The Guanylate Cyclase C-cGMP Signaling Axis Opposes Intestinal Epithelial Injury and Neoplasia.

Guanylate cyclase C (GUCY2C) is a transmembrane receptor expressed on the luminal aspect of the intestinal epithelium. Its ligands include bacterial heat-stable enterotoxins responsible for traveler\u27s diarrhea, the endogenous peptide hormones uroguanylin and guanylin, and the synthetic agents, linaclotide, plecanatide, and dolcanatide. Ligand-activated GUCY2C catalyzes the synthesis of intracellular cyclic GMP (cGMP), initiating signaling cascades underlying homeostasis of the intestinal epithelium. Mouse models of GUCY2C ablation, and recently, human populations harboring GUCY2C mutations, have revealed the diverse contributions of this signaling axis to epithelial health, including regulating fluid secretion, microbiome composition, intestinal barrier integrity, epithelial renewal, cell cycle progression, responses to DNA damage, epithelial-mesenchymal cross-talk, cell migration, and cellular metabolic status. Because of these wide-ranging roles, dysregulation of the GUCY2C-cGMP signaling axis has been implicated in the pathogenesis of bowel transit disorders, inflammatory bowel disease, and colorectal cancer. This review explores the current understanding of cGMP signaling in the intestinal epithelium and mechanisms by which it opposes intestinal injury. Particular focus will be applied to its emerging role in tumor suppression. In colorectal tumors, endogenous GUCY2C ligand expression is lost by a yet undefined mechanism conserved in mice and humans. Further, reconstitution of GUCY2C signaling through genetic or oral ligand replacement opposes tumorigenesis in mice. Taken together, these findings suggest an intriguing hypothesis that colorectal cancer arises in a microenvironment of functional GUCY2C inactivation, which can be repaired by oral ligand replacement. Hence, the GUCY2C signaling axis represents a novel therapeutic target for preventing colorectal cancer

Use of the KlADH3 promoter for the quantitative production of the murine PDE5A isoforms in the yeast Kluyveromyces lactis

Background: Phosphodiesterases (PDE) are a superfamily of enzymes that hydrolyse cyclic nucleotides (cAMP/ cGMP), signal molecules in transduction pathways regulating crucial aspects of cell life. PDEs regulate the intensity and duration of the cyclic nucleotides signal modulating the downstream biological efect. Due to this critical role associated with the extensive distribution and multiplicity of isozymes, the 11 mammalian families (PDE1 to PDE11) constitute key therapeutic targets. PDE5, one of these cGMP-specifc hydrolysing families, is the molecular target of several well known drugs used to treat erectile dysfunction and pulmonary hypertension. Kluyveromyces lactis, one of the few yeasts capable of utilizing lactose, is an attractive host alternative to Saccharomyces cerevisiae for heterologous protein production. Here we established K. lactis as a powerful host for the quantitative production of the murine PDE5 isoforms. Results: Using the promoter of the highly expressed KlADH3 gene, multicopy plasmids were engineered to produce the native and recombinant Mus musculus PDE5 in K. lactis. Yeast cells produced large amounts of the purifed A1, A2 and A3 isoforms displaying Km, Vmax and Sildenafl inhibition values similar to those of the native murine enzymes. PDE5 whose yield was nearly 1 mg/g wet weight biomass for all three isozymes (30 mg/L culture), is well tolerated by K. lactis cells without major growth defciencies and interferences with the endogenous cAMP/cGMP signal transduction pathways. Conclusions: To our knowledge, this is the frst time that the entire PDE5 isozymes family containing both regulatory and catalytic domains has been produced at high levels in a heterologous eukaryotic organism. K. lactis has been shown to be a very promising host platform for large scale production of mammalian PDEs for biochemical and structural studies and for the development of new specifc PDE inhibitors for therapeutic applications in many pathologies

Submerged culture production of 5\u27-phosphodiesterase by streptomyces albus

The flavor enhancing properties of certain 5\u27-ribonucleotides have been known for some time. In 1913, Kodama (20)* reported on the seasoning effect of inosine-5-phosphate (5’-IMP). This compound was also identified as one of the important beef flavor precursors by Batzer and LandMann (3). Kuninaka (21) found that guanosine-5-phosphate (5\u27-GMP) and xanthine-5-phosphate (5\u27-XMP) also had a flavoring effect similar to 5’-IMP. Snake venom (16) and intestional mucosa (6) were known as sources of 5\u27-phosphodiesterase capable of hydrolyzing the phosphate ester link-ages of ribonucleic acid (RNA) to produce 5\u27-ribonucleotides. Recently, it has been discovered that many microorganisms (22,23,25,26,27) produce 5\u27-phosphodiesterase. Therefore, it has become possible to use certain microorganisms as sources of 5\u27-phosphodiesterase for use in production of 5\u27-ribonucleotides from RNA. With this in mind, a strain of Strep-tomyces albus, known to produce 5\u27-phosphodiesterase in submerged cul-ture (23), was studied to determine the effects of various factors (pH, temperature, and nutrients) on growth and enzyme production and to scale up production from laboratory to pilot-plant quantity

Phosphodiesterase D is Involved in Bile Resistance in Listeria monocytogenes

Listeria monocytogenes is a deadly foodborne bacterium that is responsible for almost 20% of food-related deaths in the United States. Listeria monocytogenes contaminates ready-to-eat products such as cheese, deli meat, and ice cream. Once ingested, it invades the intestinal lining and can enter the bloodstream, causing listeriosis. There is a gap in the knowledge of the pathogenesis of L. monocytogenes in how it is able to survive in the gastrointestinal tract in the presence of bile, which has bactericidal properties. Previous studies have suggested that the second messenger cyclic-dimeric-GMP may be involved in the regulation of virulence factors of Listeria. This nucleotide is produced by diguanylate cyclases and degraded by phosphodiesterases. The purpose of this study was to determine whether phosphodiesterase D was responsible for bile survival and if oxygen availability influences the impact of this phosphodiesterase. Survival of the wild-type strain (F2365) and the pdeD mutant was analyzed in aerobic and anaerobic conditions in neutral and acidic pH with and without 1% bile to mimic locations within the body where bile would be present (i.e. duodenum and gall bladder). Results showed that the pdeD mutant was more sensitive to bile in anaerobic and acidic conditions than the wild type. In order to better understand the relationship between PdeD and bile, real-time qPCR was conducted to determine if there were differences in the expression of bsh in pdeD and F2365. Bsh is the bile salt hydrolase that is used to detoxify bile. Using the 16S gene as an internal control, it was found that there was a slight decrease in expression of bsh in pdeD than F2365, though this change was not significant. These data suggest that the phosphodiesterase D may be involved in responding to bile-induced damage, but does so independently of the bsh expression. The v reduction in bile survival exhibited by this strain suggests that the phosphodiesterase may be responsive to oxidative stress. Further research is needed to determine if the regulation of the pdeD is due to exposure to oxidative stress

Regulation of biofilm formation in Salmonella typhimurium and Escherichia coli Nissle 1917

Bacteria have the ability to grow in cell communities designated biofilms. This mode of growth is widespread and offers numerous advantages to the bacteria in terms of survival, persistence and propagation. Bacteria have developed different ways of building up a biofilm. Complex regulatory mechanisms control this sophisticated mode of growth in response to environmental conditions. This thesis focuses on the regulation of biofilm formation by the food-borne pathogen Salmonella enterica serovar Typhimurium and the probiotic strain Escherichia coli Nissle 1917. Commonly, species of the family of Enterobacteriaceae produce the biofilm extracellular matrix components cellulose and curli fimbriae at low temperature. The expression of cellulose and curli is activated by the transcriptional regulator CsgD. In this work, we demonstrated an altered pattern of biofilm regulation in E. coli Nissle 1917 (Paper I). Biogenesis of curli fimbriae was activated by CsgD at low temperature, while cellulose production at 28°C and 37°C did not require CsgD nor the di-guanylate cyclase AdrA. Cellulose production was, however, still dependent on the second messenger c-di-GMP. This regulatory pattern of cellulose and curli fimbriae production has been conserved in E. coli Nissle 1917 clonal isolates for more than 80 years implying biological significance. Production of cellulose mediated adhesion of E. coli Nissle 1917 to the gastrointestinal epithelial cell line HT-29 and to the mouse epithelium in vivo, thus possibly playing a role in colonization of the gut. A characteristic of biofilm formation is cell heterogeneity. In S. typhimurium, expression of the master regulator CsgD was bistable during biofilm development (Paper II). Bistability led to task distribution, whereby the subpopulation of cells, which expressed high amounts of CsgD, was associated with microcolony formation and the production of cellulose. CsgD expression is tightly regulated and responds to a variety of environmental conditions such as nutrient starvation and oxygen tension. Several global regulators contribute directly or indirectly to CsgD regulation. In this work, we identified novel factors involved in the complex CsgD regulation. Two lytic transglycosylases, MltE and MltC redundantly activated CsgD and rdar morphotype expression (Manuscript III). The absence of these two lytic transglycosylases could be partially compensated by the second messenger c-di-GMP. The chaperone Hfq and two Hfq dependent sRNAs, ArcZ and RyeB, also activated rdar morphotype expression by controlling the expression of CsgD (Manuscript IV). We demonstrated that ArcZ is a key regulator of biofilm formation. In addition, ArcZ played a role in the transition between sessility and motility and was involved in the timing of type 1 versus curli fimbriae surface attachment

Exploring strategies to improve volumetric hydrogen productivities of Caldicellulosiruptor species

Ongoing consumption of fossil-based fuels generates a massive amount of greenhouse gases. This may lead to global warming that is currently threatening human society and wild animal habitats. Hydrogen is an energy carrier with the highest energy content per weight compared to other all fuels and no carbon dioxide is released when combusted. Thermophilic bacteria belonging to the genus of Caldicellulosiruptor have the ability to produce hydrogen from an array of substrates such as poly-, oligo-, di-, and monosaccharides, including lignocellulosic material. Caldicellulosiruptor species have the capacity to produce hydrogen at nearly the maximum theoretical yield of 4 mol⋅mol-1 hexose. In this work, pure and co-cultures of Caldicellulosiruptor species degraded and fermented heat-treated wheat straw. The outcome indicated that the performance of C. kronotskyensis is superior and it is thus promising candidate for utilizing wheat straw through consolidated bioprocessing. Therefore, the physiology of C. kronotskyensis was further investigated using defined media containing glucose and xylose mixtures corresponding to the sugar ratio present in wheat straw hydrolysate. Interestingly, growth of C. kronotskyensis did not possess a diauxic-like growth pattern during its growth on glucose and xylose mixtures like was observed with C. saccharolyticus. This phenomenon was determined by both the volumetric productivity profile of hydrogen (QH2) and carbon dioxide (QCO2). The maximum growth rate (µmax) of C. kronotskyensis on xylose was 0.57 h-1 which is twice the µ max on glucose (0.28 h-1). C. kronotskyensis was grown on sugar mixtures i.e. xylose-cellobiose and glucose-cellobiose. The uptake of xylose and cellobiose occurred concurrently. However, for glucose and cellobiose mixtures, C. kronotskyensis consumed cellobiose faster than glucose. These results indicated that C. kronotskyensis has adapted to pentoses andoligosaccharides. Cell immobilization and co-cultures offered a promising technique for retaining cells in the system. During this work, chitosan and rubber were used as a carrier to retain biomass, thereby improving volumetric hydrogen productivity (QH2). Chitosan exhibited the property to retain C. saccharolyticus and C. owensensis but did not improve the QH2. Acrylic fibres filled in a homemade stainless-steel cage was introduced in continuous stirred tank reactors (CSTR). Notably, the highest QH2 obtained was 30 ± 0.2 mmol⋅L-1⋅h-1 at a dilution rate (D) of 0.3 h-1 with a pure culture of C. kronotskyensis with acrylic fibres and chitosan. In the co-culture of C. kronotskyensis and C. owensensis with acrylic fibres, the population dynamics indicated that C. kronotskyensis was the dominant species in the biofilm fraction, whereas C. owensensis was the dominant in the planktonic phase. Bis-(3',5')-cyclic di-guanosine-mono-phosphate (c-di-GMP) is an intracellular messenger correlated with planktonic and biofilm lifestyle. C. owensensis is a high producer of c-di-GMP, while C. kronotskyensis produced less during its fermentations. In this study, a co-culture of C. kronotskyensis and C. owensensis without carrier obtained the highest concentration of c-di-GMP at 260 ± 27.3 µM. In conclusion, this study revealed that immobilization of Caldicellulosiruptor species improved the QH2 . Secondly, it revealed the superior performance of C. kronotskyensis in relation to consolidated bioprocessing, biofilm formation and QH2. Therefore, it is recommended to carry out more research with C. kronotskyensis to pursue a breakthrough in cost-effective hydrogen production

A 5\u27-phosphodiesterase of the streptomyces species

The flavor enhancing property of inosine-5-phosphate (5’-IMP) was first reported by Kodama in 1913 (23). Batzer and LandMann (7) also identified this compound as one of the important beef flavor precusors. Kuninaka (24) from his studies on the characteristics of 5’-ribomononucleotides found that guanosine-5-phosphate and xanthine-5-phosphate also had a seasoning effect similar to 5\u27-IMP. Snake venom (21) and intestinal mucosa (13) were known as sources of 5\u27-phosphodiesterase which was able to catalyze the cleavage of phosphate ester linkage of RNA to produce 5\u27-ribomononucleotides. However, it has been discovered recently (25, 33, 35) that many microorganisms also capable of producing 5\u27-phosphodiesterase and of degrading nucleic acid into 5\u27-nucleotides. Therefore, the production of 5\u27-mononucleotides by means of a microbial enzyme has become possible. With these ideas in mind, a microorganism was isolated from soil and this study was made in an attempt: (1) to identify the microorganism which is capable of degrading ribonucleic acid (RNA) into 5\u27-ribomononucleotides; (2) to identify the variety of 5 *-ribomononucleotides produced from the hydrolysis of RNA by this organism

Diverse cyclic dinucleotide signals regulate Escherichia coli lifestyle transition

Bacteria have the ability to change their lifestyle to adapt to various environmental conditions. Cyclic dinucleotides (cDNs) are ubiquitous second messengers that can regulate fundamental lifestyle switches, such as motility versus sessility and acute versus chronic virulence, in bacteria. Investigation of the diverse established cDNs, cyclic di-GMP, cyclic di-AMP, and the recently identified hybrid molecule cyclic GAMP, expanded our knowledge of the complexity of regulation of bacterial physiology by nucleotide-based second messengers and unravel common and distinct regulatory patterns. In this thesis, we provided the molecular basis to assess the regulatory mechanisms of semiconstitutive rdar biofilm formation by Illumina Miseq or PacBio sequencing of the genomes of eight rdar biofilm forming E. coli strains (Paper I). By using phenotypic, genetic and biochemical approaches, we showed that animal commensal isolate E. coli ECOR31 expresses a semi-constitutive rdar biofilm morphotype on agar plates characterized by expression of the extracellular matrix components cellulose and curli fimbriae. This morphotype is conventionally dependent on the major biofilm regulator CsgD and positively regulated by cyclic di-GMP signaling (Paper II). As expected, flagella-dependent motility is negatively regulated by cyclic di-GMP signaling. Bioinformatic analysis suggested the presence of a dinucleotide cyclase DncV homolog, hypothesized to possess cyclic GAMP synthase activity, encoded by the E. coli ECOR31 genome. DncV synthesized 3’3’-cGAMP in vitro and in vivo and, via its catalytic activity, negatively regulated csgD expression at the transcriptional level with subsequent suppression of rdar biofilm formation and cell aggregation. DncV also suppressed swimming and swarming motility post-transcriptional of class 1 flagellar regulon genes. In liquid culture, expression of dncV restricted cell aggregation, but showed a complex temporal pattern of biofilm formation at the abiotic surface. The patatin-like phospholipase CapV is a receptor for 3’3’-cGAMP. In this thesis, we showed that expression of CapVQ329R, a single amino acid variant of CapV, induced extensive cell filamentation in E. coli MG1655 independently of the 3’3’-cGAMP synthase DncV (Paper III). Moreover, overexpression of CapVQ329R repressed swimming motility by inhibiting flagella biosynthesis and reduced rdar biofilm formation and CsgD expression, possibly through modulation of cyclic di-GMP levels. The observed phenotypes of CapVQ329R are not restricted to E. coli MG1655, but common to other E. coli strains and S. typhimurium UMR1 suggesting that conserved pathway(s) are required for their expression. Based on our genome sequences, in the last study, we investigated the molecular basis of temperature-independent expression of the rdar biofilm morphotype and subsequently csgD expression in seven semi-constitutive rdar biofilm forming E. coli strains (Paper IV). Based on the observation that amino acid variations in cyclic di-GMP turnover proteins correlated with the expression of a semi-constitutive rdar biofilm, in particular, we demonstrated that distinct amino acid changes outside of conserved signature motifs that potentially lead to an alteration in the trigger activity of the hybrid cyclic di-GMP phosphodiesterase/diguanylate cyclase YciR contributed to regulate rdar biofilm formation and csgD expression in semiconstitutive rdar biofilm forming E. coli strains. In conclusion, this thesis highlights that diverse cDN second messenger signals differentially regulate the bacterial sessile/motile lifestyle transition. Furthermore, the effect of CapVQ329R on bacterial phenotypes and physiology is an example of rapid evolution of protein functionality

Biofilm dispersion : the key to biofilm eradication or opening Pandora’s box?

Biofilms are extremely difficult to eradicate due to their decreased antibiotic susceptibility. Inducing biofilm dispersion could be a potential strategy to help combat biofilm-related infections. Mechanisms of biofilm dispersion can basically be divided into two groups, i.e. active and passive dispersion. Active dispersion depends on a decrease in the intracellular c-di-GMP levels, leading to the production of enzymes that degrade the biofilm matrix and promote dispersion. In contrast, passive dispersion relies on triggers that directly release cells from the biofilm. In the present review, several active and passive dispersion strategies are discussed. In addition, the disadvantages and possible consequences of using dispersion as a treatment approach for biofilm-related infections are also reviewed

Emerging optical materials in sensing and discovery of bioactive compounds

Optical biosensors are used in numerous applications and analytical fields. Advances in these sensor platforms offer high sensitivity, selectivity, miniaturization, and real-time analysis, among many other advantages. Research into bioactive natural products serves both to protect against potentially dangerous toxic compounds and to promote pharmacological innovation in drug discovery, as these compounds have unique chemical compositions that may be characterized by greater safety and efficacy. However, conventional methods for detecting these biomolecules have drawbacks, as they are time-consuming and expensive. As an alternative, optical biosensors offer a faster, simpler, and less expensive means of detecting various biomolecules of clinical interest. In this review, an overview of recent developments in optical biosensors for the detection and monitoring of aquatic biotoxins to prevent public health risks is first provided. In addition, the advantages and applicability of these biosensors in the field of drug discovery, including high-throughput screening, are discussed. The contribution of the investigated technological advances in the timely and sensitive detection of biotoxins while deciphering the pathways to discover bioactive compounds with great health-promoting prospects is envisaged to meet the increasing demands of healthcare systems.The authors gratefully acknowledge funding from the European Regional Development Fund (ERDF) through COMPETE 2020-POCI and Fundação para a Ciência e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio