Human gut microbes express functionally distinct endoglycosidases to metabolize the same N-glycan substrate.

Bacteroidales (syn. Bacteroidetes) are prominent members of the human gastrointestinal ecosystem mainly due to their efficient glycan-degrading machinery, organized into gene clusters known as polysaccharide utilization loci (PULs). A single PUL was reported for catabolism of high-mannose (HM) N-glycan glyco-polypeptides in the gut symbiont Bacteroides thetaiotaomicron, encoding a surface endo-ß-N-acetylglucosaminidase (ENGase), BT3987. Here, we discover an ENGase from the GH18 family in B. thetaiotaomicron, BT1285, encoded in a distinct PUL with its own repertoire of proteins for catabolism of the same HM N-glycan substrate as that of BT3987. We employ X-ray crystallography, electron microscopy, mass spectrometry-based activity measurements, alanine scanning mutagenesis and a broad range of biophysical methods to comprehensively define the molecular mechanism by which BT1285 recognizes and hydrolyzes HM N-glycans, revealing that the stabilities and activities of BT1285 and BT3987 were optimal in markedly different conditions. BT1285 exhibits significantly higher affinity and faster hydrolysis of poorly accessible HM N-glycans than does BT3987. We also find that two HM-processing endoglycosidases from the human gut-resident Alistipes finegoldii display condition-specific functional properties. Altogether, our data suggest that human gut microbes employ evolutionary strategies to express distinct ENGases in order to optimally metabolize the same N-glycan substrate in the gastroinstestinal tract.

A conserved inhibitory interdomain interaction regulates DNA-binding activities of hybrid two-component systems in Bacteroides.

Hybrid two-component systems (HTCSs) comprise a major class of transcription regulators of polysaccharide utilization genes in Bacteroides. Distinct from classical two-component systems in which signal transduction is carried out by intermolecular phosphotransfer between a histidine kinase (HK) and a cognate response regulator (RR), HTCSs contain the membrane sensor HK and the RR transcriptional regulator within a single polypeptide chain. Tethering the DNA-binding domain (DBD) of the RR with the dimeric HK domain in an HTCS could potentially promote dimerization of the DBDs and would thus require a mechanism to suppress DNA-binding activity in the absence of stimulus. Analysis of phosphorylation and DNA-binding activities of several HTCSs from Bacteroides thetaiotaomicron revealed a DBD suppression mechanism in which an inhibitory interaction between the DBD and the phosphoryl group-accepting receiver domain (REC) decreases autophosphorylation rates of HTCS-RECs and represses DNA-binding activities in the absence of phosphorylation. Sequence analyses and structure predictions identified a highly conserved sequence motif correlated with a conserved inhibitory domain arrangement of REC and DBD. The presence of the motif, as in most HTCSs, or its absence, in a small subset of HTCSs, is likely predictive of two distinct regulatory mechanisms evolved for different glycans. Substitutions within the conserved motif relieve the inhibitory interaction and result in elevated DNA-binding activities in the absence of phosphorylation. Our data suggest a fundamental regulatory mechanism shared by most HTCSs to suppress DBD activities using a conserved inhibitory interdomain arrangement to overcome the challenge of the fused HK and RR components. IMPORTANCE: Different dietary and host-derived complex carbohydrates shape the gut microbial community and impact human health. In Bacteroides, the prevalent gut bacteria genus, utilization of these diverse carbohydrates relies on different gene clusters that are under sophisticated control by various signaling systems, including the hybrid two-component systems (HTCSs). We have uncovered a highly conserved regulatory mechanism in which the output DNA-binding activity of HTCSs is suppressed by interdomain interactions in the absence of stimulating phosphorylation. A consensus amino acid motif is found to correlate with the inhibitory interaction surface while deviations from the consensus can lead to constitutive activation. Understanding of such conserved HTCS features will be important to make regulatory predictions for individual systems as well as to engineer novel systems with substitutions in the consensus to explore the glycan regulation landscape in Bacteroides.

Bacteroides thetaiotaomicron and its inactivated bacteria ameliorate colitis by inhibiting macrophage activation.

BACKGROUND: Studies have demonstrated that Bacteroides thetaiotaomicron (BT) has protective effect against colon inflammation in murine models. Macrophages play an important role in gut immunity. However, the specific mechanisms of BT on macrophage are still unelucidated. Thus, our study investigates the anti-inflammatory effect of BT and its heat-treated inactivated bacteria on experimental colitis and macrophages. METHODS: A dextran sulfate sodium (DSS)-induced acute colitis model with male C57BL/6 mice, BT (ATCC29148) strain, THP1 cell lines were used in this study. Live and heat-treated inactivated BT (IBT) solution (1 × 10^9cfu/ml) were intragastrically gavaged daily for 14 days. Colonic inflammation was determined by the disease activity index (DAI) score, colon length, histological score, and inflammatory factors. THP1 cells were induced towards M1, then treated with different concentrations of inactivated BT solution and p38 inhibitor. Western blotting, immunohistochemistry, immunofluorescence and qRT-PCR were performed to assess the levels of inflammatory cytokines and molecules of MAPK pathway including IL-6, TNF-α, IL-1ß, IL-22, p38 and phosphor-p38 expressions. Moreover, 16S rRNA sequencing of colitis murine fecal samples was applied to investigate the influence of supplementation of BT to the gut microbiota homeostasis. RESULTS: Both live and heat-treated inactivated BT decreased the DAI and histological scores as well as levels of inflammatory factors, particularly IL-6 while increasing IL-22 of DSS-induced colitis murine models. The cell experiments showed that inactivated BT downregulates IL-6 expression in THP1 via inhibiting p38 phosphorylation and affecting M1 polarization. Moreover, the 16S rRNA sequencing results showed that BT and IBT gavage could increase beta-diversity of gut flora in DSS-induced colitis mice. Furthermore, the significance test for differences between the groups showed that BT could increase Faecalebaculum, Lactobacillus and Bacteroides, while decreasing Akkermansia. CONCLUSION: In summary, our findings imply that BT and its heat-treated inactivated bacteria exert a protective effect by suppressing macrophage-induced IL-6 through the inhibition of p38 MAPK pathway and ameliorating intestinal gut dysbiosis in experimental colitis.

Chemoproteomic identification of a DPP4 homolog in Bacteroides thetaiotaomicron.

Serine hydrolases have important roles in signaling and human metabolism, yet little is known about their functions in gut commensal bacteria. Using bioinformatics and chemoproteomics, we identify serine hydrolases in the gut commensal Bacteroides thetaiotaomicron that are specific to the Bacteroidetes phylum. Two are predicted homologs of the human dipeptidyl peptidase 4 (hDPP4), a key enzyme that regulates insulin signaling. Our functional studies reveal that BT4193 is a true homolog of hDPP4 that can be inhibited by FDA-approved type 2 diabetes medications targeting hDPP4, while the other is a misannotated proline-specific triaminopeptidase. We demonstrate that BT4193 is important for envelope integrity and that loss of BT4193 reduces B. thetaiotaomicron fitness during in vitro growth within a diverse community. However, neither function is dependent on BT4193 proteolytic activity, suggesting a scaffolding or signaling function for this bacterial protease.

Linker-peptide-mediated one-step purification and immobilization of α-L-rhamnosidase from Bacteroides thetaiotaomicron for direct biotransformation from epimedin C to icariin.

Icariin, the most effective bioactive component in Epimedium, is also the index component of Epimedium quality control in Pharmacopoeia. It was a very attractive approach for bioconversion from epimedin C to icariin. However, its potential was impeded by poor stability and non-recyclable properties of free enzymes. In this study, we have fused the linker (4LP) to α-L-rhamnosidase BtRha and successfully prepared the immobilized enzyme (incubated 4LP-BtRha@Na-Y) to produce icariin from epimedin C. The activity recovery of 4LP-BtRha@Na-Y was 79.6 %, and enzyme activity was 209.8 U/g, which was 1.75-fold and 1.6-fold higher than that of immobilized BtRha (BtRha@Na-Y), respectively. The optimal reaction temperature and pH of 4LP-BtRha@Na-Y was 55 °C and 6.5, respectively. The thermal stability of immobilized enzyme was significantly improved by incubation in phosphate buffer containing 20 % glycerol and 10 % fructose. The kcat/Km value of incubated 4LP-BtRha@Na-Y was 7.98 × 105 s-1M-1, which increased by 8 % compared with free BtRha. Finally, under suitable conditions, 1 g/L epimedin C was transformed into icariin with icariin yield 75.1 %, and the relative conversion rate retained 74.9 % after reused 13 cycles. This experiment provides a new idea for one-step purification and immobilization of α-L-rhamnosidase for direct biotransformation from epimedin C to icariin, which will have great prospects in food and pharmaceutical production.

A biosynthetic pathway for the selective sulfonation of steroidal metabolites by human gut bacteria.

Members of the human gut microbiome enzymatically process many bioactive molecules in the gastrointestinal tract. Most gut bacterial modifications characterized so far are hydrolytic or reductive in nature. Here we report that abundant human gut bacteria from the phylum Bacteroidetes perform conjugative modifications by selectively sulfonating steroidal metabolites. While sulfonation is a ubiquitous biochemical modification, this activity has not yet been characterized in gut microbes. Using genetic and biochemical approaches, we identify a widespread biosynthetic gene cluster that encodes both a sulfotransferase (BtSULT, BT0416) and enzymes that synthesize the sulfonate donor adenosine 3'-phosphate-5'-phosphosulfate (PAPS), including an APS kinase (CysC, BT0413) and an ATP sulfurylase (CysD and CysN, BT0414-BT0415). BtSULT selectively sulfonates steroidal metabolites with a flat A/B ring fusion, including cholesterol. Germ-free mice monocolonized with Bacteroides thetaiotaomicron ΔBT0416 exhibited reduced gastrointestinal levels of cholesterol sulfate (Ch-S) compared with wild-type B. thetaiotaomicron-colonized mice. The presence of BtSULT and BtSULT homologues in bacteria inhibited leucocyte migration in vitro and in vivo, and abundances of cluster genes were significantly reduced in patients with inflammatory bowel disease. Together, these data provide a mechanism by which gut bacteria sulfonate steroidal metabolites and suggest that these compounds can modulate immune cell trafficking in the host.

Extracellular vesicles produced by the human commensal gut bacterium Bacteroides thetaiotaomicron affect host immune pathways in a cell-type specific manner that are altered in inflammatory bowel disease.

The gastrointestinal (GI) tract harbours a complex microbial community, which contributes to its homeostasis. A disrupted microbiome can cause GI-related diseases, including inflammatory bowel disease (IBD), therefore identifying host-microbe interactions is crucial for better understanding gut health. Bacterial extracellular vesicles (BEVs), released into the gut lumen, can cross the mucus layer and access underlying immune cells. To study BEV-host interactions, we examined the influence of BEVs generated by the gut commensal bacterium, Bacteroides thetaiotaomicron, on host immune cells. Single-cell RNA sequencing data and host-microbe protein-protein interaction networks were used to predict the effect of BEVs on dendritic cells, macrophages and monocytes focusing on the Toll-like receptor (TLR) pathway. We identified biological processes affected in each immune cell type and cell-type specific processes including myeloid cell differentiation. TLR pathway analysis highlighted that BEV targets differ among cells and between the same cells in healthy versus disease (ulcerative colitis) conditions. The in silico findings were validated in BEV-monocyte co-cultures demonstrating the requirement for TLR4 and Toll-interleukin-1 receptor domain-containing adaptor protein (TIRAP) in BEV-elicited NF-kB activation. This study demonstrates that both cell-type and health status influence BEV-host communication. The results and the pipeline could facilitate BEV-based therapies for the treatment of IBD.

Modeling human pollution in water bodies using somatic coliphages and bacteriophages that infect Bacteroides thetaiotaomicron strain GA17.

The ability to detect human fecal pollution in water is of great importance when assessing the associated health risks. Many microbial source tracking (MST) markers have been proposed to determine the origin of fecal pollution, but their application remains challenging. A range of factors, not yet sufficiently analyzed, may affect MST markers in the environment, such as dilution and inactivation processes. In this work, a statistical framework based on Monte Carlo simulations and non-linear regression was used to develop a classification procedure for use in MST studies. The predictive model tested uses only two parameters: somatic coliphages (SOMCPH), as an index of general fecal pollution, and human host-specific bacteriophages that infect Bacteroides thetaiotaomicron strain GA17 (GA17PH). Taking into account bacteriophage dilution and differential inactivation, the threshold concentration of SOMCPH was calculated to be around 500 PFU/100 mL for a limit of detection of 10 PFU/100 mL. However, this threshold can be lowered by increasing the analyzed volume sample, which in turn lowers the limit of detection. The resulting model is sufficiently accurate for application in practical cases involving MST and could be easily used with markers other than those tested here.

Gut microbiome ADP-ribosyltransferases are widespread phage-encoded fitness factors.

Bacterial ADP-ribosyltransferases (ADPRTs) have been described as toxins involved in pathogenesis through the modification of host proteins. Here, we report that ADPRTs are not pathogen restricted but widely prevalent in the human gut microbiome and often associated with phage elements. We validated their biochemical activity in a large clinical isolate collection and further examined Bxa, a highly abundant ADPRT in Bacteroides. Bxa is expressed, secreted, and enzymatically active in Bacteroides and can ADP-ribosylate non-muscle myosin II proteins. Addition of Bxa to epithelial cells remodeled the actin cytoskeleton and induced secretion of inosine. Bxa-encoding B. stercoris can use inosine as a carbon source and colonizes the gut to significantly greater numbers than a bxa-deleted strain in germ-free and altered Schaedler flora (ASF) mice. Colonization correlated with increased inosine concentrations in the feces and tissues. Altogether, our results show that ADPRTs are abundant in the microbiome and act as bacterial fitness factors.

Coculture of primary human colon monolayer with human gut bacteria.

The presence of microbes in the colon impacts host physiology. Therefore, microbes are being evaluated as potential treatments for colorectal diseases. Humanized model systems that enable robust culture of primary human intestinal cells with bacteria facilitate evaluation of potential treatments. Here, we describe a protocol that can be used to coculture a primary human colon monolayer with aerotolerant bacteria. Primary human colon cells maintained as organoids are dispersed into single-cell suspensions and then seeded on collagen-coated Transwell inserts, where they attach and proliferate to form confluent monolayers within days of seeding. The confluent monolayers are differentiated for an additional 4 d and then cocultured with bacteria. As an example application, we describe how to coculture differentiated colon cells for 8 h with four strains of Bacteroides thetaiotaomicron, each engineered to detect different colonic microenvironments via genetically embedded logic circuits incorporating deoxycholic acid and anhydrotetracycline sensors. Characterization of this coculture system reveals that barrier function remains intact in the presence of engineered B. thetaiotaomicron. The bacteria stay close to the mucus layer and respond in a microenvironment-specific manner to the inducers (deoxycholic acid and anhydrotetracycline) of the genetic circuits. This protocol thus provides a useful mucosal barrier system to assess the effects of bacterial cells that respond to the colonic microenvironment, and may also be useful in other contexts to model human intestinal barrier properties and microbiota-host interactions.

CRISPR-based functional genomics in human dendritic cells.

Dendritic cells (DCs) regulate processes ranging from antitumor and antiviral immunity to host-microbe communication at mucosal surfaces. It remains difficult, however, to genetically manipulate human DCs, limiting our ability to probe how DCs elicit specific immune responses. Here, we develop a CRISPR-Cas9 genome editing method for human monocyte-derived DCs (moDCs) that mediates knockouts with a median efficiency of >94% across >300 genes. Using this method, we perform genetic screens in moDCs, identifying mechanisms by which DCs tune responses to lipopolysaccharides from the human microbiome. In addition, we reveal donor-specific responses to lipopolysaccharides, underscoring the importance of assessing immune phenotypes in donor-derived cells, and identify candidate genes that control this specificity, highlighting the potential of our method to pinpoint determinants of inter-individual variation in immunity. Our work sets the stage for a systematic dissection of the immune signaling at the host-microbiome interface and for targeted engineering of DCs for neoantigen vaccination.

Genome sequence and annotation of Bacteroides sp aff. Thetaiotaomicron strain isolated from blood.

This study is focused on genome sequence and annotation of the Bacteroides strain isolated from the blood of a patient with descending colon cancer. According to a comparison of the 16S ribosomal RNA sequence with the National Center for Biotechnology Information database, this strain was identified as Bacteroides sp. aff. Thetaiotaomicron. The next-generation sequencing of the strain was performed in a GENEWIZ laboratory (Jiangsu, China) on Illumina HiSeq device. According to CAZy classification, metabolic pathways related to carbohydrate metabolism of this strain engage the following enzymes: 427 glycosylhydrolases, 277 glycosyltransferases, 137 carbohydrate-binding modules, 48 carbohydrate esterases, and 24 polysaccharide lyases. According to the KEGG pathway database, Bacteroides sp. aff thetaiotaomicron strain is reported to incorporate 199 pathway associated genes. Bacteroides sp. aff. Thetaiotaomicron exposes the capacity of metabolizing a variety of polysaccharides. Its genome is enriched with an expanded repertoire of enzymes for the hydrolysis of glycosidic bonds and, thus, likely to hydrolyze most of glycosidic bonds in biological polysaccharides. The advanced capabilities of the studied strain to recognize and respond to environmental signals are expressed in the rich representation of one- and two-component signal transduction systems.

Bacteroides thetaiotaomicron-derived outer membrane vesicles promote regulatory dendritic cell responses in health but not in inflammatory bowel disease.

BACKGROUND: Bacteroides thetaiotaomicron (Bt) is a prominent member of the human intestinal microbiota that, like all gram-negative bacteria, naturally generates nanosized outer membrane vesicles (OMVs) which bud off from the cell surface. Importantly, OMVs can cross the intestinal epithelial barrier to mediate microbe-host cell crosstalk involving both epithelial and immune cells to help maintain intestinal homeostasis. Here, we have examined the interaction between Bt OMVs and blood or colonic mucosa-derived dendritic cells (DC) from healthy individuals and patients with Crohn's disease (CD) or ulcerative colitis (UC). RESULTS: In healthy individuals, Bt OMVs stimulated significant (p < 0.05) IL-10 expression by colonic DC, whereas in peripheral blood-derived DC they also stimulated significant (p < 0.001 and p < 0.01, respectively) expression of IL-6 and the activation marker CD80. Conversely, in UC Bt OMVs were unable to elicit IL-10 expression by colonic DC. There were also reduced numbers of CD103+ DC in the colon of both UC and CD patients compared to controls, supporting a loss of regulatory DC in both diseases. Furthermore, in CD and UC, Bt OMVs elicited a significantly lower proportion of DC which expressed IL-10 (p < 0.01 and p < 0.001, respectively) in blood compared to controls. These alterations in DC responses to Bt OMVs were seen in patients with inactive disease, and thus are indicative of intrinsic defects in immune responses to this commensal in inflammatory bowel disease (IBD). CONCLUSIONS: Overall, our findings suggest a key role for OMVs generated by the commensal gut bacterium Bt in directing a balanced immune response to constituents of the microbiota locally and systemically during health which is altered in IBD patients. Video Abstract.

A Metabolic Pathway for Activation of Dietary Glucosinolates by a Human Gut Symbiont.

Consumption of glucosinolates, pro-drug-like metabolites abundant in Brassica vegetables, has been associated with decreased risk of certain cancers. Gut microbiota have the ability to metabolize glucosinolates, generating chemopreventive isothiocyanates. Here, we identify a genetic and biochemical basis for activation of glucosinolates to isothiocyanates by Bacteroides thetaiotaomicron, a prominent gut commensal species. Using a genome-wide transposon insertion screen, we identified an operon required for glucosinolate metabolism in B. thetaiotaomicron. Expression of BT2159-BT2156 in a non-metabolizing relative, Bacteroides fragilis, resulted in gain of glucosinolate metabolism. We show that isothiocyanate formation requires the action of BT2158 and either BT2156 or BT2157 in vitro. Monocolonization of mice with mutant BtΔ2157 showed reduced isothiocyanate production in the gastrointestinal tract. These data provide insight into the mechanisms by which a common gut bacterium processes an important dietary nutrient.

A Double-Blind, Placebo-Controlled Trial to Assess Safety and Tolerability of (Thetanix) Bacteroides thetaiotaomicron in Adolescent Crohn's Disease.

INTRODUCTION: Thetanix (gastroresistant capsules containing lyophilized Bacteroides thetaiotaomicron) is a live biotherapeutic, under development for Crohn's disease, that antagonizes transcription factor nuclear factor kappa B, reducing proinflammatory cytokines, particularly tumor necrosis factor alpha. We aimed to assess safety and tolerability in adolescents with Crohn's disease in remission. METHODS: Subjects who were 16-18 years with Crohn's in remission (weighted pediatric Crohn's disease activity index <12.5) were recruited. Each active dose comprised ∼108.2±1.4 colony forming units of B. thetaiotaomicron (randomized 4:1 active:placebo). Part A was single dose. Part B involved 7.5 days twice daily dosing. Serial stools were analyzed for calprotectin, 16S rRNA sequencing, and B. thetaiotaomicron real-time polymerase chain reaction. Bloods were taken serially. Subjects reported adverse events and recorded temperature twice daily. RESULTS: Fifteen subjects were treated-8 in part A (75% men, median 17.1 years) and 10 in part B, including 3 from part A (80% men, median 17.1 years); all 18 completed. Seventy percent took concurrent immunosuppression. Reported compliance was >99% in part B. Two subjects reported adverse events deemed related-one in part A with eructation, flatulence, and reflux; one in part B with dizziness, abdominal pain, and headache. No serious adverse events were reported. There was no significant change in median calprotectin across part B (87.8 [4.4-447] to 50.5 [5.3-572], P = 0.44 by the Fisher exact test in the active group). No significant differences were found in microbiota profiles, but diversity seemed to increase in treated subjects. DISCUSSION: Thetanix, after single and multiple doses, was well tolerated. Although the numbers in this study were small, the safety profile seems good. Future studies should explore efficacy.

Antibiotic Korormicin A Kills Bacteria by Producing Reactive Oxygen Species.

Korormicin is an antibiotic produced by some pseudoalteromonads which selectively kills Gram-negative bacteria that express the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR.) We show that although korormicin is an inhibitor of Na+-NQR, the antibiotic action is not a direct result of inhibiting enzyme activity. Instead, perturbation of electron transfer inside the enzyme promotes a reaction between O2 and one or more redox cofactors in the enzyme (likely the flavin adenine dinucleotide [FAD] and 2Fe-2S center), leading to the production of reactive oxygen species (ROS). All Pseudoalteromonas contain the nqr operon in their genomes, including Pseudoalteromonas strain J010, which produces korormicin. We present activity data indicating that this strain expresses an active Na+-NQR and that this enzyme is not susceptible to korormicin inhibition. On the basis of our DNA sequence data, we show that the Na+-NQR of Pseudoalteromonas J010 carries an amino acid substitution (NqrB-G141A; Vibrio cholerae numbering) that in other Na+-NQRs confers resistance against korormicin. This is likely the reason that a functional Na+-NQR is able to exist in a bacterium that produces a compound that typically inhibits this enzyme and causes cell death. Korormicin is an effective antibiotic against such pathogens as Vibrio cholerae, Aliivibrio fischeri, and Pseudomonas aeruginosa but has no effect on Bacteroides fragilis and Bacteroides thetaiotaomicron, microorganisms that are important members of the human intestinal microflora.IMPORTANCE As multidrug antibiotic resistance in pathogenic bacteria continues to rise, there is a critical need for novel antimicrobial agents. An essential requirement for a useful antibiotic is that it selectively targets bacteria without significant effects on the eukaryotic hosts. Korormicin is an excellent candidate in this respect because it targets a unique respiratory enzyme found only in prokaryotes, the Na+-pumping NADH:quinone oxidoreductase (Na+-NQR). Korormicin is synthesized by some species of the marine bacterium Pseudoalteromonas and is a potent and specific inhibitor of Na+-NQR, an enzyme that is essential for the survival and proliferation of many Gram-negative human pathogens, including Vibrio cholerae and Pseudomonas aeruginosa, among others. Here, we identified how korormicin selectively kills these bacteria. The binding of korormicin to Na+-NQR promotes the formation of reactive oxygen species generated by the reaction of the FAD and the 2Fe-2S center cofactors with O2.

X-ray Structures of Two Bacteroides thetaiotaomicron C11 Proteases in Complex with Peptide-Based Inhibitors.

Commensal bacteria secrete proteins and metabolites to influence host intestinal homeostasis, and proteases represent a significant constituent of the components at the host:microbiome interface. Here, we determined the structures of the two secreted C11 cysteine proteases encoded by the established gut commensal Bacteroides thetaiotaomicron. We employed mutational analysis to demonstrate the two proteases, termed "thetapain" and "iotapain", undergo in trans autoactivation after lysine and/or arginine residues, as observed for other C11 proteases. We determined the structures of the active forms of thetapain and iotapain in complex with irreversible peptide inhibitors, Ac-VLTK-AOMK and biotin-VLTK-AOMK, respectively. Structural comparisons revealed key active-site interactions important for peptide recognition are more extensive for thetapain; however, both proteases employ a glutamate residue to preferentially bind small polar residues at the P2 position. Our results will aid in the design of protease-specific probes to ultimately understand the biological role of C11 proteases in bacterial fitness, elucidate their host and/or microbial substrates, and interrogate their involvement in microbiome-related diseases.

Bacteroides thetaiotaomicron Ameliorates Colon Inflammation in Preclinical Models of Crohn's Disease.

Background: Alterations in the gut microbiota are strongly associated with the development of inflammatory bowel disease (IBD), particularly with Crohn's disease, which is characterized by reduced abundance of commensal anaerobic bacteria including members of the Bacteroides genus. Our aim was to investigate the protective effects of Bacteroides thetaiotaomicron, an abundant member of this genus, in different rodent models of IBD. Methods: We assessed the effect of B. thetaiotaomicron administration on primary readouts of colitis (weight loss, histopathology, and immune parameters) in dextran sodium sulphate (DSS) and interleukin-10 knockout (IL10KO) models of IBD. Efficacy of a freeze-dried bacterial formulation and a purified recombinant protein of B. thetaiotaomicron was also investigated. Results: B. thetaiotaomicron showed protective effects in both DSS and IL10KO rodent models, as demonstrated by significant amelioration of weight loss, colon shortening, histopathological damage and immune activation. This efficacy was not exclusive to actively growing bacterial preparations but was retained by freeze-dried cells of B. thetaiotaomicron. A pirin-like protein (PLP) of B. thetaiotaomicron, identified by microarray analysis during coculture of the bacterial strain with Caco-2 cells, reduced pro-inflammatory NF-κB signalling in these intestinal epithelial cells. Recombinant PLP partially recapitulated the effect of the whole strain in a rat DSS model. Conclusions: B. thetaiotaomicron displays strong efficacy in preclinical models of IBD and protects against weight loss, histopathological changes in the colon and inflammatory markers. These data indicate that the live strain or its products may be a novel alternative to current treatment options for Crohn's disease.