Removal of phosphoglycolate in hyperthermophilic archaea.

Many organisms that utilize the Calvin-Benson-Bassham (CBB) cycle for autotrophic growth harbor metabolic pathways to remove and/or salvage 2-phosphoglycolate, the product of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). It has been presumed that the occurrence of 2-phosphoglycolate salvage is linked to the CBB cycle, and in particular, the C2 pathway to the CBB cycle and oxygenic photosynthesis. Here, we examined 2-phosphoglycolate salvage in the hyperthermophilic archaeon Thermococcus kodakarensis, an obligate anaerobe that harbors a Rubisco that functions in the pentose bisphosphate pathway. T. kodakarensis harbors enzymes that have the potential to convert 2-phosphoglycolate to glycine and serine, and their genes were identified by biochemical and/or genetic analyses. 2-phosphoglycolate phosphatase activity increased 1.6-fold when cells were grown under microaerobic conditions compared to anaerobic conditions. Among two candidates, TK1734 encoded a phosphatase specific for 2-phosphoglycolate, and the enzyme was responsible for 80% of the 2-phosphoglycolate phosphatase activity in T. kodakarensis cells. The TK1734 disruption strain displayed growth impairment under microaerobic conditions, which was relieved upon addition of sodium sulfide. In addition, glycolate was detected in the medium when T. kodakarensis was grown under microaerobic conditions. The results suggest that T. kodakarensis removes 2-phosphoglycolate via a phosphatase reaction followed by secretion of glycolate to the medium. As the Rubisco in T. kodakarensis functions in the pentose bisphosphate pathway and not in the CBB cycle, mechanisms to remove 2-phosphoglycolate in this archaeon emerged independent of the CBB cycle.

Role of microbiota-derived corisin in coagulation activation during SARS-CoV-2 infection.

BACKGROUND: Coagulopathy is a major cause of morbidity and mortality in COVID-19 patients. Hypercoagulability in COVID-19 results in deep vein thrombosis, thromboembolic complications, and diffuse intravascular coagulation. Microbiome dysbiosis influences the clinical course of COVID-19. However, the role of dysbiosis in COVID-19-associated coagulopathy is not fully understood. OBJECTIVES: The present study tested the hypothesis that the microbiota-derived proapoptotic corisin is involved in the coagulation system activation during SARS-CoV-2 infection. METHODS: This cross-sectional study included 47 consecutive patients who consulted for symptoms of COVID-19. A mouse acute lung injury model was used to recapitulate the clinical findings. A549 alveolar epithelial, THP-1, and human umbilical vein endothelial cells were used to evaluate procoagulant and anticoagulant activity of corisin. RESULTS: COVID-19 patients showed significantly high circulating levels of corisin, thrombin-antithrombin complex, D-dimer, tumor necrosis factor-α, and monocyte-chemoattractant protein-1 with reduced levels of free protein S compared with healthy subjects. The levels of thrombin-antithrombin complex, D-dimer, and corisin were significantly correlated. A monoclonal anticorisin-neutralizing antibody significantly inhibited the inflammatory response and coagulation system activation in a SARS-CoV-2 spike protein-associated acute lung injury mouse model, and the levels of corisin and thrombin-antithrombin complex were significantly correlated. In an in vitro experiment, corisin increased the tissue factor activity and decreased the anticoagulant activity of thrombomodulin in epithelial, endothelial, and monocytic cells. CONCLUSION: The microbiota-derived corisin is significantly increased and correlated with activation of the coagulation system during SARS-CoV-2 infection, and corisin may directly increase the procoagulant activity in epithelial, endothelial, and monocytic cells.

- Invited Review - Hydrogen production and hydrogen utilization in the rumen: key to mitigating enteric methane production.

Molecular hydrogen (H2) and formate (HCOO-) are metabolic end products of many primary fermenters in the rumen ecosystem. Both play a vital role in fermentation where they are electron sinks for individual microbes in an anaerobic environment that lacks external electron acceptors. If H2 and/or formate accumulate within the rumen, the ability of primary fermenters to regenerate electron carriers may be inhibited and microbial metabolism and growth disrupted. Consequently, H2- and/or formate-consuming microbes such as methanogens and possibly homoacetogens play a key role in maintaining the metabolic efficiency of primary fermenters. There is increasing interest in identifying approaches to manipulate the rumen ecosystem for the benefit of the host and the environment. As H2 and formate are important mediators of interspecies interactions, an understanding of their production and utilization could be a significant starting point for the development of successful interventions aimed at redirecting electron flow and reducing methane emissions. We conclude by discussing in brief ruminant methane mitigation approaches as a model to help understand the fate of H2 and formate in the rumen ecosystem.

Elevated plasma and bile levels of corisin, a microbiota-derived proapoptotic peptide, in patients with severe acute cholangitis.

BACKGROUND: Acute cholangitis is a severe, life-threatening infection of the biliary system that requires early diagnosis and treatment. The Tokyo Guidelines recommend a combination of clinical, laboratory, and imaging findings for diagnosis and severity assessment, but there are still challenges in identifying severe cases that need immediate intervention. The microbiota and its derived products have been implicated in the pathogenesis of acute cholangitis. Corisin is a microbiome-derived peptide that induces cell apoptosis, acute tissue injury, and inflammation. This study aimed to evaluate the potential of plasma and bile corisin as a biomarker of acute cholangitis. METHODS: Forty patients with acute cholangitis associated with choledocholithiasis or malignant disease were enrolled. Nine patients without acute cholangitis were used as controls. Corisin was measured by enzyme immunoassays in plasma and bile samples. Patients were classified into severe and non-severe groups. The associations of plasma and bile corisin with the clinical grade of acute cholangitis and other parameters were analyzed by univariate and multivariate regression analysis. RESULTS: Plasma and bile corisin levels were significantly higher in patients with acute cholangitis than in controls. Patients with severe acute cholangitis had significantly higher plasma and bile corisin levels than those with non-severe form of the disease. Bile corisin level was significantly correlated with markers of inflammation, coagulation, fibrinolysis, and renal function. Univariate analysis revealed a significant association of bile corisin but a weak association of plasma corisin with the clinical grade of acute cholangitis. In contrast, multivariate analysis showed a significant relationship between plasma corisin level and the disease clinical grade. The receiver operating characteristic curve analysis showed low sensitivity but high specificity for plasma and bile corisin to detect the severity of acute cholangitis. The plasma and bile corisin sensitivity was increased when serum C-reactive protein level was included in the receiver operating characteristic curve analysis. CONCLUSIONS: Overall, these findings suggest that plasma and bile corisin levels may be useful biomarkers for diagnosing and monitoring acute cholangitis and that corisin may play a role in the pathophysiology of the disease by modulating inflammatory, coagulation and renal pathways.

Inhibition of a Microbiota-Derived Peptide Ameliorates Established Acute Lung Injury.

Acute lung injury (ALI) is a clinical syndrome characterized by a diffuse lung inflammation that commonly evolves into acute respiratory distress syndrome and respiratory failure. The lung microbiota is involved in the pathogenesis of ALI. Corisin, a proapoptotic peptide derived from the lung microbiota, plays a role in ALI and acute exacerbation of pulmonary fibrosis. Preventive therapeutic intervention with a monoclonal anticorisin antibody inhibits ALI in mice. However, whether inhibition of corisin with the antibody ameliorates established ALI is unknown. Here, the therapeutic effectiveness of the anticorisin antibody in already established ALI in mice was assessed. Lipopolysaccharide was used to induce ALI in mice. After causing ALI, the mice were treated with a neutralizing anticorisin antibody. Mice treated with the antibody showed significant improvement in lung radiological and histopathologic findings, decreased lung infiltration of inflammatory cells, reduced markers of lung tissue damage, and inflammatory cytokines in bronchoalveolar lavage fluid compared with untreated mice. In addition, the mice treated with anticorisin antibody showed significantly increased expression of antiapoptotic proteins with decreased caspase-3 activation in the lungs compared with control mice treated with an irrelevant antibody. In conclusion, these observations suggest that the inhibition of corisin is a novel and promising approach for treating established ALI.

Carbohydrate-binding modules facilitate the enzymatic hydrolysis of lignocellulosic biomass: Releasing reducing sugars and dissociative lignin available for producing biofuels and chemicals.

The microbial decomposition and utilization of lignocellulosic biomass present in the plant tissues are driven by a series of carbohydrate active enzymes (CAZymes) acting in concert. As the non-catalytic domains widely found in the modular CAZymes, carbohydrate-binding modules (CBMs) are intimately associated with catalytic domains (CDs) that effect the diverse hydrolytic reactions. The CBMs function as auxiliary components for the recognition, adhesion, and depolymerization of the complex substrate mediated by the associated CDs. Therefore, CBMs are deemed as significant biotools available for enzyme engineering, especially to facilitate the enzymatic hydrolysis of dense and insoluble plant tissues to acquire more fermentable sugars. This review aims at presenting the taxonomies and biological properties of the CBMs currently curated in the CAZy database. The molecular mechanisms that CBMs use in assisting the enzymatic hydrolysis of plant polysaccharides and the regulatory factors of CBM-substrate interactions are outlined in detail. In addition, guidelines for the rational designs of CBM-fused CAZymes are proposed. Furthermore, the potential to harness CBMs for industrial applications, especially in enzymatic pretreatment of the recalcitrant lignocellulose, is evaluated. It is envisaged that the ideas outlined herein will aid in the engineering and production of novel CBM-fused enzymes to facilitate efficient degradation of lignocellulosic biomass to easily fermentable sugars for production of value-added products, including biofuels.

Formation of secondary allo-bile acids by novel enzymes from gut Firmicutes.

The gut microbiome of vertebrates is capable of numerous biotransformations of bile acids, which are responsible for intestinal lipid digestion and function as key nutrient-signaling molecules. The human liver produces bile acids from cholesterol predominantly in the A/B-cis orientation in which the sterol rings are "kinked", as well as small quantities of A/B-trans oriented "flat" stereoisomers known as "primary allo-bile acids". While the complex multi-step bile acid 7α-dehydroxylation pathway has been well-studied for conversion of "kinked" primary bile acids such as cholic acid (CA) and chenodeoxycholic acid (CDCA) to deoxycholic acid (DCA) and lithocholic acid (LCA), respectively, the enzymatic basis for the formation of "flat" stereoisomers allo-deoxycholic acid (allo-DCA) and allo-lithocholic acid (allo-LCA) by Firmicutes has remained unsolved for three decades. Here, we present a novel mechanism by which Firmicutes generate the "flat" bile acids allo-DCA and allo-LCA. The BaiA1 was shown to catalyze the final reduction from 3-oxo-allo-DCA to allo-DCA and 3-oxo-allo-LCA to allo-LCA. Phylogenetic and metagenomic analyses of human stool samples indicate that BaiP and BaiJ are encoded only in Firmicutes and differ from membrane-associated bile acid 5α-reductases recently reported in Bacteroidetes that indirectly generate allo-LCA from 3-oxo-Δ4-LCA. We further map the distribution of baiP and baiJ among Firmicutes in human metagenomes, demonstrating an increased abundance of the two genes in colorectal cancer (CRC) patients relative to healthy individuals.

Tutorial: Microbiome studies in drug metabolism.

The human gastrointestinal tract is home to a dense population of microorganisms whose metabolism impacts human health and physiology. The gut microbiome encodes millions of genes, the products of which endow our bodies with unique biochemical activities. In the context of drug metabolism, microbial biochemistry in the gut influences humans in two major ways: (1) by producing small molecules that modulate expression and activity of human phase I and II pathways; and (2) by directly modifying drugs administered to humans to yield active, inactive, or toxic metabolites. Although the capacity of the microbiome to modulate drug metabolism has long been known, recent studies have explored these interactions on a much broader scale and have revealed an unprecedented scope of microbial drug metabolism. The implication of this work is that we might be able to predict the capacity of an individual's microbiome to metabolize drugs and use this information to avoid toxicity and inform proper dosing. Here, we provide a tutorial of how to study the microbiome in the context of drug metabolism, focusing on in vitro, rodent, and human studies. We then highlight some limitations and opportunities for the field.

Sas20 is a highly flexible starch-binding protein in the Ruminococcus bromii cell-surface amylosome.

Ruminococcus bromii is a keystone species in the human gut that has the rare ability to degrade dietary resistant starch (RS). This bacterium secretes a suite of starch-active proteins that work together within larger complexes called amylosomes that allow R. bromii to bind and degrade RS. Starch adherence system protein 20 (Sas20) is one of the more abundant proteins assembled within amylosomes, but little could be predicted about its molecular features based on amino acid sequence. Here, we performed a structure-function analysis of Sas20 and determined that it features two discrete starch-binding domains separated by a flexible linker. We show that Sas20 domain 1 contains an N-terminal ß-sandwich followed by a cluster of α-helices, and the nonreducing end of maltooligosaccharides can be captured between these structural features. Furthermore, the crystal structure of a close homolog of Sas20 domain 2 revealed a unique bilobed starch-binding groove that targets the helical α1,4-linked glycan chains found in amorphous regions of amylopectin and crystalline regions of amylose. Affinity PAGE and isothermal titration calorimetry demonstrated that both domains bind maltoheptaose and soluble starch with relatively high affinity (Kd ≤ 20 µM) but exhibit limited or no binding to cyclodextrins. Finally, small-angle X-ray scattering analysis of the individual and combined domains support that these structures are highly flexible, which may allow the protein to adopt conformations that enhance its starch-targeting efficiency. Taken together, we conclude that Sas20 binds distinct features within the starch granule, facilitating the ability of R. bromii to hydrolyze dietary RS.

Complete Genome Sequences of Three Staphylococcus haemolyticus Strains Isolated from the Lung of a TGFß1 Transgenic Mouse with Lung Fibrosis.

We report here the complete genome sequences of three Staphylococcus haemolyticus strains isolated from a mouse fibrotic lung tissue and exhibiting proapoptotic activity on human lung alveolar epithelial cells. The genomes were obtained from a combination of Illumina MiSeq and Oxford Nanopore MinION sequencing.

Inhibition of lung microbiota-derived proapoptotic peptides ameliorates acute exacerbation of pulmonary fibrosis.

Idiopathic pulmonary fibrosis is an incurable disease of unknown etiology. Acute exacerbation of idiopathic pulmonary fibrosis is associated with high mortality. Excessive apoptosis of lung epithelial cells occurs in pulmonary fibrosis acute exacerbation. We recently identified corisin, a proapoptotic peptide that triggers acute exacerbation of pulmonary fibrosis. Here, we provide insights into the mechanism underlying the processing and release of corisin. Furthermore, we demonstrate that an anticorisin monoclonal antibody ameliorates lung fibrosis by significantly inhibiting acute exacerbation in the human transforming growth factorß1 model and acute lung injury in the bleomycin model. By investigating the impact of the anticorisin monoclonal antibody in a general model of acute lung injury, we further unravel the potential of corisin to impact such diseases. These results underscore the role of corisin in the pathogenesis of acute exacerbation of pulmonary fibrosis and acute lung injury and provide a novel approach to treating this incurable disease.

Isolation of DiNP-Degrading Microbes from the Mouse Colon and the Influence DiNP Exposure Has on the Microbiota, Intestinal Integrity, and Immune Status of the Colon.

Di-isononyl phthalate (DiNP) is a plasticizer used to impart flexibility or stability in a variety of products including polyvinyl chloride, cable coatings, artificial leather, and footwear. Previous studies have examined the impact of DiNP on gut integrity and the colonic immune microenvironment, but this study further expands the research by examining whether DiNP exposure alters the colonic microbiota and various immune markers. Previous studies have also revealed that environmental microbes degrade various phthalates, but no studies have examined whether anaerobic gut bacteria can degrade DiNP. Thus, this study tested the hypothesis that DiNP exposure alters the gut microbiota and immune-related factors, and that anaerobic bacteria in the gut can utilize DiNP as the sole carbon source. To test this hypothesis, adult female mice were orally dosed with corn oil or various doses of DiNP for 10-14 consecutive days. After the treatment period, mice were euthanized during diestrus. Colonic contents were collected for full-length 16S rRNA gene sequencing to identify the bacteria in the colon contents. Sanger sequencing of the 16S rRNA gene was used to identify bacteria that were able to grow in Bacteroides minimal media with DiNP as the sole carbon source. Colon tissues were collected for immunohistochemistry of immune(-related) factors. An environmentally relevant dose of DiNP (200 µg/kg) significantly increased a Lachnoclostridium taxon and decreased Blautia compared to the control. Collectively, minimal changes in the colonic microbiota were observed as indicated by non-significant beta-diversities between DiNP treatments and control. Furthermore, three strains of anaerobic bacteria derived from the colon were identified to use DiNP as the sole carbon source. Interestingly, DiNP exposure did not alter protein levels of interleukin-6, tumor necrosis factor alpha, claudin-1, and mucin-1 compared to the control. Collectively, these findings show that DiNP exposure alters the gut microbiota and that the gut contains DiNP-degrading microbes.

A Microbiome-Derived Peptide Induces Apoptosis of Cells from Different Tissues.

Apoptosis is a programmed cell death involved in embryogenesis and tissue homeostasis under physiological conditions. However, abnormalities in the process of apoptosis are implicated in the pathogenesis of various diseases. The human microbiota may release products that induce apoptosis of host cells. We recently identified a novel microbiome-derived peptide called corisin that worsens lung fibrosis by inducing apoptosis of lung epithelial cells. We hypothesized that corisin and a corisin-like peptide might also induce apoptosis of cells from different tissues. We cultured podocytes, renal tubular epithelial cells, keratinocytes, retinal and intestinal cells treated with corisin and evaluated apoptosis by flow cytometry and Western blotting. Although at different grades, flow cytometry analysis and Western blotting showed that corisin and a corisin-like peptide induced apoptosis of podocytes, keratinocytes, tubular epithelial cells, retinal, and intestinal cells. In addition, we found that corisin synergistically enhances the proapoptotic activity of transforming growth factor-ß1 on podocytes. In conclusion, these results suggest that corisin and corisin-like peptides may play a role in the pathogenesis of disease in different organs by promoting apoptosis of parenchymal cells.

Subacute Exposure to an Environmentally Relevant Dose of Di-(2-ethylhexyl) Phthalate during Gestation Alters the Cecal Microbiome, but Not Pregnancy Outcomes in Mice.

Di-2-ethylhexyl phthalate (DEHP) is a plasticizer commonly found in polyvinyl chloride, medical equipment, and food packaging. DEHP has been shown to target the reproductive system and alter the gut microbiome in humans and experimental animals. However, very little is known about the impact of DEHP-induced microbiome changes and its effects during pregnancy. Thus, the objective of this study was to investigate the effects of DEHP exposure during pregnancy on the cecal microbiome and pregnancy outcomes. Specifically, this study tested the hypothesis that subacute exposure to DEHP during pregnancy alters the cecal microbiome in pregnant mice, leading to changes in birth outcomes. To test this hypothesis, pregnant dams were orally exposed to corn oil vehicle or 20 µg/kg/day DEHP for 10 days and euthanized 21 days after their last dose. Cecal contents were collected for 16S Illumina and shotgun metagenomic sequencing. Fertility studies were also conducted to examine whether DEHP exposure impacted birth outcomes. Subacute exposure to environmentally relevant doses of DEHP in pregnant dams significantly increased alpha diversity and significantly altered beta diversity. Furthermore, DEHP exposure during pregnancy significantly increased the relative abundance of Bacteroidetes and decreased the relative abundance of Firmicutes and Deferribacteres compared with controls. The affected taxonomic families included Deferribacteraceae, Lachnospiraceae, and Mucisprillum. In addition to changes in the gut microbiota, DEHP exposure significantly altered 14 functional pathways compared with the control. Finally, DEHP exposure did not significantly impact the fertility and birth outcomes compared with the control. Collectively, these data indicate that DEHP exposure during pregnancy shifts the cecal microbiome, but the shifts do not impact fertility and birth outcomes.

Early postnatal exposure to di(2-ethylhexyl) phthalate causes sex-specific disruption of gonadal development in pigs.

Di(2-ethylhexyl) phthalate (DEHP) is a chemical commonly used as a plasticizer to render polyvinyl chloride products more durable and flexible. Although exposure to DEHP has raised many health concerns due to the identification of DEHP as an endocrine disruptor, it is still used in consumer products, including polyvinyl chloride plastics, medical tubing, car interiors, and children's toys. To investigate the impact of early life exposure to DEHP on the ovary and testes, newborn piglets were orally dosed with DEHP (20 or 200 mg/kg/day) or vehicle control (tocopherol-stripped corn oil) for 21 days. Following treatment, ovaries, testes, and sera were harvested for histological assessment and measurement of steroid hormone levels. In male piglets, progesterone and pregnenolone levels were significantly lower in both treatment groups compared to control, whereas in female piglets, progesterone was significantly higher in the 20 mg group compared to control, indicating sex-specific effects in a non-monotonic manner. Follicle numbers and gene expression of steroidogenic enzymes and apoptotic factors were not altered in treated ovaries compared to controls. In DEHP-treated testes, germ cell migration was impaired and germ cell death was significantly increased compared to controls. Overall, the results of this study suggest that neonatal exposure to DEHP in pigs leads to sex-specific disruption of the reproductive system.

Degradation Products of Complex Arabinoxylans by Bacteroides intestinalis Enhance the Host Immune Response.

Bacteroides spp. of the human colonic microbiome degrade complex arabinoxylans from dietary fiber and release ferulic acid. Several studies have demonstrated the beneficial effects of ferulic acid. Here, we hypothesized that ferulic acid or the ferulic acid-rich culture supernatant of Bacteroides intestinalis, cultured in the presence of complex arabinoxylans, enhances the immune response. Ferulic acid and the culture supernatant of bacteria cultured in the presence of insoluble arabinoxylans significantly decreased the expression of tumor necrosis factor-α and increased the expression of interleukin-10 and transforming growth factor ß1 from activated dendritic cells compared to controls. The number of granulocytes in mesenteric lymph nodes, the number of spleen monocytes/granulocytes, and interleukin-2 and interleukin-12 plasma levels were significantly increased in mice treated with ferulic acid or the culture supernatant of bacteria cultured with insoluble arabinoxylans. Ferulic acid or the culture supernatant of bacteria cultured with insoluble arabinoxylans increased the expression of interleukin-12, interferon-α, and interferon-ß in intestinal epithelial cell lines. This study shows that ferulic acid or the ferulic acid-rich culture supernatant of the colonic bacterium Bacteroides intestinalis, cultured with insoluble arabinoxylans, exerts anti-inflammatory activity in dendritic cells under inflammatory conditions and enhances the Th1-type immune response under physiological conditions in mice.

Degradation of complex arabinoxylans by human colonic Bacteroidetes.

Some Bacteroidetes and other human colonic bacteria can degrade arabinoxylans, common polysaccharides found in dietary fiber. Previous work has identified gene clusters (polysaccharide-utilization loci, PULs) for degradation of simple arabinoxylans. However, the degradation of complex arabinoxylans (containing side chains such as ferulic acid, a phenolic compound) is poorly understood. Here, we identify a PUL that encodes multiple esterases for degradation of complex arabinoxylans in Bacteroides species. The PUL is specifically upregulated in the presence of complex arabinoxylans. We characterize some of the esterases biochemically and structurally, and show that they release ferulic acid from complex arabinoxylans. Growth of four different colonic Bacteroidetes members, including Bacteroides intestinalis, on complex arabinoxylans results in accumulation of ferulic acid, a compound known to have antioxidative and immunomodulatory properties.