From Hype to Hope: The Gut Microbiota in Enteric Infectious Disease.

One of the clearest functions of the gut microbiota in humans is resistance to colonization by enteric bacterial pathogens. Reconstitution of the microbiota offers an exciting therapeutic approach, but great challenges must be overcome.

Periportal macrophages protect against commensal-driven liver inflammation.

The liver is the main gateway from the gut, and the unidirectional sinusoidal flow from portal to central veins constitutes heterogenous zones, including the periportal vein (PV) and the pericentral vein zones1-5. However, functional differences in the immune system in each zone remain poorly understood. Here intravital imaging revealed that inflammatory responses are suppressed in PV zones. Zone-specific single-cell transcriptomics detected a subset of immunosuppressive macrophages enriched in PV zones that express high levels of interleukin-10 and Marco, a scavenger receptor that sequesters pro-inflammatory pathogen-associated molecular patterns and damage-associated molecular patterns, and consequently suppress immune responses. Induction of Marco+ immunosuppressive macrophages depended on gut microbiota. In particular, a specific bacterial family, Odoribacteraceae, was identified to induce this macrophage subset through its postbiotic isoallolithocholic acid. Intestinal barrier leakage resulted in inflammation in PV zones, which was markedly augmented in Marco-deficient conditions. Chronic liver inflammatory diseases such as primary sclerosing cholangitis (PSC) and non-alcoholic steatohepatitis (NASH) showed decreased numbers of Marco+ macrophages. Functional ablation of Marco+ macrophages led to PSC-like inflammatory phenotypes related to colitis and exacerbated steatosis in NASH in animal experimental models. Collectively, commensal bacteria induce Marco+ immunosuppressive macrophages, which consequently limit excessive inflammation at the gateway of the liver. Failure of this self-limiting system promotes hepatic inflammatory disorders such as PSC and NASH.

Identification of trypsin-degrading commensals in the large intestine.

Increased levels of proteases, such as trypsin, in the distal intestine have been implicated in intestinal pathological conditions1-3. However, the players and mechanisms that underlie protease regulation in the intestinal lumen have remained unclear. Here we show that Paraprevotella strains isolated from the faecal microbiome of healthy human donors are potent trypsin-degrading commensals. Mechanistically, Paraprevotella recruit trypsin to the bacterial surface through type IX secretion system-dependent polysaccharide-anchoring proteins to promote trypsin autolysis. Paraprevotella colonization protects IgA from trypsin degradation and enhances the effectiveness of oral vaccines against Citrobacter rodentium. Moreover, Paraprevotella colonization inhibits lethal infection with murine hepatitis virus-2, a mouse coronavirus that is dependent on trypsin and trypsin-like proteases for entry into host cells4,5. Consistently, carriage of putative genes involved in trypsin degradation in the gut microbiome was associated with reduced severity of diarrhoea in patients with SARS-CoV-2 infection. Thus, trypsin-degrading commensal colonization may contribute to the maintenance of intestinal homeostasis and protection from pathogen infection.

A widely distributed phosphate-insensitive phosphatase presents a route for rapid organophosphorus remineralization in the biosphere.

The regeneration of bioavailable phosphate from immobilized organophosphorus represents a key process in the global phosphorus cycle and is facilitated by enzymes known as phosphatases. Most bacteria possess at least one of three phosphatases with broad substrate specificity, known as PhoA, PhoX, and PhoD, whose activity is optimal under alkaline conditions. The production and activity of these phosphatases is repressed by phosphate availability. Therefore, they are only fully functional when bacteria experience phosphorus-limiting growth conditions. Here, we reveal a previously overlooked phosphate-insensitive phosphatase, PafA, prevalent in Bacteroidetes, which is highly abundant in nature and represents a major route for the regeneration of environmental phosphate. Using the enzyme from Flavobacterium johnsoniae, we show that PafA is highly active toward phosphomonoesters, is fully functional in the presence of excess phosphate, and is essential for growth on phosphorylated carbohydrates as a sole carbon source. These distinct properties of PafA may expand the metabolic niche of Bacteroidetes by enabling the utilization of abundant organophosphorus substrates as C and P sources, providing a competitive advantage when inhabiting zones of high microbial activity and nutrient demand. PafA, which is constitutively synthesized by soil and marine flavobacteria, rapidly remineralizes phosphomonoesters releasing bioavailable phosphate that can be acquired by neighboring cells. The pafA gene is highly diverse in plant rhizospheres and is abundant in the global ocean, where it is expressed independently of phosphate availability. PafA therefore represents an important enzyme in the context of global biogeochemical cycling and has potential applications in sustainable agriculture.

SRS-FISH: A high-throughput platform linking microbiome metabolism to identity at the single-cell level.

One of the biggest challenges in microbiome research in environmental and medical samples is to better understand functional properties of microbial community members at a single-cell level. Single-cell isotope probing has become a key tool for this purpose, but the current detection methods for determination of isotope incorporation into single cells do not allow high-throughput analyses. Here, we report on the development of an imaging-based approach termed stimulated Raman scattering-two-photon fluorescence in situ hybridization (SRS-FISH) for high-throughput metabolism and identity analyses of microbial communities with single-cell resolution. SRS-FISH offers an imaging speed of 10 to 100 ms per cell, which is two to three orders of magnitude faster than achievable by state-of-the-art methods. Using this technique, we delineated metabolic responses of 30,000 individual cells to various mucosal sugars in the human gut microbiome via incorporation of deuterium from heavy water as an activity marker. Application of SRS-FISH to investigate the utilization of host-derived nutrients by two major human gut microbiome taxa revealed that response to mucosal sugars tends to be dominated by Bacteroidales, with an unexpected finding that Clostridia can outperform Bacteroidales at foraging fucose. With high sensitivity and speed, SRS-FISH will enable researchers to probe the fine-scale temporal, spatial, and individual activity patterns of microbial cells in complex communities with unprecedented detail.

Transcriptomic analysis of polysaccharide utilization loci reveals substrate preferences in ruminal generalists Segatella bryantii TF1-3 and Xylanibacter ruminicola KHP1.

Bacteria of the genera Xylanibacter and Segatella are among the most dominant groups in the rumen microbiota. They are characterized by the ability to utilize different hemicelluloses and pectin of plant cell-wall as well as plant energy storage polysaccharides. The degradation is possible with the use of cell envelope bound multiprotein apparatuses coded in polysaccharide utilization loci (PULs), which have been shown to be substrate specific. The knowledge of PUL presence in rumen Xylanibacter and Segatella based on bioinformatic analyses is already established and transcriptomic and genetic approaches confirmed predicted PULs for a limited number of substrates. In this study, we transcriptomically identified additional different PULs in Xylanibacter ruminicola KHP1 and Segatella bryantii TF1-3. We also identified substrate preferences and found that specific growth rate and extent of growth impacted the choice of substrates preferentially used for degradation. These preferred substrates were used by both strains simultaneously as judged by their PUL upregulation. Lastly, ß-glucan and xyloglucan were used by these strains in the absence of bioinformatically and transcriptomically identifiable PUL systems.

Unveiling the role of novel carbohydrate-binding modules in laminarin interaction of multimodular proteins from marine Bacteroidota during phytoplankton blooms.

Laminarin, a ß(1,3)-glucan, serves as a storage polysaccharide in marine microalgae such as diatoms. Its abundance, water solubility and simple structure make it an appealing substrate for marine bacteria. Consequently, many marine bacteria have evolved strategies to scavenge and decompose laminarin, employing carbohydrate-binding modules (CBMs) as crucial components. In this study, we characterized two previously unassigned domains as laminarin-binding CBMs in multimodular proteins from the marine bacterium Christiangramia forsetii KT0803T, thereby introducing the new laminarin-binding CBM families CBM102 and CBM103. We identified four CBM102s in a surface glycan-binding protein (SGBP) and a single CBM103 linked to a glycoside hydrolase module from family 16 (GH16_3). Our analysis revealed that both modular proteins have an elongated shape, with GH16_3 exhibiting greater flexibility than SGBP. This flexibility may aid in the recognition and/or degradation of laminarin, while the constraints in SGBP could facilitate the docking of laminarin onto the bacterial surface. Exploration of bacterial metagenome-assembled genomes (MAGs) from phytoplankton blooms in the North Sea showed that both laminarin-binding CBM families are widespread among marine Bacteroidota. The high protein abundance of CBM102- and CBM103-containing proteins during phytoplankton blooms further emphasizes their significance in marine laminarin utilization.

The catabolic specialization of the marine bacterium Polaribacter sp. Q13 to red algal ß1,3/1,4-mixed-linkage xylan.

Catabolism of algal polysaccharides by marine bacteria is a significant process of marine carbon cycling. ß1,3/1,4-Mixed-linkage xylan (MLX) is a class of xylan in the ocean, widely present in the cell walls of red algae. However, the catabolic mechanism of MLX by marine bacteria remains elusive. Recently, we found that a marine Bacteroidetes strain, Polaribacter sp. Q13, is a specialist in degrading MLX, which secretes a novel MLX-specific xylanase. Here, the catabolic specialization of strain Q13 to MLX was studied by multiomics and biochemical analyses. Strain Q13 catabolizes MLX with a canonical starch utilization system (Sus), which is encoded by a single xylan utilization locus, XUL-Q13. In this system, the cell surface glycan-binding protein SGBP-B captures MLX specifically, contributing to the catabolic specificity. The xylanolytic enzyme system of strain Q13 is unique, and the enzymatic cascade dedicates the stepwise hydrolysis of the ß1,3- and ß1,4-linkages in MLX in the extracellular, periplasmic, and cytoplasmic spaces. Bioinformatics analysis and growth observation suggest that other marine Bacteroidetes strains harboring homologous MLX utilization loci also preferentially utilize MLX. These results reveal the catabolic specialization of MLX degradation by marine Bacteroidetes, leading to a better understanding of the degradation and recycling of MLX driven by marine bacteria.IMPORTANCERed algae contribute substantially to the primary production in marine ecosystems. The catabolism of red algal polysaccharides by marine bacteria is important for marine carbon cycling. Mixed-linkage ß1,3/1,4-xylan (MLX, distinct from hetero-ß1,4-xylans from terrestrial plants) is an abundant red algal polysaccharide, whose mechanism of catabolism by marine bacteria, however, remains largely unknown. This study reveals the catabolism of MLX by marine Bacteroidetes, promoting our understanding of the degradation and utilization of algal polysaccharides by marine bacteria. This study also sets a foundation for the biomass conversion of MLX.

Phytoplankton exudates and lysates support distinct microbial consortia with specialized metabolic and ecophysiological traits.

Blooms of marine phytoplankton fix complex pools of dissolved organic matter (DOM) that are thought to be partitioned among hundreds of heterotrophic microbes at the base of the food web. While the relationship between microbial consumers and phytoplankton DOM is a key component of marine carbon cycling, microbial loop metabolism is largely understood from model organisms and substrates. Here, we took an untargeted approach to measure and analyze partitioning of four distinct phytoplankton-derived DOM pools among heterotrophic populations in a natural microbial community using a combination of ecogenomics, stable isotope probing (SIP), and proteomics. Each 13C-labeled exudate or lysate from a diatom or a picocyanobacterium was preferentially assimilated by different heterotrophic taxa with specialized metabolic and physiological adaptations. Bacteroidetes populations, with their unique high-molecular-weight transporters, were superior competitors for DOM derived from diatom cell lysis, rapidly increasing growth rates and ribosomal protein expression to produce new relatively high C:N biomass. Proteobacteria responses varied, with relatively low levels of assimilation by Gammaproteobacteria populations, while copiotrophic Alphaproteobacteria such as the Roseobacter clade, with their diverse array of ABC- and TRAP-type transporters to scavenge monomers and nitrogen-rich metabolites, accounted for nearly all cyanobacteria exudate assimilation and produced new relatively low C:N biomass. Carbon assimilation rates calculated from SIP data show that exudate and lysate from two common marine phytoplankton are being used by taxonomically distinct sets of heterotrophic populations with unique metabolic adaptations, providing a deeper mechanistic understanding of consumer succession and carbon use during marine bloom events.

Indoleacrylic acid produced by Parabacteroides distasonis alleviates type 2 diabetes via activation of AhR to repair intestinal barrier.

BACKGROUND: Anti-inflammatory therapy is an effective strategy in the treatment of type 2 diabetes (T2D). Studies found that inflammatory responses in vivo were strongly associated with defects in the mucosal barrier function of the gut epithelium. While some microbial strains could help repair the intestinal mucosa and maintain the integrity of the intestinal barrier, the specific mechanisms remain to be fully elucidated. The present study investigated the effects of Parabacteroides distasonis (P. distasonis) on the intestinal barrier and the inflammation level in T2D rats and explored the specific mechanisms. RESULTS: By analyzing the intestinal barrier function, the inflammatory conditions, and the gut microbiome, we found that P. distasonis could attenuate insulin resistance by repairing the intestinal barrier and reducing inflammation caused by the disturbed gut microbiota. We quantitatively profiled the level of tryptophan and indole derivatives (IDs) in rats and fermentation broth of the strain, demonstrating that indoleacrylic acid (IA) was the most significant factor correlated with the microbial alterations among all types of endogenous metabolites. Finally, we used molecular and cell biological techniques to determine that the metabolic benefits of P. distasonis were mainly attributed to its ability to promote IA generation, active the aryl hydrocarbon receptor (AhR) signaling pathway, and increase the expression level of interleukin-22 (IL-22), thus enhancing the expression of intestinal barrier-related proteins. CONCLUSIONS: Our study revealed the effects of P. distasonis in the treatment of T2D via intestinal barrier repairment and inflammation reduction and highlighted a host-microbial co-metabolite indoleacrylic acid that could active AhR to perform its physiological effects. Our study provided new therapeutic strategies for metabolic diseases by targeting the gut microbiota and tryptophan metabolism.

Comparative Transcriptomics of Fat Bodies between Symbiotic and Quasi-Aposymbiotic Adult Females of Blattella germanica with Emphasis on the Metabolic Integration with Its Endosymbiont Blattabacterium and Its Immune System.

We explored the metabolic integration of Blattella germanica and its obligate endosymbiont Blattabacterium cuenoti by the transcriptomic analysis of the fat body of quasi-aposymbiotic cockroaches, where the endosymbionts were almost entirely removed with rifampicin. Fat bodies from quasi-aposymbiotic insects displayed large differences in gene expression compared to controls. In quasi-aposymbionts, the metabolism of phenylalanine and tyrosine involved in cuticle sclerotization and pigmentation increased drastically to compensate for the deficiency in the biosynthesis of these amino acids by the endosymbionts. On the other hand, the uricolytic pathway and the biosynthesis of uric acid were severely decreased, probably because the reduced population of endosymbionts was unable to metabolize urea to ammonia. Metabolite transporters that could be involved in the endosymbiosis process were identified. Immune system and antimicrobial peptide (AMP) gene expression was also reduced in quasi-aposymbionts, genes encoding peptidoglycan-recognition proteins, which may provide clues for the maintenance of the symbiotic relationship, as well as three AMP genes whose involvement in the symbiotic relationship will require additional analysis. Finally, a search for AMP-like factors that could be involved in controlling the endosymbiont identified two orphan genes encoding proteins smaller than 200 amino acids underexpressed in quasi-aposymbionts, suggesting a role in the host-endosymbiont relationship.

Biochemical characterization of the first step in sulfonolipid biosynthesis in Alistipes finegoldii.

Sulfonolipids are unusual lipids found in the outer membranes of Gram-negative bacteria in the phylum Bacteroidetes. Sulfonolipid and its deacylated derivative, capnine, are sulfur analogs of ceramide-1-phosphate and sphingosine-1-phosphate, respectively; thus, sulfonolipid biosynthesis is postulated to be similar to the sphingolipid biosynthetic pathway. Here, we identify the first enzyme in sulfonolipid synthesis in Alistipes finegoldii as the product of the alfi_1224 gene, cysteate acyl-acyl carrier protein (ACP) transferase (SulA). We show SulA catalyzes the condensation of acyl-ACP and cysteate (3-sulfo-alanine) to form 3-ketocapnine. Acyl-CoA is a poor substrate. We show SulA has a bound pyridoxal phosphate (PLP) cofactor that undergoes a spectral redshift in the presence of cysteate, consistent with the transition of the lysine-aldimine complex to a substrate-aldimine complex. Furthermore, the SulA crystal structure shows the same prototypical fold found in bacterial serine palmitoyltransferases (Spts), enveloping the PLP cofactor bound to Lys251. We observed the SulA and Spt active sites are identical except for Lys281 in SulA, which is an alanine in Spt. Additionally, SulA(K281A) is catalytically inactive but binds cysteate and forms the external aldimine normally, highlighting the structural role of the Lys281 side chain in walling off the active site from bulk solvent. Finally, the electropositive groove on the protein surface adjacent to the active site entrance provides a landing pad for the electronegative acyl-ACP surface. Taken together, these data identify the substrates, products, and mechanism of SulA, the PLP-dependent condensing enzyme that catalyzes the first step in sulfonolipid synthesis in a gut commensal bacterium.

Reisomerization of retinal represents a molecular switch mediating Na+ uptake and release by a bacterial sodium-pumping rhodopsin.

Sodium-pumping rhodopsins (NaRs) are membrane transporters that utilize light energy to pump Na+ across the cellular membrane. Within the NaRs, the retinal Schiff base chromophore absorbs light, and a photochemically induced transient state, referred to as the "O intermediate", performs both the uptake and release of Na+. However, the structure of the O intermediate remains unclear. Here, we used time-resolved cryo-Raman spectroscopy under preresonance conditions to study the structure of the retinal chromophore in the O intermediate of an NaR from the bacterium Indibacter alkaliphilus. We observed two O intermediates, termed O1 and O2, having distinct chromophore structures. We show O1 displays a distorted 13-cis chromophore, while O2 contains a distorted all-trans structure. This finding indicated that the uptake and release of Na+ are achieved not by a single O intermediate but by two sequential O intermediates that are toggled via isomerization of the retinal chromophore. These results provide crucial structural insight into the unidirectional Na+ transport mediated by the chromophore-binding pocket of NaRs.

Transferable Immunoglobulin A-Coated Odoribacter splanchnicus in Responders to Fecal Microbiota Transplantation for Ulcerative Colitis Limits Colonic Inflammation.

BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) is an emerging treatment modality for ulcerative colitis (UC). Several randomized controlled trials have shown efficacy for FMT in the treatment of UC, but a better understanding of the transferable microbiota and their immune impact is needed to develop more efficient microbiome-based therapies for UC. METHODS: Metagenomic analysis and strain tracking was performed on 60 donor and recipient samples receiving FMT for active UC. Sorting and sequencing of immunoglobulin (Ig) A-coated microbiota (called IgA-seq) was used to define immune-reactive microbiota. Colonization of germ-free or genetically engineered mice with patient-derived strains was performed to determine the mechanism of microbial impact on intestinal immunity. RESULTS: Metagenomic analysis defined a core set of donor-derived transferable bacterial strains in UC subjects achieving clinical response, which predicted response in an independent trial of FMT for UC. IgA-seq of FMT recipient samples and gnotobiotic mice colonized with donor microbiota identified Odoribacter splanchnicus as a transferable strain shaping mucosal immunity, which correlated with clinical response and the induction of mucosal regulatory T cells. Colonization of mice with O splanchnicus led to an increase in Foxp3+/RORγt+ regulatory T cells, induction of interleukin (IL) 10, and production of short chain fatty acids, all of which were required for O splanchnicus to limit colitis in mouse models. CONCLUSIONS: This work provides the first evidence of transferable, donor-derived strains that correlate with clinical response to FMT in UC and reveals O splanchnicus as a key component promoting both metabolic and immune cell protection from colitis. These mechanistic features will help enable strategies to enhance the efficacy of microbial therapy for UC. Clinicaltrials.gov ID NCT02516384.

Marine Bacteroidetes enzymatically digest xylans from terrestrial plants.

Marine Bacteroidetes that degrade polysaccharides contribute to carbon cycling in the ocean. Organic matter, including glycans from terrestrial plants, might enter the oceans through rivers. Whether marine bacteria degrade structurally related glycans from diverse sources including terrestrial plants and marine algae was previously unknown. We show that the marine bacterium Flavimarina sp. Hel_I_48 encodes two polysaccharide utilization loci (PULs) which degrade xylans from terrestrial plants and marine algae. Biochemical experiments revealed activity and specificity of the encoded xylanases and associated enzymes of these PULs. Proteomics indicated that these genomic regions respond to glucuronoxylans and arabinoxylans. Substrate specificities of key enzymes suggest dedicated metabolic pathways for xylan utilization. Some of the xylanases were active on different xylans with the conserved ß-1,4-linked xylose main chain. Enzyme activity was consistent with growth curves showing Flavimarina sp. Hel_I_48 uses structurally different xylans. The observed abundance of related xylan-degrading enzyme repertoires in genomes of other marine Bacteroidetes indicates similar activities are common in the ocean. The here presented data show that certain marine bacteria are genetically and biochemically variable enough to access parts of structurally diverse xylans from terrestrial plants as well as from marine algal sources.

Lignocellulose degradation by rumen bacterial communities: New insights from metagenome analyses.

Ruminant animals house a dense and diverse community of microorganisms in their rumen, an enlarged compartment in their stomach, which provides a supportive environment for the storage and microbial fermentation of ingested feeds dominated by plant materials. The rumen microbiota has acquired diverse and functionally overlapped enzymes for the degradation of plant cell wall polysaccharides. In rumen Bacteroidetes, enzymes involved in degradation are clustered into polysaccharide utilization loci to facilitate coordinated expression when target polysaccharides are available. Firmicutes use free enzymes and cellulosomes to degrade the polysaccharides. Fibrobacters either aggregate lignocellulose-degrading enzymes on their cell surface or release them into the extracellular medium in membrane vesicles, a mechanism that has proven extremely effective in the breakdown of recalcitrant cellulose. Based on current metagenomic analyses, rumen Bacteroidetes and Firmicutes are categorized as generalist microbes that can degrade a wide range of polysaccharides, while other members adapted toward specific polysaccharides. Particularly, there is ample evidence that Verrucomicrobia and Spirochaetes have evolved enzyme systems for the breakdown of complex polysaccharides such as xyloglucans, peptidoglycans, and pectin. It is concluded that diversity in degradation mechanisms is required to ensure that every component in feeds is efficiently degraded, which is key to harvesting maximum energy by host animals.

Pectin alleviates the pulmonary inflammatory response induced by PM2.5 from a pig house by modulating intestinal microbiota.

This study aimed to investigate whether dietary fiber pectin can alleviate PM2.5-induced pulmonary inflammation and the potential mechanism. PM2.5 samples were collected from a nursery pig house. The mice were divided into three groups: the control group, PM2.5 group and PM2.5 + pectin group. The mice in the PM2.5 group were intratracheally instilled with PM2.5 suspension twice a week for four consecutive weeks, and those in the PM2.5 + pectin group were subject to the same PM2.5 exposure, but fed with a basal diet supplemented with 5% pectin. The results showed that body weight and feed intake were not different among the treatments (p > 0.05). However, supplementation with pectin relieved PM2.5-induced pulmonary inflammation, presenting as slightly restored lung morphology, decreased mRNA expression levels of IL-1ß, IL-6 and IL-17 in the lung, decreased MPO content in bronchoalveolar lavage fluid (BLAF), and even decreased protein levels of IL-1ß and IL-6 in the serum (p < 0.05). Dietary pectin altered the composition of the intestinal microbiota, increasing the relative abundance of Bacteroidetes and decreasing the ratio of Firmicutes/Bacteroidetes. At the genus level, short-chain fatty acid (SCFA)-producing bacteria, such as Bacteroides, Anaerotruncus, Prevotella 2, Parabacteroides, Ruminococcus 2 and Butyricimonas, were enriched in the PM2.5 +pectin group. Accordingly, dietary pectin increased the concentrations of SCFAs, including acetate, propionate, butyrate and valerate, in mice. In conclusion, dietary fermentable fiber pectin can relieve PM2.5-induced pulmonary inflammation via alteration of intestinal microbiota composition and SCFA production. This study provides a new insight into reducing the health risk associated with PM2.5 exposure.

Molecular Docking of Lac_CB10: Highlighting the Great Potential for Bioremediation of Recalcitrant Chemical Compounds by One Predicted Bacteroidetes CopA-Laccase.

Laccases are multicopper oxidases (MCOs) with a broad application spectrum, particularly in second-generation ethanol biotechnology and the bioremediation of xenobiotics and other highly recalcitrant compounds. Synthetic pesticides are xenobiotics with long environmental persistence, and the search for their effective bioremediation has mobilized the scientific community. Antibiotics, in turn, can pose severe risks for the emergence of multidrug-resistant microorganisms, as their frequent use for medical and veterinary purposes can generate constant selective pressure on the microbiota of urban and agricultural effluents. In the search for more efficient industrial processes, some bacterial laccases stand out for their tolerance to extreme physicochemical conditions and their fast generation cycles. Accordingly, to expand the range of effective approaches for the bioremediation of environmentally important compounds, the prospection of bacterial laccases was carried out from a custom genomic database. The best hit found in the genome of Chitinophaga sp. CB10, a Bacteroidetes isolate obtained from a biomass-degrading bacterial consortium, was subjected to in silico prediction, molecular docking, and molecular dynamics simulation analyses. The putative laccase CB10_180.4889 (Lac_CB10), composed of 728 amino acids, with theoretical molecular mass values of approximately 84 kDa and a pI of 6.51, was predicted to be a new CopA with three cupredoxin domains and four conserved motifs linking MCOs to copper sites that assist in catalytic reactions. Molecular docking studies revealed that Lac_CB10 had a high affinity for the molecules evaluated, and the affinity profiles with multiple catalytic pockets predicted the following order of decreasing thermodynamically favorable values: tetracycline (-8 kcal/mol) > ABTS (-6.9 kcal/mol) > sulfisoxazole (-6.7 kcal/mol) > benzidine (-6.4 kcal/mol) > trimethoprim (-6.1 kcal/mol) > 2,4-dichlorophenol (-5.9 kcal/mol) mol. Finally, the molecular dynamics analysis suggests that Lac_CB10 is more likely to be effective against sulfisoxazole-like compounds, as the sulfisoxazole-Lac_CB10 complex exhibited RMSD values lower than 0.2 nm, and sulfisoxazole remained bound to the binding site for the entire 100 ns evaluation period. These findings corroborate that LacCB10 has a high potential for the bioremediation of this molecule.

Dihydroceramides Derived from Bacteroidetes Species Sensitize TRPV1 Channels.

Bacterial colonization of open wounds is common, and patients with infected wounds often report significantly elevated pain sensitivity at the wound site. Transient Receptor Potential Vanilloid Type 1 (TRPV1) channels are known to play an important role in pain signaling and may be sensitized under pro-inflammatory conditions. Bacterial membrane components, such as phosphoethanolamine dihydroceramide (PEDHC), phosphoglycerol dihydroceramide (PGDHC), and lipopolysaccharide (LPS), are released in the environment from the Gram-negative bacteria of the Bacteroidetes species colonizing the infected wounds. Here, we used intracellular calcium imaging and patch-clamp electrophysiology approaches to determine whether bacterially derived PEDHC, PGDHC, or LPS can modulate the activity of the TRPV1 channels heterologously expressed in HEK cells. We found that PEDHC and PGDHC can sensitize TRPV1 in a concentration-dependent manner, whereas LPS treatment does not significantly affect TRPV1 activity in HEK cells. We propose that sensitization of TRPV1 channels by Bacteroidetes-derived dihydroceramides may at least in part underlie the increased pain sensitivity associated with wound infections.

Analysis of Effector and Immunity Proteins of the GA2 Type VI Secretion Systems of Gut Bacteroidales.

Three distinct genetic architectures (GAs) of Type VI secretion systems (T6SSs) have been described in gut Bacteroidales species, each with unique genes and characteristics. Unlike the GA3 T6SSs, potent antagonism has not yet been demonstrated for the GA1 or GA2 T6SSs. We previously showed that the GA2 T6SS loci are contained on integrative and conjugative elements and that there are five subtypes. Collectively, GA2 are the most prevalent Bacteroidales T6SSs in the human populations analyzed. In this study, we provide a comprehensive bioinformatic analysis of the three variable regions of GA2 T6SS loci, which encode toxic effector and immunity proteins. In total, we identified 63 distinct effectors encoded within 31 nonredundant GA2 loci, 18 of which do not have described motifs or predicted functions. We provide experimental evidence for toxin activity for four different GA2 effectors, showing that each functions only when present in the periplasm, and experimentally confirm their cognate immunity proteins. Our data demonstrate that each GA2 locus encodes at least three distinct effectors with targets in both the cytoplasm and the periplasm. The data also suggest that the effectors of a given locus are loaded onto the tube by different mechanisms, which may allow all three effectors encoded within a single GA2 locus with distinct antibacterial activity to be loaded onto a single T6 tube, increasing the antagonistic effect. IMPORTANCE Humans are colonized with many gut Bacteroidales species at high density, allowing for extensive opportunities for contact-dependent antagonism. To begin to understand the antagonistic potential of the GA2 T6SSs of the gut Bacteroidales, we performed bioinformatic and experimental analyses of the three divergent regions containing the toxin effector and immunity genes. We show that each GA2 T6SS locus encodes at least three distinct toxic effectors including toxins linked to Rhs and Hcp with cytoplasmic targets, and unlinked effectors with targets in the periplasm. The diversity and modality of effectors exceeds that of the GA1 or GA3 T6SS loci (M. J. Coyne, K. G. Roelofs, and L. E. Comstock, BMC Genomics 17:58, 2016, https://doi.org/10.1186/s12864-016-2377-z) and suggests that these T6SSs have the potential to be potent antibacterial weapons in the human gut.