Histologic Distribution of Lung and Bronchus Tumors: A Population-Based Study in the Upper Peninsula of Michigan.

INTRODUCTION: Lung cancer remains the leading cause of cancer-related mortality in the United States, with cigarette smoking recognized as the most important modifiable risk factor. The distinct smoking rates and occupational landscape in the Upper Peninsula of Michigan underscore the necessity of investigating the multifactorial influences on the prevalence and distribution of lung and bronchus cancer within this population. METHODS: This study, conducted from January 2012 to December 2022, included 1035 patients diagnosed with lung or bronchus tumors who were first seen and/or received the first course of treatment at Upper Peninsula Health Systems (UPHS) - Marquette, the largest hospital system in the Upper Peninsula of Michigan and one of only two radiation oncology treatment centers in the Upper Peninsula. RESULTS: This study demonstrated that the histologic trend of lung and bronchus cancers in a sample of 1035 patients in the Upper Peninsula of Michigan closely resembles that of national averages. Participants with a lifetime history of smoking made up 943 (91.1%) cases of patients diagnosed with lung or bronchus cancers in this study. Lifetime non-smokers only made up 53 (5.1%) cases of patients diagnosed with lung or bronchus cancers. The median age at diagnosis of participants in this study was 70 years. CONCLUSION: Our study provides significant insights into the histologic distribution of lung and bronchus cancers within the Upper Peninsula of Michigan, addressing a notable gap in the current literature for this rural and medically underserved population. The histologic distribution of lung and bronchus cancers in this region aligns with national trends. Furthermore, the distinct rates of cigarette smoking in the Upper Peninsula emphasize the critical role of smoking cessation efforts in reducing the burden of lung and bronchus cancers in this region.

Loss of Bacteroides thetaiotaomicron bile acid-altering enzymes impacts bacterial fitness and the global metabolic transcriptome.

IMPORTANCE: Recent work on bile salt hydrolases (BSHs) in Gram-negative bacteria, such as Bacteroides, has primarily focused on how they can impact host physiology. However, the benefits bile acid metabolism confers to the bacterium that performs it are not well understood. In this study, we set out to define if and how Bacteroides thetaiotaomicron (B. theta) uses its BSHs and hydroxysteroid dehydrogenase to modify bile acids to provide a fitness advantage for itself in vitro and in vivo. Genes encoding bile acid-altering enzymes were able to impact how B. theta responds to nutrient limitation in the presence of bile acids, specifically carbohydrate metabolism, affecting many polysaccharide utilization loci. This suggests that B. theta may be able to shift its metabolism, specifically its ability to target different complex glycans including host mucin, when it comes into contact with specific bile acids in the gut.

A Comparison of In Vivo Bone Tissue Generation Using Calcium Phosphate Bone Substitutes in a Novel 3D Printed Four-Chamber Periosteal Bioreactor.

Autologous bone replacement remains the preferred treatment for segmental defects of the mandible; however, it cannot replicate complex facial geometry and causes donor site morbidity. Bone tissue engineering has the potential to overcome these limitations. Various commercially available calcium phosphate-based bone substitutes (Novabone®, BioOss®, and Zengro®) are commonly used in dentistry for small bone defects around teeth and implants. However, their role in ectopic bone formation, which can later be applied as vascularized graft in a bone defect, is yet to be explored. Here, we compare the above-mentioned bone substitutes with autologous bone with the aim of selecting one for future studies of segmental mandibular repair. Six female sheep, aged 7-8 years, were implanted with 40 mm long four-chambered polyether ether ketone (PEEK) bioreactors prepared using additive manufacturing followed by plasma immersion ion implantation (PIII) to improve hydrophilicity and bioactivity. Each bioreactor was wrapped with vascularized scapular periosteum and the chambers were filled with autologous bone graft, Novabone®, BioOss®, and Zengro®, respectively. The bioreactors were implanted within a subscapular muscle pocket for either 8 weeks (two sheep), 10 weeks (two sheep), or 12 weeks (two sheep), after which they were removed and assessed by microCT and routine histology. Moderate bone formation was observed in autologous bone grafts, while low bone formation was observed in the BioOss® and Zengro® chambers. No bone formation was observed in the Novabone® chambers. Although the BioOss® and Zengro® chambers contained relatively small amounts of bone, endochondral ossification and retained hydroxyapatite suggest their potential in new bone formation in an ectopic site if a consistent supply of progenitor cells and/or growth factors can be ensured over a longer duration.

Multiple TonB homologs are important for carbohydrate utilization by Bacteroides thetaiotaomicron.

IMPORTANCE: The human gut microbiota, including Bacteroides, is required for the degradation of otherwise undigestible polysaccharides. The gut microbiota uses polysaccharides as an energy source, and fermentation products such as short-chain fatty acids are beneficial to the human host. This use of polysaccharides is dependent on the proper pairing of a TonB protein with polysaccharide-specific TonB-dependent transporters; however, the formation of these protein complexes is poorly understood. In this study, we examine the role of 11 predicted TonB homologs in polysaccharide uptake. We show that two proteins, TonB4 and TonB6, may be functionally redundant. This may allow for the development of drugs targeting Bacteroides species containing only a TonB4 homolog with limited impact on species encoding the redundant TonB6.

Using Multidimensional Separations to Distinguish Isomeric Amino Acid-Bile Acid Conjugates and Assess Their Presence and Perturbations in Model Systems.

Bile acids play key roles in nutrient uptake, inflammation, signaling, and microbiome composition. While previous bile acid analyses have primarily focused on profiling 5 canonical primary and secondary bile acids and their glycine and taurine amino acid-bile acid (AA-BA) conjugates, recent studies suggest that many other microbial conjugated bile acids (or MCBAs) exist. MCBAs are produced by the gut microbiota and serve as biomarkers, providing information about early disease onset and gut health. Here we analyzed 8 core bile acids synthetically conjugated with 22 proteinogenic and nonproteogenic amino acids totaling 176 MCBAs. Since many of the conjugates were isomeric and only 42 different m/z values resulted from the 176 MCBAs, a platform coupling liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) was used for their separation. Their molecular characteristics were then used to create an in-house extended bile acid library for a combined total of 182 unique compounds. Additionally, ∼250 rare bile acid extracts were also assessed to provide additional resources for bile acid profiling and identification. This library was then applied to healthy mice dosed with antibiotics and humans having fecal microbiota transplantation (FMT) to assess the MCBA presence and changes in the gut before and after each perturbation.

The microbial-derived bile acid lithocholate and its epimers inhibit Clostridioides difficile growth and pathogenicity while sparing members of the gut microbiota.

Clostridioides difficile is a Gram-positive, spore-forming anaerobe that causes clinical diseases ranging from diarrhea and pseudomembranous colitis to toxic megacolon and death. C. difficile infection (CDI) is associated with antibiotic usage, which disrupts the indigenous gut microbiota and causes the loss of microbial-derived secondary bile acids that normally provide protection against C. difficile colonization. Previous work has shown that the secondary bile acid lithocholate (LCA) and its epimer isolithocholate (iLCA) have potent inhibitory activity against clinically relevant C. difficile strains. To further characterize the mechanisms by which LCA and its epimers iLCA and isoallolithocholate (iaLCA) inhibit C. difficile, we tested their minimum inhibitory concentration against C. difficile R20291 and a commensal gut microbiota panel. We also performed a series of experiments to determine the mechanism of action by which LCA and its epimers inhibit C. difficile through bacterial killing and effects on toxin expression and activity. Additionally, we tested the cytotoxicity of these bile acids through Caco-2 cell apoptosis and viability assays to gauge their effects on the host. Here, we show that the epimers iLCA and iaLCA strongly inhibit C. difficile growth in vitro while sparing most commensal Gram-negative gut microbes. We also show that iLCA and iaLCA have bactericidal activity against C. difficile, and these epimers cause significant bacterial membrane damage at subinhibitory concentrations. Finally, we observe that iLCA and iaLCA decrease the expression of the large cytotoxin tcdA, while LCA significantly reduces toxin activity. Although iLCA and iaLCA are both epimers of LCA, they have distinct mechanisms for inhibiting C. difficile. LCA epimers, iLCA and iaLCA, represent promising compounds that target C. difficile with minimal effects on members of the gut microbiota that are important for colonization resistance. IMPORTANCE In the search for a novel therapeutic that targets Clostridioides difficile, bile acids have become a viable solution. Epimers of bile acids are particularly attractive as they may provide protection against C. difficile while leaving the indigenous gut microbiota largely unaltered. This study shows that LCA epimers isolithocholate (iLCA) and LCA epimers isoallolithocholate (iaLCA) specifically are potent inhibitors of C. difficile, affecting key virulence factors including growth, toxin expression, and activity. As we move toward the use of bile acids as therapeutics, further work will be required to determine how best to deliver these bile acids to a target site within the host intestinal tract.

Loss of Bacteroides thetaiotaomicron bile acid altering enzymes impact bacterial fitness and the global metabolic transcriptome.

Bacteroides thetaiotaomicron (B. theta) is a Gram-negative gut bacterium that encodes enzymes that alter the bile acid pool in the gut. Primary bile acids are synthesized by the host liver and are modified by gut bacteria. B. theta encodes two bile salt hydrolases (BSHs), as well as a hydroxysteroid dehydrogenase (HSDH). We hypothesize that B. theta modifies the bile acid pool in the gut to provide a fitness advantage for itself. To investigate each gene's role, different combinations of genes encoding bile acid altering enzymes (bshA, bshB, and hsdhA) were knocked out by allelic exchange, including a triple KO. Bacterial growth and membrane integrity assays were done in the presence and absence of bile acids. To explore if B. theta's response to nutrient limitation changes due to the presence of bile acid altering enzymes, RNASeq analysis of WT and triple KO strains in the presence and absence of bile acids was done. WT B. theta is more sensitive to deconjugated bile acids (CA, CDCA, and DCA) compared to the triple KO, which also decreased membrane integrity. The presence of bshB is detrimental to growth in conjugated forms of CDCA and DCA. RNA-Seq analysis also showed bile acid exposure impacts multiple metabolic pathways in B. theta, but DCA significantly increases expression of many genes in carbohydrate metabolism, specifically those in polysaccharide utilization loci or PULs, in nutrient limited conditions. This study suggests that bile acids B. theta encounters in the gut may signal the bacteria to increase or decrease its utilization of carbohydrates. Further study looking at the interactions between bacteria, bile acids, and the host may inform rationally designed probiotics and diets to ameliorate inflammation and disease. Importance: Recent work on BSHs in Gram-negative bacteria, such as Bacteroides, has primarily focused on how they can impact host physiology. However, the benefits bile acid metabolism confers to the bacterium that performs it is not well understood. In this study we set out to define if and how B. theta uses its BSHs and HSDH to modify bile acids to provide a fitness advantage for itself in vitro and in vivo. Genes encoding bile acid altering enzymes were able to impact how B. theta responds to nutrient limitation in the presence of bile acids, specifically carbohydrate metabolism, affecting many polysaccharide utilization loci (PULs). This suggests that B. theta may be able to shift its metabolism, specifically its ability to target different complex glycans including host mucin, when it comes into contact with specific bile acids in the gut. This work will aid in our understanding of how to rationally manipulate the bile acid pool and the microbiota to exploit carbohydrate metabolism in the context of inflammation and other GI diseases.

Multiple TonB Homologs are Important for Carbohydrate Utilization by Bacteroides thetaiotaomicron.

The human gut microbiota is able to degrade otherwise undigestible polysaccharides, largely through the activity of the Bacteroides. Uptake of polysaccharides into Bacteroides is controlled by TonB-dependent transporters (TBDT) whose transport is energized by an inner membrane complex composed of the proteins TonB, ExbB, and ExbD. Bacteroides thetaiotaomicron (B. theta) encodes 11 TonB homologs which are predicted to be able to contact TBDTs to facilitate transport. However, it is not clear which TonBs are important for polysaccharide uptake. Using strains in which each of the 11 predicted tonB genes are deleted, we show that TonB4 (BT2059) is important but not essential for proper growth on starch. In the absence of TonB4, we observed an increase in abundance of TonB6 (BT2762) in the membrane of B. theta, suggesting functional redundancy of these TonB proteins. Growth of the single deletion strains on pectin galactan, chondroitin sulfate, arabinan, and levan suggests a similar functional redundancy of the TonB proteins. A search for highly homologous proteins across other Bacteroides species and recent work in B. fragilis suggests that TonB4 is widely conserved and may play a common role in polysaccharide uptake. However, proteins similar to TonB6 are found only in B. theta and closely related species suggesting that the functional redundancy of TonB4 and TonB6 may be limited across the Bacteroides. This study extends our understanding of the protein network required for polysaccharide utilization in B. theta and highlights differences in TonB complexes across Bacteroides species.

The microbial derived bile acid lithocholate and its epimers inhibit Clostridioides difficile growth and pathogenicity while sparing members of the gut microbiota.

C. difficile infection (CDI) is associated with antibiotic usage, which disrupts the indigenous gut microbiota and causes the loss of microbial derived secondary bile acids that normally provide protection against C. difficile colonization. Previous work has shown that the secondary bile acid lithocholate (LCA) and its epimer isolithocholate (iLCA) have potent inhibitory activity against clinically relevant C. difficile strains. To further characterize the mechanisms by which LCA and its epimers iLCA and isoallolithocholate (iaLCA) inhibit C. difficile, we tested their minimum inhibitory concentration (MIC) against C. difficile R20291, and a commensal gut microbiota panel. We also performed a series of experiments to determine the mechanism of action by which LCA and its epimers inhibit C. difficile through bacterial killing and effects on toxin expression and activity. Here we show that epimers iLCA and iaLCA strongly inhibit C. difficile growth in vitro while sparing most commensal Gram-negative gut microbes. We also show that iLCA and iaLCA have bactericidal activity against C. difficile, and these epimers cause significant bacterial membrane damage at subinhibitory concentrations. Finally, we observe that iLCA and iaLCA decrease the expression of the large cytotoxin tcdA while LCA significantly reduces toxin activity. Although iLCA and iaLCA are both epimers of LCA, they have distinct mechanisms for inhibiting C. difficile . LCA epimers, iLCA and iaLCA, represent promising compounds that target C. difficile with minimal effects on members of the gut microbiota that are important for colonization resistance. Importance: In the search for a novel therapeutic that targets C. difficile , bile acids have become a viable solution. Epimers of bile acids are particularly attractive as they may provide protection against C. difficile while leaving the indigenous gut microbiota largely unaltered. This study shows that iLCA and iaLCA specifically are potent inhibitors of C. difficile , affecting key virulence factors including growth, toxin expression and activity. As we move toward the use of bile acids as therapeutics, further work will be required to determine how best to deliver these bile acids to a target site within the host intestinal tract.

Bile salt hydrolases shape the bile acid landscape and restrict Clostridioides difficile growth in the murine gut.

Bile acids (BAs) mediate the crosstalk between human and microbial cells and influence diseases including Clostridioides difficile infection (CDI). While bile salt hydrolases (BSHs) shape the BA pool by deconjugating conjugated BAs, the basis for their substrate selectivity and impact on C. difficile remain elusive. Here we survey the diversity of BSHs in the gut commensals Lactobacillaceae, which are commonly used as probiotics, and other members of the human gut microbiome. We structurally pinpoint a loop that predicts BSH preferences for either glycine or taurine substrates. BSHs with varying specificities were shown to restrict C. difficile spore germination and growth in vitro and colonization in pre-clinical in vivo models of CDI. Furthermore, BSHs reshape the pool of microbial conjugated bile acids (MCBAs) in the murine gut, and these MCBAs can further restrict C. difficile virulence in vitro. The recognition of conjugated BAs by BSHs defines the resulting BA pool, including the expansive MCBAs. This work provides insights into the structural basis of BSH mechanisms that shape the BA landscape and promote colonization resistance against C. difficile.

Dataset for the Victorian energy transition including technical, social, economic, and environmental detail.

This data relates to existing and planned electricity generation projects in Victoria, Australia. Planning Victoria, part of the Victorian Government, registered most projects. The technical performance data for the projects includes the electricity generated, input fuel, losses in the transmission of electricity, energy storage options, and transparency between grid operators and stakeholders. The social data related to the projects include health data for the effect of Victorian coal plant pollution, including a rich dataset on the health effects of a coal fire in (Jennens, 2021). A dataset for all the health effects of coal plants in Victoria was then compiled. The social data also includes a measure of customer engagement with electricity distribution companies in Victoria and public satisfaction with renewable projects in Victoria. The economic cost data includes capital costs, operation and maintenance costs, and externality costs related to greenhouse gasses emitted. The environmental data used a life cycle assessment and the critical materials needed for the electricity generation projects. The analysis performed was an nBL assessment (Foliente, 2007) which uses a comparative analysis of the four bottom lines (environmental, social, economic and technology). An nBL assessment is similar to a Triple Bottom Line assessment but includes additional parameters. The data used for this nBL analysis was for three scenarios (business as usual, a renewable generation future with electricity imported from other states and a renewable generation future with all electricity generated in Victoria). The first step in managing the raw data was to normalise, standardise and aggregate the data. These steps were done for the four bottom lines and the three scenarios. The reuse potential of this data is high as it is for a pipeline of projects that will continue to evolve. This data would also have the potential for other researchers to compare the Victorian electricity transition with other places internationally.

Physical properties of root cementum: Part 29. The effects of LED-mediated photobiomodulation on orthodontically induced root resorption and pain: a pilot split-mouth randomized controlled trial.

OBJECTIVES: To examine the effects of light-emitting diode (LED)-mediated photobiomodulation (PBM) on orthodontic root resorption and pain. METHODS: Twenty patients (3 males, 17 females, mean age 15 years 6 months) needing bilateral maxillary first premolar extractions for orthodontic treatment were included in this single-centre, split-mouth randomized controlled trial. Both premolars received 150 g of buccal tipping force for 28 days. One side was randomly assigned to receive intraoral 850 nm wavelength, 60 mW/cm2 power, continuous LED illumination via OrthoPulse device (Biolux Research Ltd, Vancouver, British Columbia, Canada) for 5 minutes/day. The other side served as control. After 28 days, both premolars were extracted and scanned with micro-computed tomography for primary outcome assessment of root resorption crater volume measurements. For secondary outcome assessment, visual analogue scale pain questionnaires were used for both sides at 24 hours, 48 hours, 72 hours, and 7 days. Randomization was generated using www.randomization.com and allocation was concealed in sequentially numbered, opaque, sealed envelopes. Blinding was not possible during the experiment due to the use of tape to block light on control side of the devices. Assessors were blinded during outcome assessments. RESULTS: All 40 premolars from 20 patients were included. There was no significant difference in the mean total root resorption between the LED PBM and control sides (mean 0.216 versus 0.284 mm3, respectively, P = 0.306). The LED side was associated with less pain at 24 hours (P = 0.023) and marginally more pain at subsequent time points, which was not statistically significant. No harms were observed. LIMITATIONS: Short study duration and the inability to blind patients and clinician during clinical part of study. CONCLUSION: This 28-day randomized split-mouth controlled trial showed that daily, LED-mediated PBM application, when applied for 5 minute/day, does not influence orthodontic root resorption. It is associated with significantly less pain 24 hours after the application of orthodontic force, but no difference thereafter. These results should be tested on patients undergoing a full course of orthodontic treatment. TRIAL REGISTRATION: Clinical Trials Registry ACTRN12616000652471.

Development of a bioactive and radiopaque bismuth doped baghdadite ceramic for bone tissue engineering.

Baghdadite (Ca3ZrSi2O9, BAG), is a Zr-doped calcium silicate that has outstanding bioactivity both in vitro and in vivo. Bioceramic scaffolds should be sufficiently radiopaque to be distinguishable in vivo from surrounding bone structures. To enhance the radiopacity of BAG, this study investigated the effect of incorporating bismuth ions into its crystalline structure (BixCa3-xZrSi2O9, x = 0, 0.1, 0.2, 0.5; BAG, Bi0.1-BAG, Bi0.2-BAG, Bi0.5-BAG, respectively). Monophasic baghdadite was retained after bismuth ion incorporation up to x = 0.2 at calcination temperatures of 1350 °C. When pressed and sintered, energy dispersive x-ray spectroscopy showed that BAG and Bi0.1-BAG retained crystalline homogeneity, but Bi0.2-BAG formed zirconium-rich crystalline regions. BAG, Bi0.1-BAG and Bi0.2-BAG exhibited non-degradation after 56 days of immersion in culture medium. Bi0.1-BAG exhibited the lowest change in culture medium pH (+0.0), compared to BAG (+0.7) and Bi0.2-BAG (+0.2) after 56 days of culture media immersion. Bi0.1-BAG exhibited similar strength and modulus to BAG (σ: 200-290 MPa; E: 4-5 GPa), and significantly higher compressive strength and modulus versus Bi0.2-BAG (σ: 150-200 MPa; E: 3.5-4 GPa) across 56 days of aqueous immersion. In vitro studies using primary human bone derived cells (HOBs) demonstrated a significant increase in HOBs proliferation when cultured on Bi0.1-BAG for seven days compared to BAG and Bi0.2-BAG. Importantly, Bi0.1-BAG showed increased radiopacity by ~33%, when compared to BAG, and by ~115% when compared to biphasic calcium phosphate. The properties of Bi0.1-BAG show promise for its use as a bioactive ceramic with sufficient radiopacity for treatment of bone defects.

CCM2L (Cerebral Cavernous Malformation 2 Like) Deletion Aggravates Cerebral Cavernous Malformation Through Map3k3-KLF Signaling Pathway.

[Figure: see text].

Lactobacillus bile salt hydrolase substrate specificity governs bacterial fitness and host colonization.

Primary bile acids (BAs) are a collection of host-synthesized metabolites that shape physiology and metabolism. BAs transit the gastrointestinal tract and are subjected to a variety of chemical transformations encoded by indigenous bacteria. The resulting microbiota-derived BA pool is a mediator of host-microbiota interactions. Bacterial bile salt hydrolases (BSHs) cleave the conjugated glycine or taurine from BAs, an essential upstream step for the production of deconjugated and secondary BAs. Probiotic lactobacilli harbor a considerable number and diversity of BSHs; however, their contribution to Lactobacillus fitness and colonization remains poorly understood. Here, we define and compare the functions of multiple BSHs encoded by Lactobacillus acidophilus and Lactobacillus gasseri Our genetic and biochemical characterization of lactobacilli BSHs lend to a model of Lactobacillus adaptation to the gut. These findings deviate from previous notions that BSHs generally promote colonization and detoxify bile. Rather, we show that BSH enzymatic preferences and the intrinsic chemical features of various BAs determine the toxicity of these molecules during Lactobacillus growth. BSHs were able to alter the Lactobacillus transcriptome in a BA-dependent manner. Finally, BSHs were able to dictate differences in bacterial competition in vitro and in vivo, defining their impact on BSH-encoding bacteria within the greater gastrointestinal tract ecosystem. This work emphasizes the importance of considering the enzymatic preferences of BSHs alongside the conjugated/deconjugated BA-bacterial interaction. These results deepen our understanding of the BA-microbiome axis and provide a framework to engineer lactobacilli with improved bile resistance and use probiotics as BA-altering therapeutics.

Clostridioides difficile exploits toxin-mediated inflammation to alter the host nutritional landscape and exclude competitors from the gut microbiota.

Clostridioides difficile is a bacterial pathogen that causes a range of clinical disease from mild to moderate diarrhea, pseudomembranous colitis, and toxic megacolon. Typically, C. difficile infections (CDIs) occur after antibiotic treatment, which alters the gut microbiota, decreasing colonization resistance against C. difficile. Disease is mediated by two large toxins and the expression of their genes is induced upon nutrient depletion via the alternative sigma factor TcdR. Here, we use tcdR mutants in two strains of C. difficile and omics to investigate how toxin-induced inflammation alters C. difficile metabolism, tissue gene expression and the gut microbiota, and to determine how inflammation by the host may be beneficial to C. difficile. We show that C. difficile metabolism is significantly different in the face of inflammation, with changes in many carbohydrate and amino acid uptake and utilization pathways. Host gene expression signatures suggest that degradation of collagen and other components of the extracellular matrix by matrix metalloproteinases is a major source of peptides and amino acids that supports C. difficile growth in vivo. Lastly, the inflammation induced by C. difficile toxin activity alters the gut microbiota, excluding members from the genus Bacteroides that are able to utilize the same essential nutrients released from collagen degradation.

Complete Genome Sequence of Lactobacillus johnsonii NCK2677, Isolated from Mice.

We report the closed genome sequence of a Lactobacillus johnsonii strain (NCK2677) that was isolated from a cefoperazone-treated mouse model designed for the study of Clostridioides difficile infection. Illumina and Nanopore sequencing reads were assembled into a circular 1,951,416-bp chromosome with a G+C content of 34.7%, containing 1,865 genes.

Surface glycan-binding proteins are essential for cereal beta-glucan utilization by the human gut symbiont Bacteroides ovatus.

The human gut microbiota, which underpins nutrition and systemic health, is compositionally sensitive to the availability of complex carbohydrates in the diet. The Bacteroidetes comprise a dominant phylum in the human gut microbiota whose members thrive on dietary and endogenous glycans by employing a diversity of highly specific, multi-gene polysaccharide utilization loci (PUL), which encode a variety of carbohydrases, transporters, and sensor/regulators. PULs invariably also encode surface glycan-binding proteins (SGBPs) that play a central role in saccharide capture at the outer membrane. Here, we present combined biophysical, structural, and in vivo characterization of the two SGBPs encoded by the Bacteroides ovatus mixed-linkage ß-glucan utilization locus (MLGUL), thereby elucidating their key roles in the metabolism of this ubiquitous dietary cereal polysaccharide. In particular, molecular insight gained through several crystallographic complexes of SGBP-A and SGBP-B with oligosaccharides reveals that unique shape complementarity of binding platforms underpins specificity for the kinked MLG backbone vis-à-vis linear ß-glucans. Reverse-genetic analysis revealed that both the presence and binding ability of the SusD homolog BoSGBPMLG-A are essential for growth on MLG, whereas the divergent, multi-domain BoSGBPMLG-B is dispensable but may assist in oligosaccharide scavenging from the environment. The synthesis of these data illuminates the critical role SGBPs play in concert with other MLGUL components, reveals new structure-function relationships among SGBPs, and provides fundamental knowledge to inform future (meta)genomic, biochemical, and microbiological analyses of the human gut microbiota.