, 108 genes were retrieved. Among these were known symbiosis-associated genes such as nif (nitrogenase), and genes which are not know as symbiosis-associated genes such as can (carbonic anhydrase, CAN). The role of CAN, which supplies carbonate ions necessary for carboxylases and acidifies the cytoplasm, was thus analyzed by staining cells with pH-responsive dyes; assaying for CO2 levels in N-fixing propionate-fed cells (that require a propionate-CoA carboxylase to yield succinate-CoA), fumarate-fed cells and N-replete propionate-fed cells; conducting proteomics on N-fixing fumarate and propionate-fed cells and direct measurement of organic acids in nodules and in roots. The interiors of both in vitro and nodular vesicles were found to be at a lower pH than that of hyphae. CO2 levels in N2-fixing propionate-fed cultures were lower than in N-replete ones. Proteomics of propionate-fed cells showed carbamoyl-phosphate synthase (CPS) as the most overabundant enzyme relative to fumarate-fed cells. CPS combines carbonate and ammonium in the first step of the citrulline pathway, something which would help manage acidity and NH4+. Nodules were found to have sizeable amounts of pyruvate and acetate in addition to TCA intermediates. This points to CAN reducing the vesicles’ pH to prevent the escape of NH3 and to control ammonium assimilation by GS and GOGAT, two enzymes that work in different ways in vesicles and hyphae. Genes with related functions (carboxylases, biotin operon and citrulline-aspartate ligase) appear to have undergone decay in non-symbiotic lineages.

Correct identification of the microorganisms present in a complex sample is a crucial issue. Proteotyping based on tandem mass spectrometry can help establish an inventory of organisms present in a sample. Evaluation of bioinformatics strategies and tools for mining the recorded datasets is essential to establish confidence in the results obtained and to improve these pipelines in terms of sensitivity and accuracy. Here, we propose several tandem mass spectrometry datasets recorded on an artificial reference consortium comprising 24 bacterial species. This assemblage of environmental and pathogenic bacteria covers 20 different genera and 5 bacterial phyla. The dataset comprises difficult cases, such as the Shigella flexneri species, which is closely related to Escherichia coli, and several highly sequenced clades. Different acquisition strategies simulate real-life scenarios: from rapid survey sampling to exhaustive analysis. We provide access to individual proteomes of each bacterium separately to provide a rational basis for evaluating the assignment strategy of MS/MS spectra when recorded from complex mixtures. This resource should provide an interesting common reference for developers who wish to compare their proteotyping tools and for those interested in evaluating protein assignment when dealing with complex samples, such as microbiomes.

Many prokaryotes are covered by a two-dimensional array of proteinaceous subunits. This surface layers (S-layer) is incompletely characterized for many microorganisms. Here, we studied Bacillus cereus AH187. A genome analysis identified two genes encoding the S-layer proteins SL2 and EA1, which we experimentally confirmed to encode the two protein components of the S-layer covering the surface of B. cereus. Shotgun proteomics analysis indicated that SL2 is the major component of the B. cereus S-layer at the beginning of exponential growth, whereas EA1 becomes more abundant than SL2 during later stages of stationary growth. Microscopy analysis revealed the spatial organization of SL2 and EA1 at the surface of B. cereus to depend on their temporal-dynamics during growth. Our results also show that a mutant strain lacking functional SL2 and EA1 proteins has distinct surface properties compared to its parental strain, in terms of stiffness and hydrophilicity during the stationary growth phase. Surface properties, self-aggregation capacity, and bacterial adhesion were observed to correlate. We conclude that the dynamics of SL2 and EA1 expression is a key determinant of the surface properties of B. cereus AH187, and that the S-layer could contribute to B. cereus survival in starvation conditions.

The rapidly expanding utilization of lanthanides (Ln) for the development of new technologies, green energies, and agriculture has raised concerns regarding their impacts on the environment and human health. The absence of characterization of the underlying cellular and molecular mechanisms regarding their toxicity is a caveat in the apprehension of their environmental impacts. We performed genomic phenotyping and molecular physiology analyses of Saccharomyces cerevisiae mutants exposed to La and Yb to uncover genes and pathways affecting Ln resistance and toxicity. Ln responses strongly differed from well-known transition metal and from common responses mediated by oxidative compounds. Shared response pathways to La and Yb exposure were associated to lipid metabolism, ion homeostasis, vesicular trafficking, and endocytosis, which represents a putative way of entry for Ln. Cell wall organization and related signaling pathways allowed for the discrimination of light and heavy Ln. Mutants in cell wall integrity-related proteins (e.g., Kre1p, Kre6p) or in the activation of secretory pathway and cell wall proteins (e.g., Kex2p, Kex1p) were resistant to Yb but sensitive to La. Exposure of WT yeast to the serine protease inhibitor tosyl phenylalanyl chloromethyl ketone mimicked the phenotype of kex2∆ under Ln, strengthening these results. Our data also suggest that the relative proportions of chitin and phosphomannan could modulate the proportion of functional groups (phosphates and carboxylates) to which La and Yb could differentially bind. Moreover, we showed that kex2∆, kex1∆, kre1∆, and kre6∆ strains were all sensitive to light Ln (La to Eu), while being increasingly resistant to heavier Ln. Finally, shotgun proteomic analyses identified modulated proteins in kex2∆ exposed to Ln, among which several plasmalemma ion transporters that were less abundant and that could play a role in Yb uptake. By combining these different approaches, we unraveled that cell wall components not only act in Ln adsorption but are also active signal effectors allowing cells to differentiate light and heavy Ln. This work paves the way for future investigations to the better understanding of Ln toxicity in higher eukaryotes.

Shotgun proteomics has proven to be an attractive alternative for identifying a pathogen and characterizing the antimicrobial resistance genes it produces. Because of its performance, proteotyping of microorganisms by tandem mass spectrometry is expected to become an essential tool in modern healthcare. Proteotyping microorganisms that have been isolated from the environment by culturomics is also a cornerstone for the development of new biotechnological applications. Phylopeptidomics is a new strategy that estimates the phylogenetic distances between the organisms present in the sample and calculates the ratio of their shared peptides, thus improving the quantification of their contributions to the biomass. Here, we established the limit of detection of tandem mass spectrometry proteotyping based on MS/MS data recorded for several bacteria. The limit of detection for Salmonella bongori with our experimental set-up is 4 × 104 colony-forming units from a sample volume of 1 mL. This limit of detection is directly related to the amount of protein per cell and therefore depends on the shape and size of the microorganism. We have demonstrated that identification of bacteria by phylopeptidomics is independent of their growth stage and that the limit of detection of the method is not degraded in presence of additional bacteria in the same proportion.

Scientific examination of the heart of Blessed Pauline Jaricot—a French missionary figure—was carried out in 2022. As tandem mass spectrometry proteotyping has proven to be valuable to obtain the broad taxonomic repertoire of a given sample without any a priori information, we aimed at exploring the conditions of preservation of the relics and possible conditions of death. Metaproteomics and high-resolution microtomography imaging approaches were combined. A dataset comprising 6731 high-resolution MS/MS spectra was acquired and 968 of these spectra could be assigned to specific peptidic biomolecules. Based on the taxonomical information encompassed by the identified peptide sequences, 5 phyla were identified amongst eukaryota (94% of the biomass): Ascomycota (55%), with the species Aspergillus versicolor, Trichophyton mentagrophytes and Aspergillus glaucus, corresponding to expected cadaverous fungal flora; Chordata (42%), represented by a unique species, Homo sapiens; Streptophyta (3%); and Arthropoda (traces). Bacteria (6% of the biomass) were poorly represented. No trace of embalming substance could be retrieved, nor any pathogens. Imaging evidenced no heart defect nor embalming traces. No evidence that was inconsistent with natural and spontaneous conservation could be retrieved. This study prefigures the power of modern molecular techniques such as paleoproteotyping coupled to microtomography to gain insight into historical relics.

Abstract Background There is an important need for the development of fast and robust methods to quantify the diversity and temporal dynamics of microbial communities in complex environmental samples. Because tandem mass spectrometry allows rapid inspection of protein content, metaproteomics is increasingly used for the phenotypic analysis of microbiota across many fields, including biotechnology, environmental ecology, and medicine. Results Here, we present a new method for identifying the biomass contribution of any given organism based on a signature describing the number of peptide sequences shared with all other organisms, calculated by mathematical modeling and phylogenetic relationships. This so-called “phylopeptidomics” principle allows for the calculation of the relative ratios of peptide-specified taxa by the linear combination of such signatures applied to an experimental metaproteomic dataset. We illustrate its efficiency using artificial mixtures of two closely related pathogens of clinical interest, and with more complex microbiota models. Conclusions This approach paves the way to a new vision of taxonomic changes and accurate label-free quantitative metaproteomics for fine-tuned functional characterization. Video abstract

ABSTRACTSevere acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and is continuing to spread rapidly around the globe. No effective vaccine is currently available to prevent COVID-19, and intense efforts are being invested worldwide into vaccine development. In this context, all technology platforms must overcome several challenges resulting from the use of an incompletely characterized new virus. These include finding the right conditions for virus amplification for the development of vaccines based on inactivated or attenuated whole viral particles. Here, we describe a shotgun tandem mass spectrometry workflow, the data produced can be used to guide optimization of the conditions for viral amplification. In parallel, we analysed the changes occurring in the host cell proteome following SARS-CoV-2 infection to glean information on the biological processes modulated by the virus that could be further explored as potential drug targets to deal with the pandemic.

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The BR2 nuclear research reactor in Mol, Belgium, runs in successive phases of operation (cycles) and shutdown, whereby a water basin surrounding the reactor vessel undergoes periodic changes in physico-chemical parameters such as flow rate, temperature, and radiation. The aim of this study was to explore the microbial community in this unique environment and to investigate its long-term dynamics using a 16S rRNA amplicon sequencing approach. Results from two sampling campaigns spanning several months showed a clear shift in community profiles: cycles were mostly dominated by two Operational Taxonomic Units (OTUs) assigned to unclassified Gammaproteobacterium and Pelomonas, whereas shutdowns were dominated by an OTU assigned to Methylobacterium. Although 1 year apart, both campaigns showed similar results, indicating that the system remained stable over this 2-year period. The community shifts were linked with changes in physico-chemical parameters by Non-metric Multidimensional Scaling (NMDS) and correlation analyses. In addition, radiation was hypothesized to cause a decrease in cell number, whereas temperature had the opposite effect. Chemoautotrophic use of H2 and dead cell recycling are proposed to be used as a strategies for nutrient retrieval in this extremely oligotrophic environment.

The Coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in a major health crisis worldwide with its continuously emerging new strains, resulting in new viral variants that drive “waves” of infection. PCR or antigen detection assays have been routinely used to detect clinical infections; however, the emergence of these newer strains has presented challenges in detection. One of the alternatives has been to detect and characterize variant-specific peptide sequences from viral proteins using mass spectrometry (MS)-based methods. MS methods can potentially help in both diagnostics and vaccine development by understanding the dynamic changes in the viral proteome associated with specific strains and infection waves. In this study, we developed an accessible, flexible, and shareable bioinformatics workflow that was implemented in the Galaxy Platform to detect variant-specific peptide sequences from MS data derived from the clinical samples. We demonstrated the utility of the workflow by characterizing published clinical data from across the world during various pandemic waves. Our analysis identified six SARS-CoV-2 variant-specific peptides suitable for confident detection by MS in commonly collected clinical samples.

The wastewater from hospitals, pharmaceutical industries and more generally human and animal dejections leads to environmental releases of antibiotics that cause severe problems for all living organisms. The aim of this study was to investigate the capacity of three fungal strains to biotransform the fluoroquinolone levofloxacin. The degradation processes were analyzed in solid and liquid media. Among the three fungal strains tested, Coriolopsis gallica strain CLBE55 (BRFM 3473) showed the highest removal efficiency, with a 15% decrease in antibiogram zone of inhibition for Escherichia coli cultured in solid medium and 25% degradation of the antibiotic in liquid medium based on high-performance liquid chromatography (HPLC). Proteomic analysis suggested that laccases and dye-decolorizing peroxidases such as extracellular enzymes could be involved in levofloxacin degradation, with a putative major role for laccases. Degradation products were proposed based on mass spectrometry analysis, and annotation suggested that the main product of biotransformation of levofloxacin by Coriolopsis gallica is an N-oxidized derivative.

In this article of EBioMedicine, Santosh Renuse and colleagues1 show the relevance of combining immunoaffinity capture with targeted mass spectrometry measurement to detect SARS-CoV-2 nucleocapsid proteins in nasopharyngeal swab samples. The COVID-19 pandemic has confirmed the need to improve the toolbox available to diagnose respiratory infections. Rapid, reliable, and highly specific detection is essential if we are to mount immediate preventive and therapeutic responses. This report stands out from previous studies as it implements immunocapture along with robust validation for a large cohort of subjects. The results presented show that mass spectrometry is definitively at a crossroads for large-scale clinical applications.

Microbacterium oleivorans A9 cells were exposed or not to 10 µM uranyl nitrate as resting cells in sodium chloride solution. Bacteria exposed to U(VI) and controls were harvested after 0.5, 4, and 24 h of toxicant exposure. Bacteria were subjected to high-throughput proteomics analysis using a Q-Exactive HF high resolution tandem mass spectrometer incorporating an ultra-high-field orbitrap analyzer. MS/MS spectra were assigned with a protein sequence database derived from a draft genome obtained by Illumina sequencing and systematic six-reading frame translation of all the contigs. Proteins identified in bacteria exposed to U(VI) and controls at the three time points allow defining the proteome dynamics upon uranium stress. The data reported here are related to a published study regarding the proteome dynamics of M. oleivorans A9 upon uranium stress by Gallois et al. (in press) entitled “Proteogenomic insights into uranium tolerance of a Chernobyl׳s Microbacterium bacterial isolate”. The data accompanying the manuscript describing the database searches and comparative analysis have been deposited to the ProteomeXchange with identifier PXD005794.

Massive mortality outbreaks affecting Pacific oysters (Crassostrea gigas) spat/juveniles are often associated with the detection of a herpesvirus called ostreid herpesvirus type 1 (OsHV-1). In this work, experimental infection trials of C. gigas spat with OsHV-1 were conducted using two contrasted Pacific oyster families for their susceptibility to viral infection. Live oysters were sampled at 12, 26, and 144 h post infection (hpi) to analyze host-pathogen interactions using comparative proteomics. Shotgun proteomics allowed the detection of seven viral proteins in infected oysters, some of them with potential immunomodulatoy functions. Viral proteins were mainly detected in susceptible oysters sampled at 26 hpi, which correlates with the mortality and viral load observed in this oyster family. Concerning the Pacific oyster proteome, more than 3,000 proteins were identified and contrasted proteomic responses were observed between infected A- and P-oysters, sampled at different post-injection times. Gene ontology (GO) and KEGG pathway enrichment analysis performed on significantly modulated proteins uncover the main immune processes (such as RNA interference, interferon-like pathway, antioxidant defense) which contribute to the defense and resistance of Pacific oysters to viral infection. In the more susceptible Pacific oysters, results suggest that OsHV-1 manipulate the molecular machinery of host immune response, in particular the autophagy system. This immunomodulation may lead to weakening and consecutively triggering death of Pacific oysters. The identification of several highly modulated and defense-related Pacific oyster proteins from the most resistant oysters supports the crucial role played by the innate immune system against OsHV-1 and the viral infection. Our results confirm the implication of proteins involved in an interferon-like pathway for efficient antiviral defenses and suggest that proteins involved in RNA interference process prevent viral replication in C. gigas. Overall, this study shows the interest of multi-omic approaches applied on groups of animals with differing sensitivities and provides novel insight into the interaction between Pacific oyster and OsHV-1 with key proteins involved in viral infection resistance.

The vast majority of marine microorganisms and their functions are yet to be explored. The considerable diversity they encompass is an endless source of knowledge and wealth that can be valued on an industrial scale, emphasizing the need to develop rapid and efficient identification and characterization techniques. In this study, we identified 26 microbial isolates from coastal water of the NW Mediterranean Sea, using phylopeptidomics, a cutting-edge tandem mass spectrometry proteotyping technique. Taxonomical identification at the species level was successfully conducted for all isolates. The presence of strains belonging to the newly described Balneolaeota phylum, yet uncharacterized at the proteomics scale, was noted. The very first proteomics-based investigation of a representative of the Balneolaeota phylum, Balneola vulgaris, is proposed, demonstrating the use of our proteotyping workflow for the rapid identification and in-depth molecular characterization, in a single MS/MS analytical run. Tandem mass spectrometry proteotyping is a valuable asset for culturomic programs as the methodology is able to quickly classify the most atypical isolates.

Omics are the most promising approaches to investigate microbes for which no genetic tools exist such as the nitrogen-fixing symbiotic Frankia. A proteogenomic analysis of symbiotic Frankia alni was done by comparing those proteins more and less abundant in Alnus glutinosa nodules relative to N-replete pure cultures with propionate as the carbon source and ammonium as the nitrogen-source. There were 250 proteins that were significantly overabundant in nodules at a fold change (FC) ≥ 2 threshold, and 1429 with the same characteristics in in vitro nitrogen-replete pure culture. Nitrogenase, SuF (Fe–Su biogenesis) and hopanoid lipids synthesis determinants were the most overabundant proteins in symbiosis. Nitrogenase was found to constitute 3% of all Frankia proteins in nodules. Sod (superoxide dismutase) was overabundant, indicating a continued oxidative stress, while Kats (catalase) were not. Several transporters were overabundant including one for dicarboxylates and one for branched amino acids. The present results confirm the centrality of nitrogenase in the actinorhizal symbiosis.

Lassa virus (LASV), an Old World arenavirus, is responsible for hemorrhagic fevers in western Africa. The privileged tropism of LASV for endothelial cells combined with a dysregulated inflammatory response are the main cause of the increase in vascular permeability observed during the disease. Mopeia virus (MOPV) is another arenavirus closely related to LASV but nonpathogenic for non-human primates (NHPs) and has never been described in humans. MOPV is more immunogenic than LASV in NHPs and in vitro in human immune cell models, with more intense type I IFN and adaptive cellular responses. Here, we compared the transcriptomic and proteomic responses of human umbilical vein endothelial cells (HUVECs) to infection with the two viruses to further decipher the mechanisms involved in their differences in immunogenicity and pathogenicity. Both viruses replicated durably and efficiently in HUVECs, but the responses they induced were strikingly different. Modest activation was observed at an early stage of LASV infection and then rapidly shut down. By contrast, MOPV induced a late but more intense response, characterized by the expression of genes and proteins mainly associated with the type I IFN response and antigen processing/presentation. Such a response is consistent with the higher immunogenicity of MOPV relative to LASV, whereas the lack of an innate response induced in HUVECs by LASV is consistent with its uncontrolled systemic dissemination through the vascular endothelium.

This study unveils the single and combined drought and heat impacts on the photosynthetic performance of Coffea arabica cv. Icatu and C. canephora cv. Conilon Clone 153 (CL153). Well-watered (WW) potted plants were gradually submitted to severe water deficit (SWD) along 20 days under adequate temperature (25/20°C, day/night), and thereafter exposed to a gradual temperature rise up to 42/30°C, followed by a 14-day water and temperature recovery. Single drought affected all gas exchanges (including Amax) and most fluorescence parameters in both genotypes. However, Icatu maintained Fv/Fm and RuBisCO activity, and reinforced electron transport rates, carrier contents, and proton gradient regulation (PGR5) and chloroplast NADH dehydrogenase-like (NDH) complex proteins abundance. This suggested negligible non-stomatal limitations of photosynthesis that were accompanied by a triggering of protective cyclic electron transport (CEF) involving both photosystems (PSs). These findings contrasted with declines in RuBisCO and PSs activities, and cytochromes (b559, f, b563) contents in CL153. Remarkable heat tolerance in potential photosynthetic functioning was detected in WW plants of both genotypes (up to 37/28°C or 39/30°C), likely associated with CEF in Icatu. Yet, at 42/30°C the tolerance limit was exceeded. Reduced Amax and increased Ci values reflected non-stomatal limitations of photosynthesis, agreeing with impairments in energy capture (F0 rise), PSII photochemical efficiency, and RuBisCO and Ru5PK activities. In contrast to PSs activities and electron carrier contents, enzyme activities were highly heat sensitive. Until 37/28°C, stresses interaction was largely absent, and drought played the major role in constraining photosynthesis functioning. Harsher conditions (SWD, 42/30°C) exacerbated impairments to PSs, enzymes, and electron carriers, but uncontrolled energy dissipation was mitigated by photoprotective mechanisms. Most parameters recovered fully between 4 and 14 days after stress relief in both genotypes, although some aftereffects persisted in SWD plants. Icatu was more drought tolerant, with WW and SWD plants usually showing a faster and/or greater recovery than CL153. Heat affected both genotypes mostly at 42/30°C, especially in SWD and Icatu plants. Overall, photochemical components were highly tolerant to heat and to stress interaction in contrast to enzymes that deserve special attention by breeding programs to increase coffee sustainability in climate change scenarios.