Serine proteases at the cutting edge of IBD: Focus on gastrointestinal inflammation.

Serine proteases have been long recognized to coordinate many physiological processes and play key roles in regulating the inflammatory response. Accordingly, their dysregulation has been regularly associated with several inflammatory disorders and suggested as a central mechanism in the pathophysiology of digestive inflammation. So far, studies addressing the proteolytic homeostasis in the gut have mainly focused on host serine proteases as candidates of interest, while largely ignoring the potential contribution of their bacterial counterparts. The human gut microbiota comprises a complex ecosystem that contributes to host health and disease. Yet, our understanding of microbially produced serine proteases and investigation of whether they are causally linked to IBD is still in its infancy. In this review, we highlight recent advances in the emerging roles of host and bacterial serine proteases in digestive inflammation. We also discuss the application of available tools in the gut to monitor disease-related serine proteases. An exhaustive representation and understanding of such functional potential would help in closing existing gaps in mechanistic knowledge.

Gut Serpinome: Emerging Evidence in IBD.

Inflammatory bowel diseases (IBD) are incurable disorders whose prevalence and global socioeconomic impact are increasing. While the role of host genetics and immunity is well documented, that of gut microbiota dysbiosis is increasingly being studied. However, the molecular basis of the dialogue between the gut microbiota and the host remains poorly understood. Increased activity of serine proteases is demonstrated in IBD patients and may contribute to the onset and the maintenance of the disease. The intestinal proteolytic balance is the result of an equilibrium between the proteases and their corresponding inhibitors. Interestingly, the serine protease inhibitors (serpins) encoded by the host are well reported; in contrast, those from the gut microbiota remain poorly studied. In this review, we provide a concise analysis of the roles of serine protease in IBD physiopathology and we focus on the serpins from the gut microbiota (gut serpinome) and their relevance as a promising therapeutic approach.

Microbial impact on cholesterol and bile acid metabolism: current status and future prospects.

Recently, the gut microbiota has emerged as a crucial factor that influences cholesterol metabolism. Ever since, significant interest has been shown in investigating these host-microbiome interactions to uncover microbiome-mediated functions on cholesterol and bile acid (BA) metabolism. Indeed, changes in gut microbiota composition and, hence, its derived metabolites have been previously reported to subsequently impact the metabolic processes and have been linked to several diseases. In this context, associations between a disrupted gut microbiome, impaired BA metabolism, and cholesterol dysregulation have been highlighted. Extensive advances in metagenomic and metabolomic studies in this field have allowed us to further our understanding of the role of intestinal bacteria in metabolic health and disease. However, only a few have provided mechanistic insights into their impact on cholesterol metabolism. Identifying the myriad functions and interactions of these bacteria to maintain cholesterol homeostasis remain an important challenge in such a field of research. In this review, we discuss the impact of gut microbiota on cholesterol metabolism, its association with disease settings, and the potential of modulating gut microbiota as a promising therapeutic target to lower hypercholesterolemia.

Siropins, novel serine protease inhibitors from gut microbiota acting on human proteases involved in inflammatory bowel diseases.

BACKGROUND: In eukaryotes, the serpins constitute a wide family of protease inhibitors regulating many physiological pathways. Many reports stressed the key role of serpins in several human physiopathologies including mainly the inflammatory bowel diseases. In this context, eukaryotic serpins were largely studied and their use to limit inflammation was reported. In comparison to that, bacterial serpins and mainly those from human gut microbiota remain poorly studied. RESULTS: The two genes encoding for putative serpins from the human gut bacterium Eubacterium sireaum, display low sequence identities. These genes were overexpressed and the encoded proteins, named Siropins, were purified. Activity studies demonstrated that both purified proteins inhibited serine proteases but surprisingly they preferentially inhibited two human serine proteases (Human Neutrophil Elastase and Proteinase3). The biochemical characterization of these Siropins revealed that Siropin 1 was the most active and stable at low pH values while Siropin 2 was more thermoactive and thermostable. Kinetic analysis allowed the determination of the stoichiometry of inhibition (SI) which was around 1 and of the association rate constants of 7.7 × 104 for the Human Neutrophil Elastase and 2.6 × 105 for the Proteinase3. Moreover, both Siropins displayed the ability to inhibit proteases usually present in fecal waters. Altogether our data indicate the high efficiency of Siropins and their probable involvement in the control of the overall intestine protease activity. CONCLUSIONS: Here we report the purification and the biochemical characterization of two novel serpins originated from Eubacterium sireaum, a human gastro-intestinal tract commensal bacteria. These proteins that we called Siropins, efficiently inhibited two human proteases reported to be associated with inflammatory bowel diseases. The determination of the biochemical properties of these enzymes revealed different temperature and pH behaviours that may reflect adaptation of this human commensal bacterium to different ecological environments. To the best of our knowledge, it is the first bacterial serpins showing an attractive inhibition of fecal proteases recovered from a mice group with chemically induced inflammation. Altogether our data highlight the interesting potential of Siropins, and serpins from the human gut microbiota in general, to be used as new alternative to face inflammatory diseases.

The secreted L-arabinose isomerase displays anti-hyperglycemic effects in mice.

BACKGROUND: The L-arabinose isomerase is an intracellular enzyme which converts L-arabinose into L-ribulose in living systems and D-galactose into D-tagatose in industrial processes and at industrial scales. D-tagatose is a natural ketohexose with potential uses in pharmaceutical and food industries. The D-galactose isomerization reaction is thermodynamically equilibrated, and leads to secondary subproducts at high pH. Therefore, an attractive L-arabinose isomerase should be thermoactive and acidotolerant with high catalytic efficiency. While many reports focused on the set out of a low cost process for the industrial production of D-tagatose, these procedures remain costly. When compared to intracellular enzymes, the production of extracellular ones constitutes an interesting strategy to increase the suitability of the biocatalysts. RESULTS: The L-arabinose isomerase (L-AI) from Lactobacillus sakei was expressed in Lactococcus lactis in fusion with the signal peptide of usp45 (SP(Usp45)). The L-AI protein and activity were detected only in the supernatant of the induced cultures of the recombinant L. lactis demonstrating the secretion in the medium of the intracellular L. sakei L-AI in an active form. Moreover, we showed an improvement in the enzyme secretion using either (1) L. lactis strains deficient for their two major proteases, ClpP and HtrA, or (2) an enhancer of protein secretion in L. lactis fused to the recombinant L-AI with the SP(Usp45). Th L-AI enzyme secreted by the recombinant L. lactis strains or produced intracellularly in E. coli, showed the same functional properties than the native enzyme. Furthermore, when mice are fed with the L. lactis strain secreting the L-AI and galactose, tagatose was produced in vivo and reduced the glycemia index. CONCLUSIONS: We report for the first time the secretion of the intracellular L-arabinose isomerase in the supernatant of food grade L. lactis cultures with hardly display other secreted proteins. The secreted L-AI originated from the food grade L. sakei 23 K was active and showed the same catalytic and structural properties as the intracellular enzyme. The L. lactis strains secreting the L-arabinose isomerase has the ability to produce D-tagatose in vivo and conferred an anti-hyperglycemic effect to mice.

Gut microbiota in cats with inflammatory bowel disease and low-grade intestinal T-cell lymphoma.

In cats and humans, several physiological and environmental factors have been shown to alter the gut microbiota of healthy individuals. Cats share several diseases with humans such as inflammatory bowel diseases and low-grade intestinal T-cell lymphoma. The physiopathology of these chronic enteropathies is poorly understood but may involve disequilibrium of the gut microbiota composition and disruption of normal microbiome activity profiles. These disorders are increasingly diagnosed in the feline species due to improved medicalization and easier access to endoscopy in veterinary practice. This review addresses the current data on the gut microbiota of cats in health and in chronic enteropathies. Such functional analysis will help the advancement of innovative diagnostic tools and targeted therapeutic strategies.

Bile Acids: Key Players in Inflammatory Bowel Diseases?

Inflammatory bowel diseases (IBDs) have emerged as a public health problem worldwide with a limited number of efficient therapeutic options despite advances in medical therapy. Although changes in the gut microbiota composition are recognized as key drivers of dysregulated intestinal immunity, alterations in bile acids (BAs) have been shown to influence gut homeostasis and contribute to the pathogenesis of the disease. In this review, we explore the interactions involving BAs and gut microbiota in IBDs, and discuss how the gut microbiota-BA-host axis may influence digestive inflammation.

Domestic Environment and Gut Microbiota: Lessons from Pet Dogs.

Accumulating data show the involvement of intestinal microbiota in the development and maintenance of numerous diseases. Many environmental factors influence the composition and function of the gut microbiota. An animal model subjected to the same environmental constraints that will allow better characterization of the microbiota-host dialogue is awaited. The domestic dog has physiological, dietary and pathological characteristics similar to those of humans and shares the domestic environment and lifestyle of its owner. This review exposes how the domestication of dogs has brought them closer to humans based on their intrinsic and extrinsic similarities which were discerned through examining and comparing the current knowledge and data on the intestinal microbiota of humans and canines in the context of several spontaneous pathologies, including inflammatory bowel disease, obesity and diabetes mellitus.

Bile Salt Hydrolases: At the Crossroads of Microbiota and Human Health.

The gut microbiota has been increasingly linked to metabolic health and disease over the last few decades. Several factors have been suggested to be involved in lipid metabolism and metabolic responses. One mediator that has gained great interest as a clinically important enzyme is bile salt hydrolase (BSH). BSH enzymes are widely distributed in human gastrointestinal microbial communities and are believed to play key roles in both microbial and host physiology. In this review, we discuss the current evidence related to the role of BSHs in health and provide useful insights that may pave the way for new therapeutic targets in human diseases.

SP-1, a Serine Protease from the Gut Microbiota, Influences Colitis and Drives Intestinal Dysbiosis in Mice.

Increased protease activity has been linked to the pathogenesis of IBD. While most studies have been focusing on host proteases in gut inflammation, it remains unclear how to address the potential contribution of their bacterial counterparts. In the present study, we report a functional characterization of a newly identified serine protease, SP-1, from the human gut microbiota. The serine protease repertoire of gut Clostridium was first explored, and the specificity of SP-1 was analyzed using a combinatorial chemistry method. Combining in vitro analyses and a mouse model of colitis, we show that oral administration of recombinant bacteria secreting SP-1 (i) compromises the epithelial barrier, (ii) alters the microbial community, and (ii) exacerbates colitis. These findings suggest that gut microbial protease activity may constitute a valuable contributor to IBD and could, therefore, represent a promising target for the treatment of the disease.

Fecal Serine Protease Profiling in Inflammatory Bowel Diseases.

Serine proteases are extensively known to play key roles in many physiological processes. However, their dysregulation is often associated to several diseases including inflammatory bowel diseases (IBD). Here, we used specific substrates to monitor fecal protease activities in a large cohort of healthy and IBD patients. Of interest, serine protease activity was 10-fold higher in IBD fecal samples compared to healthy controls. Moreover, functional analysis of these fecal proteolytic activities revealed that the most increased activities are trypsin-like, elastase-like and cathepsin G-like. We also show for the first time, an increase of proteinase 3-like activity in these samples compared to controls. Results presented here will guide further investigations to better understand the relevance of these peptidases in IBD.

Serine protease inhibitors and human wellbeing interplay: new insights for old friends.

Serine Protease Inhibitors (Serpins) control tightly regulated physiological processes and their dysfunction is associated to various diseases. Thus, increasing interest is given to these proteins as new therapeutic targets. Several studies provided functional and structural data about human serpins. By comparison, only little knowledge regarding bacterial serpins exists. Through the emergence of metagenomic studies, many bacterial serpins were identified from numerous ecological niches including the human gut microbiota. The origin, distribution and function of these proteins remain to be established. In this report, we shed light on the key role of human and bacterial serpins in health and disease. Moreover, we analyze their function, phylogeny and ecological distribution. This review highlights the potential use of bacterial serpins to set out new therapeutic approaches.

para-Sulphonato-calix[n]arene capped silver nanoparticles challenge the catalytic efficiency and the stability of a novel human gut serine protease inhibitor.

The Eubacterium saburreum serine protease inhibitor from the human gut microbiota inhibits the eukaryotic pancreatic elastase associated with acute pancreatitis. Interestingly, the inhibition efficiency and stability are markedly increased by the para-sulphonato-calix[8]arene capped silver nanoparticles. Moreover, this enzyme is distinguishable by its high inhibitory effect at broad pH range between 2-10 and temperatures from 10 to 40 °C, in the presence of para-sulphonato-calix[8]arene capped silver nanoparticles the enzyme remains active even at 70 °C.

Relevant Patented Biotechnological Applications of Ecotin: An Update.

BACKGROUND: Ecotins are serine protease inhibitors which are generally found in the periplasmic compartment. These inhibitors act on a wide range of serine proteases with different efficiencies. Actually, only few Ecotins were studied, and the main characterized proteins were derived from Escherichia coli. Functional studies of this latter protein allowed the development of numerous patents related to Ecotin relevant biotechnological applications. OBJECTIVE: This review aims to give an update on the relevant Ecotins already described and to provide a concise overview concerning the relevant patented applications of these serine protease inhibitors. METHOD: In this review, we focus on the analysis of Ecotin diversity and their distribution using Pfam protein data base. Moreover, we report a detailed overview regarding the biotechnological applications of the Ecotins based on all patents associated to Ecotins and their biotechnological applications searched in European Patent Office (Espacenet), United States Patent and National Patent Collections (WIPO) patents databases. RESULTS: On the basis of this analysis, we demonstrate that Ecotins are mostly present in bacteria. Study of Ecotin sequences and their biochemical properties reveals that they are a small serine protease inhibitor group. The high stability and specificity of Ecotins promote their biotechnological uses in several fields. The original structural organization of Ecotin-protease complexes and their flexibility lead to several patented applications. CONCLUSION: This review showed that Ecotins have many attractive biotechnological applications. Potential of Ecotins needs to be more investigated seeing the limited available data related to this protein family. Thus, further functional analyses will promote the use of Ecotins.

Biotechnological Applications of Serine Proteases: A Patent Review.

BACKGROUND: Serine proteases have long been recognized to play key roles in various physiological processes. However, their disequilibrium can be linked to several diseases. Taking into account their wide diversity and specificity, they have been actively investigated by many industrial, academic and pharmaceutical industries. OBJECTIVE: This review aims to provide a concise analysis of the described serine proteases as well as their relevant biotechnological and therapeutic applications. METHOD: Here, we give an overview of the recent knowledge on serine proteases with a particular focus on their biotechnological applications reported in European Patent Office (Espacenet), United States Patent and National Patent Collections (WIPO) patent databases. RESULTS: Serine proteases are probably the enzymes that have been mostly studied over the past few decades. However, despite their increasing interest, no significant patent so far has dealt with the identity of overactive serine proteases in disease settings. CONCLUSION: This review displays that serine proteases have several relevant industrial uses. New potential applications of such proteins require more functional analyses seeing the key role of serine proteases in many biomedical and biotechnological processes.

Purification of glycocalicin from human plasma.

Glycocalicin (GC) is a large extracellular proteolytic fragment of glycoprotein Ib, a membrane platelet component playing an essential role in the physiological processes of platelet adhesion and aggregation. GC contains the binding sites for thrombin and von Willebrand factor. GC circulates normally in vivo in significant concentrations and the plasma level of this protein reflects a complex function of factors including platelet count or platelet turnover. It can therefore serve as a good indicator for many diseases like hypoplastic thrombocytopenia and idiopathic thrombocytopenic purpura. For this reason, several purification assays have been previously described. In this work, we describe a novel analytical method for GC purification from human platelets based on preparative HPLC gel filtration followed by immuno-affinity chromatography on NHS activated column conjugated with specific antibody. Pure GC was obtained from tiny amount of starting material. Our protocol of GC purification is simple, fast and provides a pure end product.

Bacillus phytases: Current status and future prospects.

Phytases catalyze the hydrolysis of phytic acid in a stepwise manner to lower inositol phosphates, myo-inositol (having important role in metabolism and signal transduction pathways), and inorganic phosphate. These enzymes have been widely used in animal feed in order to improve phosphorus nutrition and to decrease pollution in animal waste. Compared to previously described phytases, the phytase (PhyL) from Bacillus licheniformis ATCC 14580 has attractive biochemical properties which can increase the profitability of several biotechnological procedures (animal nutrition, humain health…etc). Due to its amino acid sequence with critical substitutions, the PhyL could be a model to enhance other phytases features, in terms of thermal stability and high activity. Otherwise, an engineered PhyL, with low pH optimum, will represent a challenge within the class of ß- propeller phytases.

Discriminatory antibacterial effects of calix[n]arene capped silver nanoparticles with regard to gram positive and gram negative bacteria.

Silver nanoparticles capped with nine different sulphonated calix[n]arenes were tested for their anti-bacterial effects against B. subtilis and E. coli at an apparent concentration of 100 nM in calix[n]arene. The results show the para-sulphonato-calix[n]arenes are active against Gram positive bacteria and the derivatives having sulphonate groups at both para and alkyl terminal positions are active against Gram negative bacteria. The calix[6]arene derivative with only O-alkyl sulphonate groups shows bactericidal activity.