Inhibition of ATGL alleviates MASH via impaired PPARα signalling that favours hydrophilic bile acid composition in mice.

BACKGROUND AND AIMS: Adipose triglyceride lipase (ATGL) is an attractive therapeutic target in insulin resistance and metabolic dysfunction-associated steatotic liver disease (MASLD). This study investigated the effects of pharmacological ATGL inhibition on the development of metabolic dysfunction-associated steatohepatitis (MASH) and fibrosis in mice. METHODS: Streptozotocin-injected male mice were fed an HFD to induce MASH. Mice receiving the ATGL inhibitor, Atglistatin (ATGLi), were compared to controls using liver histology, lipidomics, metabolomics, 16s rRNA, and RNA sequencing. Human ileal organoids, HepG2 cells, and Caco2 cells treated with the human ATGL inhibitor NG-497, HepG2 ATGL knockdown cells, gel-shift, and luciferase assays were analysed for mechanistic insights. We validated its benefits on steatohepatitis and fibrosis in a low-methionine choline-deficient mouse model. RESULTS: ATGLi improved serum liver enzymes, hepatic lipid content, and histological liver injury. Mechanistically, ATGLi attenuated PPARα signalling, favouring hydrophilic bile acid (BA) synthesis with increased Cyp7a1, Cyp27a1, Cyp2c70, and reduced Cyp8b1 expression. Additionally, reduced intestinal Cd36 and Abca1, along with increased Abcg5 expression, were consistent with reduced levels of hepatic TAG-species containing PUFAs like linoleic acids as well as reduced cholesterol levels in the liver and plasma. Similar changes in gene expression associated with PPARα signaling and intestinal lipid transport were observed in ileal organoids treated with NG-497. Furthermore, HepG2 ATGL knockdown cells revealed reduced expression of PPARα target genes and upregulation of genes involved in hydrophilic BA synthesis, consistent with reduced PPARα binding and luciferase activity in the presence of the ATGL inhibitors. CONCLUSIONS: Inhibition of ATGL attenuates PPARα signalling, translating into hydrophilic BAs, interfering with dietary lipid absorption, and improving metabolic disturbances. The validation with NG-497 opens a new therapeutic perspective for MASLD. IMPACT AND IMPLICATIONS: The global prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is a crucial public health concern. Since adherence to behavioural interventions is limited, pharmacological strategies are necessary, as highlighted by the recent FDA approval of resmetirom. However, since our current mechanistic understanding and pathophysiology-oriented therapeutic options for MASLD are still limited, novel mechanistic insights are urgently needed. Our present work uncovers that pharmacological inhibition of ATGL, the key enzyme in lipid hydrolysis using Atglistatin (ATGLi), improves metabolic dysfunction-associated steatohepatitis (MASH), fibrosis, and associated key features of metabolic dysfunction in a mouse model of MASH and MCD-induced liver fibrosis. Mechanistically, we demonstrated that attenuation of PPARα signalling in the liver and gut favours hydrophilic bile acid composition, ultimately interfering with dietary lipid absorption. One of the drawbacks of ATGLi is its lack of efficacy against human ATGL, thus limiting its clinical applicability. Against this backdrop, we could show that ATGL inhibition using the human inhibitor NG-497 in human primary ileum-derived organoids, Caco2 cells, and HepG2 cells translated into therapeutic mechanisms similar to ATGLi. Collectively, these findings open a new avenue for MASLD treatment development by inhibiting human ATGL activity.

Contrasting drivers of belowground nitrogen cycling in a montane grassland exposed to a multifactorial global change experiment with elevated CO2 , warming, and drought.

Depolymerization of high-molecular weight organic nitrogen (N) represents the major bottleneck of soil N cycling and yet is poorly understood compared to the subsequent inorganic N processes. Given the importance of organic N cycling and the rise of global change, we investigated the responses of soil protein depolymerization and microbial amino acid consumption to increased temperature, elevated atmospheric CO2 , and drought. The study was conducted in a global change facility in a managed montane grassland in Austria, where elevated CO2 (eCO2 ) and elevated temperature (eT) were stimulated for 4 years, and were combined with a drought event. Gross protein depolymerization and microbial amino acid consumption rates (alongside with gross organic N mineralization and nitrification) were measured using 15 N isotope pool dilution techniques. Whereas eCO2  showed no individual effect, eT had distinct effects which were modulated by season, with a negative effect of eT on soil organic N process rates in spring, neutral effects in summer, and positive effects in fall. We attribute this to a combination of changes in substrate availability and seasonal temperature changes. Drought led to a doubling of organic N process rates, which returned to rates found under ambient conditions within 3 months after rewetting. Notably, we observed a shift in the control of soil protein depolymerization, from plant substrate controls under continuous environmental change drivers (eT and eCO2 ) to controls via microbial turnover and soil organic N availability under the pulse disturbance (drought). To the best of our knowledge, this is the first study which analyzed the individual versus combined effects of multiple global change factors and of seasonality on soil organic N processes and thereby strongly contributes to our understanding of terrestrial N cycling in a future world.

Prophages in Vibrio.

Although tailed bacteriophages (phages) of the class Caudoviricetes are thought to constitute the most abundant and ecologically relevant group of phages that can integrate their genome into the host chromosome, it is becoming increasingly clear that other prophages are widespread. Here, we show that prophages derived from filamentous and tailless phages with genome sizes below 16 kb make up the majority of prophages in marine bacteria of the genus Vibrio. To estimate prophage prevalence unaffected by database biases, we combined comparative genomics and chemical induction of 58 diverse Vibrio cyclitrophicus isolates, resulting in 107 well-curated prophages. Complemented with computationally predicted prophages, we obtained 1,158 prophages from 931 naturally co-existing strains of the family Vibrionaceae. Prophages resembling tailless and filamentous phages predominated, accounting for 80% of all prophages in V. cyclitrophicus and 60% across the Vibrionaceae. In our experimental model, prophages of all three viral realms actively replicated upon induction indicating their ability to transfer to new hosts. Indeed, prophages were rapidly gained and lost, as suggested by variable prophage content between closely related V. cyclitrophicus. Prophages related to filamentous and tailless phages were integrated into only three genomic locations and restored the function of their integration site. Despite their small size, they contained highly diverse accessory genes that may contribute to host fitness, such as phage defense systems. We propose that, like their well-studied tailed equivalent, tailless and filamentous temperate phages are active and highly abundant drivers of host ecology and evolution in marine Vibrio, which have been largely overlooked.

Sulfur-oxidizing symbionts colonize the digestive tract of their Lucinid hosts.

Like many marine invertebrates, marine lucinid clams have an intimate relationship with beneficial sulfur-oxidizing bacteria located within specialized gill cells known as bacteriocytes. Most previous research has focused on the symbionts in the gills of these (and other) symbiotic bivalves, often assuming that the symbionts only persistently colonize the gills, at least in the adult stage. We used 16S rRNA gene sequencing and digital polymerase chain reaction with symbiont-specific primers targeting the soxB gene on the foot, mantle, visceral mass, and gills of the lucinid clam Loripes orbiculatus. We also used fluorescence in situ hybridization with symbiont-specific probes to examine symbiont distribution at the level of the whole holobiont. Despite 40 years of research on these symbioses, we detected previously unknown populations of symbiont cells in several organs, including the digestive tract. As in the well-studied gills, symbionts in the digestive tract may be housed within host cells. A 14-month starvation experiment without hydrogen sulfide to power symbiont metabolism caused a larger reduction in symbiont numbers in the gills compared to the visceral mass, raising the possibility that symbionts in the digestive tract are persistent and may have a distinct physiology and role in the symbiosis compared with the gill symbionts. Our results highlight the unexpectedly complex relationships between marine lucinid clams and their symbionts and challenge the view that chemosynthetic symbionts are restricted to the gills of these hosts.

Co-occurring nitrifying symbiont lineages are vertically inherited and widespread in marine sponges.

Ammonia-oxidizing archaea and nitrite-oxidizing bacteria are common members of marine sponge microbiomes. They derive energy for carbon fixation and growth from nitrification-the aerobic oxidation of ammonia to nitrite and further to nitrate-and are proposed to play essential roles in the carbon and nitrogen cycling of sponge holobionts. In this study, we characterize two novel nitrifying symbiont lineages, Candidatus Nitrosokoinonia and Candidatus Nitrosymbion in the marine sponge Coscinoderma matthewsi using a combination of molecular tools, in situ visualization, and physiological rate measurements. Both represent a new genus in the ammonia-oxidizing archaeal class Nitrososphaeria and the nitrite-oxidizing bacterial order Nitrospirales, respectively. Furthermore, we show that larvae of this viviparous sponge are densely colonized by representatives of Ca. Nitrosokoinonia and Ca. Nitrosymbion indicating vertical transmission. In adults, the representatives of both symbiont genera are located extracellularly in the mesohyl. Comparative metagenome analyses and physiological data suggest that ammonia-oxidizing archaeal symbionts of the genus Ca. Nitrosokoinonia strongly rely on endogenously produced nitrogenous compounds (i.e. ammonium, urea, nitriles/cyanides, and creatinine) rather than on exogenous ammonium sources taken up by the sponge. Additionally, the nitrite-oxidizing bacterial symbionts of the genus Ca. Nitrosymbion may reciprocally support the ammonia-oxidizers with ammonia via the utilization of sponge-derived urea and cyanate. Comparative analyses of published environmental 16S rRNA gene amplicon data revealed that Ca. Nitrosokoinonia and Ca. Nitrosymbion are widely distributed and predominantly associated with marine sponges and corals, suggesting a broad relevance of our findings.

Down-regulation of the bacterial protein biosynthesis machinery in response to weeks, years, and decades of soil warming.

How soil microorganisms respond to global warming is key to infer future soil-climate feedbacks, yet poorly understood. Here, we applied metatranscriptomics to investigate microbial physiological responses to medium-term (8 years) and long-term (>50 years) subarctic grassland soil warming of +6°C. Besides indications for a community-wide up-regulation of centralmetabolic pathways and cell replication, we observed a down-regulation of the bacterial protein biosynthesis machinery in the warmed soils, coinciding with a lower microbial biomass, RNA, and soil substrate content. We conclude that permanently accelerated reaction rates at higher temperatures and reduced substrate concentrations result in cellular reduction of ribosomes, the macromolecular complexes carrying out protein biosynthesis. Later efforts to test this, including a short-term warming experiment (6 weeks, +6°C), further supported our conclusion. Down-regulating the protein biosynthesis machinery liberates energy and matter, allowing soil bacteria to maintain high metabolic activities and cell division rates even after decades of warming.

Increased microbial expression of organic nitrogen cycling genes in long-term warmed grassland soils.

Global warming increases soil temperatures and promotes faster growth and turnover of soil microbial communities. As microbial cell walls contain a high proportion of organic nitrogen, a higher turnover rate of microbes should also be reflected in an accelerated organic nitrogen cycling in soil. We used a metatranscriptomics and metagenomics approach to demonstrate that the relative transcription level of genes encoding enzymes involved in the extracellular depolymerization of high-molecular-weight organic nitrogen was higher in medium-term (8 years) and long-term (>50 years) warmed soils than in ambient soils. This was mainly driven by increased levels of transcripts coding for enzymes involved in the degradation of microbial cell walls and proteins. Additionally, higher transcription levels for chitin, nucleic acid, and peptidoglycan degrading enzymes were found in long-term warmed soils. We conclude that an acceleration in microbial turnover under warming is coupled to higher investments in N acquisition enzymes, particularly those involved in the breakdown and recycling of microbial residues, in comparison with ambient conditions.

Understanding the Response of Nitrifying Communities to Disturbance in the McMurdo Dry Valleys, Antarctica.

Polar ecosystems are generally limited in nitrogen (N) nutrients, and the patchy availability of N is partly determined by biological pathways, such as nitrification, which are carried out by distinctive prokaryotic functional groups. The activity and diversity of microorganisms are generally strongly influenced by environmental conditions. However, we know little of the attributes that control the distribution and activity of specific microbial functional groups, such as nitrifiers, in extreme cold environments and how they may respond to change. To ascertain relationships between soil geochemistry and the ecology of nitrifying microbial communities, we carried out a laboratory-based manipulative experiment to test the selective effect of key geochemical variables on the activity and abundance of ammonia-oxidizing communities in soils from the McMurdo Dry Valleys of Antarctica. We hypothesized that nitrifying communities, adapted to different environmental conditions within the Dry Valleys, will have distinct responses when submitted to similar geochemical disturbances. In order to test this hypothesis, soils from two geographically distant and geochemically divergent locations, Miers and Beacon Valleys, were incubated over 2 months under increased conductivity, ammonia concentration, copper concentration, and organic matter content. Amplicon sequencing of the 16S rRNA gene and transcripts allowed comparison of the response of ammonia-oxidizing Archaea (AOA) and ammonia-oxidizing Bacteria (AOB) to each treatment over time. This approach was combined with measurements of 15NH4+ oxidation rates using 15N isotopic additions. Our results showed a higher potential for nitrification in Miers Valley, where environmental conditions are milder relative to Beacon Valley. AOA exhibited better adaptability to geochemical changes compared to AOB, particularly to the increase in copper and conductivity. AOA were also the only nitrifying group found in Beacon Valley soils. This laboratorial manipulative experiment provided new knowledge on how nitrifying groups respond to changes on key geochemical variables of Antarctic desert soils, and we believe these results offer new insights on the dynamics of N cycling in these ecosystems.

Composition and activity of nitrifier communities in soil are unresponsive to elevated temperature and CO2, but strongly affected by drought.

Nitrification is a fundamental process in terrestrial nitrogen cycling. However, detailed information on how climate change affects the structure of nitrifier communities is lacking, specifically from experiments in which multiple climate change factors are manipulated simultaneously. Consequently, our ability to predict how soil nitrogen (N) cycling will change in a future climate is limited. We conducted a field experiment in a managed grassland and simultaneously tested the effects of elevated atmospheric CO2, temperature, and drought on the abundance of active ammonia-oxidizing bacteria (AOB) and archaea (AOA), comammox (CMX) Nitrospira, and nitrite-oxidizing bacteria (NOB), and on gross mineralization and nitrification rates. We found that N transformation processes, as well as gene and transcript abundances, and nitrifier community composition were remarkably resistant to individual and interactive effects of elevated CO2 and temperature. During drought however, process rates were increased or at least maintained. At the same time, the abundance of active AOB increased probably due to higher NH4+ availability. Both, AOA and comammox Nitrospira decreased in response to drought and the active community composition of AOA and NOB was also significantly affected. In summary, our findings suggest that warming and elevated CO2 have only minor effects on nitrifier communities and soil biogeochemical variables in managed grasslands, whereas drought favors AOB and increases nitrification rates. This highlights the overriding importance of drought as a global change driver impacting on soil microbial community structure and its consequences for N cycling.

Microbial growth and carbon use efficiency show seasonal responses in a multifactorial climate change experiment.

Microbial growth and carbon use efficiency (CUE) are central to the global carbon cycle, as microbial remains form soil organic matter. We investigated how future global changes may affect soil microbial growth, respiration, and CUE. We aimed to elucidate the soil microbial response to multiple climate change drivers across the growing season and whether effects of multiple global change drivers on soil microbial physiology are additive or interactive. We measured soil microbial growth, CUE, and respiration at three time points in a field experiment combining three levels of temperature and atmospheric CO2, and a summer drought. Here we show that climate change-driven effects on soil microbial physiology are interactive and season-specific, while the coupled response of growth and respiration lead to stable microbial CUE (average CUE = 0.39). These results suggest that future research should focus on microbial growth across different seasons to understand and predict effects of global changes on soil carbon dynamics.

Actinobacteria and Cyanobacteria Diversity in Terrestrial Antarctic Microenvironments Evaluated by Culture-Dependent and Independent Methods.

Bacterial diversity from McMurdo Dry Valleys in Antarctica, the coldest desert on earth, has become more easily assessed with the development of High Throughput Sequencing (HTS) techniques. However, some of the diversity remains inaccessible by the power of sequencing. In this study, we combine cultivation and HTS techniques to survey actinobacteria and cyanobacteria diversity along different soil and endolithic micro-environments of Victoria Valley in McMurdo Dry Valleys. Our results demonstrate that the Dry Valleys actinobacteria and cyanobacteria distribution is driven by environmental forces, in particular the effect of water availability and endolithic environments clearly conditioned the distribution of those communities. Data derived from HTS show that the percentage of cyanobacteria decreases from about 20% in the sample closest to the water source to negligible values on the last three samples of the transect with less water availability. Inversely, actinobacteria relative abundance increases from about 20% in wet soils to over 50% in the driest samples. Over 30% of the total HTS data set was composed of actinobacterial strains, mainly distributed by 5 families: Sporichthyaceae, Euzebyaceae, Patulibacteraceae, Nocardioidaceae, and Rubrobacteraceae. However, the 11 actinobacterial strains isolated in this study, belonged to Micrococcaceae and Dermacoccaceae families that were underrepresented in the HTS data set. A total of 10 cyanobacterial strains from the order Synechococcales were also isolated, distributed by 4 different genera (Nodosilinea, Leptolyngbya, Pectolyngbya, and Acaryochloris-like). In agreement with the cultivation results, Leptolyngbya was identified as dominant genus in the HTS data set. Acaryochloris-like cyanobacteria were found exclusively in the endolithic sample and represented 44% of the total 16S rRNA sequences, although despite our efforts we were not able to properly isolate any strain from this Acaryochloris-related group. The importance of combining cultivation and sequencing techniques is highlighted, as we have shown that culture-dependent methods employed in this study were able to retrieve actinobacteria and cyanobacteria taxa that were not detected in HTS data set, suggesting that the combination of both strategies can be usefull to recover both abundant and rare members of the communities.

The history of aerobic ammonia oxidizers: from the first discoveries to today.

Nitrification, the oxidation of ammonia to nitrite and nitrate, has long been considered a central biological process in the global nitrogen cycle, with its first description dated 133 years ago. Until 2005, bacteria were considered the only organisms capable of nitrification. However, the recent discovery of a chemoautotrophic ammonia-oxidizing archaeon, Nitrosopumilus maritimus, changed our concept of the range of organisms involved in nitrification, highlighting the importance of ammonia-oxidizing archaea (AOA) as potential players in global biogeochemical nitrogen transformations. The uniqueness of these archaea justified the creation of a novel archaeal phylum, Thaumarchaeota. These recent discoveries increased the global scientific interest within the microbial ecology society and have triggered an analysis of the importance of bacterial vs archaeal ammonia oxidation in a wide range of natural ecosystems. In this mini review we provide a chronological perspective of the current knowledge on the ammonia oxidation pathway of nitrification, based on the main physiological, ecological and genomic discoveries.