Environmentally Friendly Degradation and Detoxification of Rifampicin by a Bacterial Laccase and Hydrogen Peroxide.

Antibiotics are micropollutants accumulating in our rivers and wastewaters, potentially leading to bacterial antibiotic resistance, a worldwide problem to which there is no current solution. Here, we have developed an environmentally friendly two-step process to transform the antibiotic rifampicin (RIF) into non-antimicrobial compounds. The process involves an enzymatic oxidation step by the bacterial CotA-laccase and a hydrogen peroxide bleaching step. NMR identified rifampicin quinone as the main product of the enzymatic oxidation. Growth of Escherichia coli strains in the presence of final degradation products (FP) and minimum inhibitory concentration (MIC) measurements confirmed that FP are non-anti-microbial compounds, and bioassays suggest that FP is not toxic to eukaryotic organisms. Moreover, competitive fitness assays between susceptible and RIF-resistant bacteria show that susceptible bacteria is strongly favoured in the presence of FP. Our results show that we have developed a robust and environmentally friendly process to effectively remediate rifampicin from antibiotic contaminated environments.

New bifunctional 1,3-diamine organocatalysts derived from (+)-camphoric acid for asymmetric Michael addition of 1,3-dicarbonyl compounds to nitroolefins.

Novel 1,3-diamine-derived bifunctional thiourea and squaramide organocatalysts were synthesized from (+)-camphoric acid. These catalysts were easily obtained in up to two to five synthetic steps, in a flexible approach that facilitates their structure variation. Their catalytic activity was examined in the asymmetric Michael addition of 1,3-dicarbonyl compounds to several trans-ß-nitrostyrenes. Yields up to 98% and enantiomeric excesses up to 74% and high diastereoselectivities when applicable (dr up to 93:7) were obtained in these reactions showing that 1,3-diamine-derived bifunctional thioureas are efficient organocatalysts.

Biosynthesis of mycobacterial methylmannose polysaccharides requires a unique 1-O-methyltransferase specific for 3-O-methylated mannosides.

Mycobacteria are a wide group of organisms that includes strict pathogens, such as Mycobacterium tuberculosis, as well as environmental species known as nontuberculous mycobacteria (NTM), some of which-namely Mycobacterium avium-are important opportunistic pathogens. In addition to a distinctive cell envelope mediating critical interactions with the host immune system and largely responsible for their formidable resistance to antimicrobials, mycobacteria synthesize rare intracellular polymethylated polysaccharides implicated in the modulation of fatty acid metabolism, thus critical players in cell envelope assembly. These are the 6-O-methylglucose lipopolysaccharides (MGLP) ubiquitously detected across the Mycobacterium genus, and the 3-O-methylmannose polysaccharides (MMP) identified only in NTM. The polymethylated nature of these polysaccharides renders the intervening methyltransferases essential for their optimal function. Although the knowledge of MGLP biogenesis is greater than that of MMP biosynthesis, the methyltransferases of both pathways remain uncharacterized. Here, we report the identification and characterization of a unique S-adenosyl-l-methionine-dependent sugar 1-O-methyltransferase (MeT1) from Mycobacterium hassiacum that specifically blocks the 1-OH position of 3,3'-di-O-methyl-4α-mannobiose, a probable early precursor of MMP, which we chemically synthesized. The high-resolution 3D structure of MeT1 in complex with its exhausted cofactor, S-adenosyl-l-homocysteine, together with mutagenesis studies and molecular docking simulations, unveiled the enzyme's reaction mechanism. The functional and structural properties of this unique sugar methyltransferase further our knowledge of MMP biosynthesis and provide important tools to dissect the role of MMP in NTM physiology and resilience.

An Efficient Synthesis of Optically Active [4-13C] Labelled Quorum Sensing Signal Autoinducer-2.

A new synthetic route for the quorum sensing signal Autoinducer-2 (AI-2) is described and used for the preparation of [4-13C]-AI-2 starting from [1-13C]-bromoacetic acid. The key step in this process was the enantioselective reduction of an intermediate ketone. This synthesis provides, selectively, both enantiomers of the labelled or unlabelled parent compound, (R) or (S)-4,5-dihydroxypentane-2,3-dione (DPD) and was used for an improved synthesis of [1-13C]-AI-2.

Synthesis and biological effects of small molecule enhancers for improved recombinant protein production in plant cell cultures.

Histone deacetylases are involved in chromatin remodelling and thus play a vital role in the epigenetic regulation of gene expression. HDAC inhibitors alter the acetylation status of histone and non-histone proteins to regulate various cellular events such as transcription. Novel HDAC inhibitors were designed and synthesised to promote higher levels of recombinant protein production in tobacco cell cultures. The effect of these chemical enhancers on the epigenetic profiles in plant cells has been evaluated by molecular docking, in vitro and in vivo studies. The addition of these novel enhancers led to an increase in histone H3 acetylation levels that promoted an increase in the accumulation levels of the recombinant protein in cell culture. These results can pave the way for the application of these enhancers to improve the production of high value products in plant cell based systems.

Synthesis of d-desthiobiotin-AI-2 as a novel chemical probe for autoinducer-2 quorum sensing receptors.

In processes regulated by quorum sensing (QS) bacteria respond to the concentration of autoinducers in the environment to engage in group behaviours. Autoinducer-2 (AI-2) is unique as it can foster interspecies communication. Currently, two AI-2 receptors are known, LuxP and LsrB, but bacteria lacking these receptors can also respond to AI-2. In this work, we present an efficient and reproducible synthesis of a novel chemical probe, d-desthiobiotin-AI-2. This probe binds both LuxP and LsrB receptors from different species of bacteria. Thus, this probe is able to bind receptors that recognise the two known biologically active forms of AI-2, presenting the plasticity essential for the identification of novel unknown AI-2 receptors. Moreover, a protocol to pull down receptors bound to d-desthiobiotin-AI-2 with anti-biotin antibodies has also been established. Altogether, this work highlights the potential of conjugating chemical signals to biotinylated derivatives to identify and tag signal receptors involved in quorum sensing or other chemical signalling processes.

Synthesis and biological activity of a potent optically pure autoinducer-2 quorum sensing agonist.

Quorum sensing (QS) regulates population-dependent bacterial behaviours, such as toxin production, biofilm formation and virulence. Autoinducer-2 (AI-2) is to date the only signalling molecule known to foster inter-species bacterial communication across distantly related bacterial species. In this work, the synthesis of pure enantiomers of C4-propoxy-HPD and C4-ethoxy-HPD, known AI-2 analogues, has been developed. The optimised synthesis is efficient, reproducible and short. The (4S) enantiomer of C4-propoxy-HPD was the most active compound being approximately twice as efficient as (4S)-DPD and ten-times more potent than the (4R) enantiomer. Additionally, the specificity of this analogue to bacteria with LuxP receptors makes it a good candidate for clinical applications, because it is not susceptible to scavenging by LsrB-containing bacteria that degrade the natural AI-2. All in all, this study provides a new brief and effective synthesis of isomerically pure analogues for QS modulation that include the most active AI-2 agonist described so far.

Syntheses of the plant auxin conjugate 2-O-(indole-3-acetyl)-myo-inositol IAInos.

The plant hormone conjugate 2-O-(indole-3-acetyl)-myo-inositol (IAInos) has been selectively prepared for the first time by two routes from myo-inositol. One of the syntheses depended upon the construction of the 3-indoleacetyl group by a Fischer indole synthesis on an unreactive axial hydroxyl group, while the other via a direct acylation of the equatorially orientated hydroxy group created by conformational constraint of the cyclohexane ring. The latter synthesis produced IAInos in 5 steps and 29% overall yield.

LsrF, a coenzyme A-dependent thiolase, catalyzes the terminal step in processing the quorum sensing signal autoinducer-2.

The quorum sensing signal autoinducer-2 (AI-2) regulates important bacterial behaviors, including biofilm formation and the production of virulence factors. Some bacteria, such as Escherichia coli, can quench the AI-2 signal produced by a variety of species present in the environment, and thus can influence AI-2-dependent bacterial behaviors. This process involves uptake of AI-2 via the Lsr transporter, followed by phosphorylation and consequent intracellular sequestration. Here we determine the metabolic fate of intracellular AI-2 by characterizing LsrF, the terminal protein in the Lsr AI-2 processing pathway. We identify the substrates of LsrF as 3-hydroxy-2,4-pentadione-5-phosphate (P-HPD, an isomer of AI-2-phosphate) and coenzyme A, determine the crystal structure of an LsrF catalytic mutant bound to P-HPD, and identify the reaction products. We show that LsrF catalyzes the transfer of an acetyl group from P-HPD to coenzyme A yielding dihydroxyacetone phosphate and acetyl-CoA, two key central metabolites. We further propose that LsrF, despite strong structural homology to aldolases, acts as a thiolase, an activity previously undescribed for this family of enzymes. With this work, we have fully characterized the biological pathway for AI-2 processing in E. coli, a pathway that can be used to quench AI-2 and control quorum-sensing-regulated bacterial behaviors.

Identification and quantification of novel cranberry-derived plasma and urinary (poly)phenols.

Cranberries are a rich source of (poly)phenols, in particular proanthocyanidins, anthocyanins, flavonols, and phenolic acids. However, little is known about their bioavailability in humans. We investigated the absorption, metabolism, and excretion of cranberry (poly)phenols in plasma and urine of healthy young men after consumption of a cranberry juice (787 mg (poly)phenols). A total of 60 cranberry-derived phenolic metabolites were identified using UPLC-Q-TOF-MS analysis with authentic standards. These included sulfates of pyrogallol, valerolactone, benzoic acids, phenylacetic acids, glucuronides of flavonols, as well as sulfates and glucuronides of cinnamic acids. The most abundant plasma metabolites were small phenolic compounds, in particular hippuric acid, catechol-O-sulfate, 2,3-dihydroxybenzoic acid, phenylacetic acid, isoferulic acid, 4-methylcatechol-O-sulfate, α-hydroxyhippuric acid, ferulic acid 4-O-sulfate, benzoic acid, 4-hydroxyphenyl acetic acid, dihydrocaffeic acid 3-O-sulfate, and vanillic acid-4-O-sulfate. Some benzoic acids, cinnamic acids, and flavonol metabolites appeared in plasma early, at 1-2 h post-consumption. Others such as phenylacetic acids, benzaldehydes, pyrogallols, catechols, hippuric and dihydrocinnamic acid derivatives appear in plasma later (Tmax 4-22 h). The 24 h urinary recovery with respect to the amount of (poly)phenols consumed was 6.2%. Our extensive description of the bioavailability of cranberry (poly)phenols lays important groundwork necessary to start understanding the fate of these compounds in humans.

A unique glyceryl diglycoside identified in the thermophilic, radiation-resistant bacterium Rubrobacter xylanophilus.

The solute pool of the actinobacterium Rubrobacter xylanophilus has been investigated as a function of the growth temperature and concentration of NaCl in the medium (Empadinhas et al. Extremophiles 11: 667-673, 2007). Changing the carbon source from glucose to maltose in a minimal growth medium led to the accumulation of an unknown organic compound whose structure was investigated by NMR and confirmed by chemical synthesis in the present study as: (2R)-2-(1-O-α-D-mannopyranosyl)-3-(1-O-α-D-glucopyranosyl)-D-glycerate (MGlyG). In addition to this newly identified diglycoside, the solute pool of R. xylanophilus included trehalose, mannosylglycerate, di-myo-inositol phosphate and di-N-acetyl-glucosamine phosphate. The structure of MGlyG was established by NMR and confirmed by chemical synthesis. The availability of g-amounts of the synthetic material allowed us to perform stabilization tests on three model enzymes (malate dehydrogenase, staphylococcal nuclease, and lysozyme), and compare the efficacy of MGlyG with other natural glyceryl glycosides, such as α-D-mannosyl-D-glycerate, α-D-glucosyl-D-glycerate and α-D-glucosyl-(1 → 6)-α-D-glucosyl-(1 → 2)-D-glycerate.

Phenolic sulfates as new and highly abundant metabolites in human plasma after ingestion of a mixed berry fruit purée.

Bioavailability studies are vital to assess the potential impact of bioactive compounds on human health. Although conjugated phenolic metabolites derived from colonic metabolism have been identified in the urine, the quantification and appearance of these compounds in plasma is less well studied. In this regard, it is important to further assess their potential biological activity in vivo. To address this gap, a cross-over intervention study with a mixed fruit purée (blueberry, blackberry, raspberry, strawberry tree fruit and Portuguese crowberry) and a standard polyphenol-free meal was conducted in thirteen volunteers (ten females and three males), who received each test meal once, and plasma metabolites were identified by HPLC-MS/MS. Sulfated compounds were chemically synthesised and used as standards to facilitate quantification. Gallic and caffeic acid conjugates were absorbed rapidly, reaching a maximum concentration between 1 and 2 h. The concentrations of sulfated metabolites resulting from the colonic degradation of more complex polyphenols increased in plasma from 4 h, and pyrogallol sulfate and catechol sulfate reached concentrations ranging from 5 to 20 µm at 6 h. In conclusion, phenolic sulfates reached high concentrations in plasma, as opposed to their undetected parent compounds. These compounds have potential use as biomarkers of polyphenol intake, and their biological activities need to be considered.

Use of aziridines for the stereocontrolled synthesis of (-)-LL-C10037α, (+)-MT35214, and (+)-4-epi-MT35214.

Strategies for the synthesis of the title compounds have been developed using a diastereoselective aziridination reaction of 4-O-substituted cyclohexenones. Aziridination using a chiral amine permitted resolution of a 4-hydroxycyclohexane derivative, and this resulted in the synthesis of both enantiomers of the title compound. Alternatively, the chiral 4-hydroxycyclohexenone starting material was derived from quinic acid. In both cases stereoselective epoxidation and opening of the aziridine ring with hydrazoic acid afforded the 2-azidocyclohexenone, which was transformed to the 2-acetamido group present in the natural product.

Circulating low-molecular-weight (poly)phenol metabolites in the brain: unveiling in vitro and in vivo blood-brain barrier transport.

Circulating metabolites resulting from colonic metabolism of dietary (poly)phenols are highly abundant in the bloodstream, though still marginally explored, particularly concerning their brain accessibility. Our goal is to disclose (poly)phenol metabolites' blood-brain barrier (BBB) transport, in vivo and in vitro, as well as their role at BBB level. For three selected metabolites, benzene-1,2-diol-3-sulfate/benzene-1,3-diol-2-sulfate (pyrogallol-sulfate - Pyr-sulf), benzene-1,3-diol-6-sulfate (phloroglucinol-sulfate - Phlo-sulf), and phenol-3-sulfate (resorcinol-sulfate - Res-sulf), BBB transport was assessed in human brain microvascular endothelial cells (HBMEC). Their potential in modulating in vitro BBB properties at circulating concentrations was also studied. Metabolites' fate towards the brain, liver, kidney, urine, and blood was disclosed in Wistar rats upon injection. Transport kinetics in HBMEC highlighted different BBB permeability rates, where Pyr-sulf emerged as the most in vitro BBB permeable metabolite. Pyr-sulf was also the most potent regarding BBB properties improvement, namely increased beta(ß)-catenin membrane expression and reduction of zonula occludens-1 membrane gaps. Whereas no differences were observed for transferrin, increased expression of caveolin-1 upon Pyr-sulf and Res-sulf treatments was found. Pyr-sulf was also capable of modulating gene and protein expression of some solute carrier transporters. Notably, each of the injected metabolites exhibited a unique tissue distribution in vivo, with the remarkable ability to almost immediately reach the brain.

Self-recycling and partially conservative replication of mycobacterial methylmannose polysaccharides.

The steep increase in nontuberculous mycobacteria (NTM) infections makes understanding their unique physiology an urgent health priority. NTM synthesize two polysaccharides proposed to modulate fatty acid metabolism: the ubiquitous 6-O-methylglucose lipopolysaccharide, and the 3-O-methylmannose polysaccharide (MMP) so far detected in rapidly growing mycobacteria. The recent identification of a unique MMP methyltransferase implicated the adjacent genes in MMP biosynthesis. We report a wide distribution of this gene cluster in NTM, including slowly growing mycobacteria such as Mycobacterium avium, which we reveal to produce MMP. Using a combination of MMP purification and chemoenzymatic syntheses of intermediates, we identified the biosynthetic mechanism of MMP, relying on two enzymes that we characterized biochemically and structurally: a previously undescribed α-endomannosidase that hydrolyses MMP into defined-sized mannoligosaccharides that prime the elongation of new daughter MMP chains by a rare α-(1→4)-mannosyltransferase. Therefore, MMP biogenesis occurs through a partially conservative replication mechanism, whose disruption affected mycobacterial growth rate at low temperature.

Mechanistic insights into glycoside 3-oxidases involved in C-glycoside metabolism in soil microorganisms.

C-glycosides are natural products with important biological activities but are recalcitrant to degradation. Glycoside 3-oxidases (G3Oxs) are recently identified bacterial flavo-oxidases from the glucose-methanol-coline (GMC) superfamily that catalyze the oxidation of C-glycosides with the concomitant reduction of O2 to H2O2. This oxidation is followed by C-C acid/base-assisted bond cleavage in two-step C-deglycosylation pathways. Soil and gut microorganisms have different oxidative enzymes, but the details of their catalytic mechanisms are largely unknown. Here, we report that PsG3Ox oxidizes at 50,000-fold higher specificity (kcat/Km) the glucose moiety of mangiferin to 3-keto-mangiferin than free D-glucose to 2-keto-glucose. Analysis of PsG3Ox X-ray crystal structures and PsG3Ox in complex with glucose and mangiferin, combined with mutagenesis and molecular dynamics simulations, reveal distinctive features in the topology surrounding the active site that favor catalytically competent conformational states suitable for recognition, stabilization, and oxidation of the glucose moiety of mangiferin. Furthermore, their distinction to pyranose 2-oxidases (P2Oxs) involved in wood decay and recycling is discussed from an evolutionary, structural, and functional viewpoint.

Stereochemical diversity of AI-2 analogs modulates quorum sensing in Vibrio harveyi and Escherichia coli.

Bacteria coordinate population-dependent behaviors such as virulence by intra- and inter-species communication (quorum sensing). Autoinducer-2 (AI-2) regulates inter-species quorum sensing. AI-2 derives from the spontaneous cyclisation of linear (S)-4,5-dihydroxypentanedione (DPD) into two isomeric forms in dynamic equilibrium. Different species of bacteria have different classes of AI-2 receptors (LsrB and LuxP) which bind to different cyclic forms. In the present work, DPD analogs with a new stereocenter at C-5 (4,5-dihydroxyhexanediones (DHDs)) have been synthesized and their biological activity tested in two bacteria. (4S,5R)-DHD is a synergistic agonist in Escherichia coli (which contains the LsrB receptor), while it is an agonist in Vibrio harveyi (LuxP), displaying the strongest agonistic activity reported so far (EC(50)=0.65µM) in this organism. Thus, modification at C-5 opens the way to novel methods to manipulate quorum sensing as a method for controlling bacteria.

Phenolic Compounds from Virgin Olive Oil: Approaches for Their Synthesis and Analogues.

Virgin olive oil (VOO) is the main fat consumed by populations in the Mediterranean basin, and phenolic compounds, minor components of this fat, are known to be responsible for diverse health benefits when consumed in a regular diet. According to numerous investigations, these benefits are mostly related to phenols such as tyrosol and hydroxytyrosol and secoiridoid derivatives such as ligstroside, oleuropein, oleocanthal and oleacein. These compounds are present in low concentrations, and for some of them, standards are not commercially available, hampering studies on the mechanisms underlying their biological activity. In order to contribute to a better knowledge of the bioactivity of these compounds and their metabolites, they must be available with high purity and in sufficient amounts for the assays. Chemical synthesis has been considered a convenient way to obtain these compounds. This Review will focus on the synthesis of representative VOO compounds, namely, ligstroside, oleuropein, oleocanthal, oleacein and analogues.

An efficient synthesis of the precursor of AI-2, the signalling molecule for inter-species quorum sensing.

Autoinducer-2 (AI-2) is a signalling molecule for bacterial inter-species communication. A synthesis of (S)-4,5-dihydroxypentane-2,3-dione (DPD), the precursor of AI-2, is described starting from methyl glycolate. The key step was an asymmetric reduction of a ketone with (S)-Alpine borane. This new method was highly reproducible affording DPD for biological tests without contaminants. The biological activity was tested with the previously available assays and compared with a new method using an Escherichia coli reporter strain thus avoiding the use of the pathogenic Salmonella reporter.

A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples.

Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.