The Inhibitory Effects of Plant Derivate Polyphenols on the Main Protease of SARS Coronavirus 2 and Their Structure–Activity Relationship

The main protease (M^pro) is a major protease having an important role in viral replication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel coronavirus that caused the pandemic of 2020. Here, active M^pro was obtained as a 34.5 kDa protein by overexpression in E. coli BL21 (DE3). The optimal pH and temperature of M^pro were 7.5 and 37 °C, respectively. M^pro displayed a K_m value of 16 μM with Dabcyl-KTSAVLQ↓SGFRKME-Edans. Black garlic extract and 49 polyphenols were studied for their inhibitory effects on purified M^pro. The IC₅₀ values were 137 μg/mL for black garlic extract and 9–197 μM for 15 polyphenols. The mixtures of tannic acid with puerarin, daidzein, and/or myricetin enhanced the inhibitory effects on M^pro. The structure–activity relationship of these polyphenols revealed that the hydroxyl group in C3′, C4′, C5′ in the B-ring, C3 in the C-ring, C7 in A-ring, the double bond between C2 and C3 in the C-ring, and glycosylation at C8 in the A-ring contributed to inhibitory effects of flavonoids on M^pro.     

Coupling of enzymatic and immunoassay steps to detect E. coli: a new, highly sensitive tandem technique for the analysis of low levels of bacteria

A tandem technique for the detection of very low levels E. coli within about 2 h is demonstrated. The technique couples the widely employed microbial enzymatic detection methods with an immunoassay step. The bacterial marker enzyme, E. coli β-D-galactosidase, was used in conjunction with synthetic enzyme substrates to produce products that could be measured with a highly sensitive enzyme-labelled immunosorbent assay (ELISA). The commercially available 4-methylumbelliferyl-β-D-galactoside and a newly prepared substrate, 4-methylcoumarin-3-propionate-7-O-β-D-galactoside, were used with an ELISA for 7-hydroxy-4-methylcoumarin to demonstrate the detection of low levels of E. coli. The 2 h test indicates that a few viable bacteria cells could be detected by the tandem procedure. The end point of the test is an ELISA with colorimetric measurement step. The novel approach retains the essential features of the microbial enzymatic detection procedures and provides a highly sensitive detection system that can be used for rapid screening or quantification of viable microbial cells in water samples. The tandem test is generic for commonly employed glycosidases and other marker enzymes for which 4-methylumbillerone substrates are available.     

Chaperones mainly suppress primary nucleation during formation of functional amyloid required for bacterial biofilm formation

Unlike misfolding in neurodegenerative diseases, aggregation of functional amyloids involved in bacterial biofilm, e.g. CsgA (E. coli) and FapC (Pseudomonas), is carefully regulated. However, it is unclear whether functional aggregation is inhibited by chaperones targeting pathological misfolding and if so by what mechanism. Here we analyze how four entirely different human chaperones or protein modulators (transthyretin, S100A9, Bri2 BRICHOS and DNAJB6) and bacterial CsgC affect CsgA and FapC fibrillation. CsgA is more susceptible to inhibition than FapC and the chaperones vary considerably in the efficiency of their inhibition. However, mechanistic analysis reveals that all predominantly target primary nucleation rather than elongation or secondary nucleation, while stoichiometric considerations suggest that DNAJB6 and CsgC target nuclei rather than monomers. Inhibition efficiency broadly scales with the chaperones'' affinity for monomeric CsgA and FapC. The chaperones tend to target the most aggregation-prone regions of CsgA, but do not display such tendencies towards the more complex FapC sequence. Importantly, the most efficient inhibitors (Bri2 BRICHOS and DNAJB6) significantly reduce bacterial biofilm formation. This commonality of chaperone action may reflect the simplicity of functional amyloid formation, driven largely by primary nucleation, as well as the ability of non-bacterial chaperones to deploy their proteostatic capacities across biological kingdoms.

Unlike misfolding in neurodegenerative diseases, aggregation of functional amyloids involved in bacterial biofilm, e.g. CsgA (E. coli) and FapC (Pseudomonas), is carefully regulated.     

Functional Characterization of Porcine NK-Lysin: A Novel Immunomodulator That Regulates Intestinal Inflammatory Response

Porcine NK-Lysine (PNKL) is a new antimicrobial peptide (AMP) identified in the small intestine. In this study, PNKL protein was obtained through heterologous expression in Escherichia coli and was estimated by SDS-PAGE at 33 kDa. The antibacterial activities of PNKL were determined using various bacterial strains and showed broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria. Furthermore, E. coli K88-challenged IPEC-J2 cells were used to determine PNKL influences on inflammatory responses. Hemolytic assays showed that PNKL had no detrimental impact on cell viability. Interestingly, PNKL elevated the viability of IPEC-J2 cells exposure to E. coli K88. PNKL significantly decreased the cell apoptosis rate, and improved the distribution and abundance of tight junction protein ZO-1 in IPEC-J2 cells upon E. coli K88-challenge. Importantly, PNKL not only down regulated the expressions of inflammatory cytokines such as the IL-6 and TNF-α, but also down regulated the expressions of NF-κB, Caspase3, and Caspase9 in the E. coli K88-challenged cells. These results suggest a novel function of natural killer (NK)-lysin, and the anti-bacterial and anti-inflammatory properties of PNKL may allow it a potential substitute for conventionally used antibiotics or drugs.     

Asymmetric reduction of ketones using recombinant E. coli cells that produce a versatile carbonyl reductase with high enantioselectivity and broad substrate specificity

The gene encoding a versatile biocatalyst that shows high enantioselectivity for a variety of ketones, SCR (Saccharomyces cerevisiae carbonyl reductase), has been identified, cloned, and expressed in Escherichia coli. Two types of expression systems with high NADPH-regenerating capacities have been constructed. One is the tandem system, where the genes encoding SCR and GDH (glucose dehydrogenase) are located in the same plasmid, and the other is the two-plasmid system, where each of the SCR and GDH genes is located in separate plasmids that can coexist in one E. coli cell. Asymmetric reduction of ketones with the recombinant E. coli cells gave synthetically useful 20 alcohols, 11 of which were enantiomerically pure. The productivity of one of these products was as high as 41 g/L.     

Antigenic specificity of Escherichia coli alkaline phosphatase studied with monoclonal antibodies: Immunological characterization of E. coli and Shigella strains

Monoclonal antibodies (MoAb) to the alkaline phosphatase of Escherichia coli were produced from spleen cells of BALB/c mice primed with purified alkaline phosphatase of E. coli and SP₂O/Ag-14 myeloma cells. Five stable clones were established. They all produced antibodies which reacted by enzyme-linked immunosorbent assay (ELISA) with alkaline phosphatase of all E. coli (25 strains) independently of their origin (drinking water, saline water, surface water, faecal or clinical origin), and with that of four Shigella species (7 strains) tested. Four of these MoAb gave a positive reaction with 52 % (MoAb 4G10), 73 % (MoAb 4F8, MoAb 4G6) and 89 % (MoAb 3C8) of 14 other bacterial species (30 strains) studied, while one (MoAb 2E5) did not react with alkaline phosphatase of these unrelated bacterial strains and thus appeared specific for E. coli and Shigella species. This MoAb was still detectable in ascitic fluids at 1/500,000 in ELISA, and detected all E. coli strains in an indirect immunofluorescence assay at 1/100. It could therefore be used as a reagent for routine detection of E. coli in drinking water, food or clinical specimens.     

Efficient Antibody Assembly in <Emphasis Type="Italic">E. coli</Emphasis> Periplasm by Disulfide Bond Folding Factor Co-expression and Culture Optimization

Molecular chaperones and protein folding factors of bacterial periplasmic space play important roles in assisting disulfide bond formation and proper protein folding. In this study, effects of disulfide bond protein (Dsb) families were investigated on assembly of 3F3 Fab, an antibody inhibitor targeting matrix metalloproteinase-14 (MMP-14). By optimizing DsbA/C co-expression, promoter for 3F3 Fab, host strains, and culture media and conditions, a high yield of 30-mg purified 3F3 Fab per liter culture was achieved. Produced 3F3 Fab exhibited binding affinity of 34 nM and inhibition potency of 970 nM. This established method of DsbA/C co-expression can be applied to produce other important disulfide bond-dependent recombinant proteins in E. coli periplasm.     

The direct electrocatalysis of Escherichia coli through electroactivated excretion in microbial fuel cell

Previous reports showed that Escherichia coli (E. coli) undergone an electrochemical activation process can catalyze glucose oxidation in microbial fuel cell (MFC) in the absence of extraneous mediators [T. Zhang, C.Z. Cui, S.L. Chen, X.P. Ai, H.X. Yang, P. Shen, Z.R. Peng, Chem. Commun. (2006) 2257; T. Zhang, Y.L. Zeng, S.L. Chen, X.P. Ai, H.X. Yang, Electrochem. Commun. 9 (2007) 349]. This paper investigates the electron transfer mechanism associated with the direct bioelectrocatalysis of electroactivated E. coli in MFC. It is shown that covering the electrode surface with a filter membrane with pore smaller than the size of E. coli cells hardly changes the fuel cell discharge behaviors and the voltammetric responses in the culture of electroactivated E. coli, indicating that the electron transfer between the electrode and E. coli cells is carried out by soluble compounds in the culture. The GC–MS results indicate that these compounds are excreted by E. coli in MFC, i.e., they are exploitable metabolites.     

The effect of A uvr mutation on rescue of U.V. irradiated Lon strains of Escherichia coli

Abstract— Treatments such as plating at elavated temperature, plating in the presence of pantoyl lactone, plating on minimal medium, plating on complete medium after intense white illumination, or plating on complete medium after holding in buffer, are known to increase survival of U.V. irradiated Lon strains of Escherichia coli. The effect of these treatments on Hcr Lon⁺ and Hcr Lon strains was studied. Pantoyl lactone and elavated temperature recovery seem specifically related to the Lon defect. Holding in buffer does not enhance survival in an Hcr strain, and only exerts a small effect in an Hcr Lon strain. White irradiation and minimal medium may greatly enhance recovery of both Hcr and Lon strains. However, an Hcr Lon strain is sensitive to white irradiation itself.     

β-Galactosidase-Catalyzed Synthesis of Galactosyl Chlorphenesin and Its Characterization

We synthesized galactosyl chlorphenesin (CPN-G) using β-gal-containing Escherichia coli (E. coli) cells in which the conversion yield of chlorphenesin (CPN) to CPN-G reached about 64 % during 12 h. CPN-G was identified and characterized using high-performance liquid chromatography, liquid chromatography-mass spectrometry, Fourier transform-infrared spectrometry, and nuclear magnetic resonance analysis (¹H and ¹³C). We verified that a galactose was covalently bound to a CPN alcohol group during CPN-G synthesis throughout these analyses. In particular, by the hydrolysis of CPN-G using β-gal, it was confirmed that a galactose was bound to CPN. The minimal inhibitory concentration (MIC) results showed that the CPN-G MICs were fairly similar to those of CPN. HACAT cell viability was significantly higher in CPN-G-treated cells than in CPN-treated cells at concentrations of 0.0–20.0 mM. Finally, we accomplished the synthesis of less toxic CPN-G, compared with CPN, using β-gal-containing E. coli cells as whole cells without changes in the MICs against microorganisms.     

Molecular chaperones, folding catalysts, and the recovery of active recombinant proteins fromE. coli

The high-level expression of recombinant gene products in the gramnegative bacteriumEscherichia coli often results in the misfolding of the protein of interest and its subsequent degradation by cellular proteases or its deposition into biologically inactive aggregates known as inclusion bodies. It has recently become clear that in vivo protein folding is an energy-dependent process mediated by two classes of folding modulators. Molecular chaperones, such as the DnaK-DnaJ-GrpE and GroEL-GroES systems, suppress off-pathway aggregation reactions and facilitate proper folding through ATP-coordinated cycles of binding and release of folding intermediates. On the other hand, folding catalysts (foldases) accelerate rate-limiting steps along the protein folding pathway such as thecis/trans isomerization of peptidyl-prolyl bonds and the formation and reshuffling of disulfide bridges. Manipulating the cytoplasmic folding environment by increasing the intracellular concentration of all or specific folding modulators, or by inactivating genes encoding these proteins, holds great promise in facilitating the production and purification of heterologous proteins. Purified folding modulators and artificial systems that mimic their mode of action have also proven useful in improving the in vitro refolding yields of chemically denatured polypeptides. This review examines the usefulness and limitations of molecular chaperones and folding catalysts in both in vivo and in vitro folding processes.     

ASSIMILATION,DISTRIBUTION, AND METABOLISM OF (75Se)-SELENITE,SELENATE, AND SELENOAMINO ACIDS BY ESCHERICHIA COLI

Abstract

Assimilation of selenium (Se) by Escherichia coli as (⁷⁵Se)-selenite, selenate, selenomethionine, selenocystine and Se?CH₃-selenocystine revealed that (a) selenoamino acids from a culture media are more completely assimilated than selenite or selenate and (b) that the amount of selenite is assimilated three to four times selenate. Most (>95%) of the Se assimilated by E. coli could not be solubilized by sonication and ethanol extraction but much (28% to 70%) of the Se, except Se from selenomethionine, was removed by alkaline dialysis. Se from selenocystine and from Se?CH₃-selenocystine dialyzed from intact cells, whereas Se from selenite and selenate did not. Dialyzable Se is that Se probably present in selenotrisulfide (R?S?Se?S?R) bonds or bound nonspecifically. Analysis of the soluble Se metabolites from selenite, selenate, selenomethionine and selenocystine showed that E. coli produces at least one major metabolic product common to all substrates which upon chromatography appeared to be selenocysteic acid. In monogastric animals selenite and selenate Se does not enter the primary protein structure as amino acids yet metabolites of selenite, selenate and selenocystine produced by E. coli could enter the primary protein structure of animals in minute amounts.     

SARS-CoV-2 Main Protease Active Site Ligands in the Human Metabolome

In late 2019, a global pandemic occurred. The causative agent was identified as a member of the Coronaviridae family, called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, we present an analysis on the substances identified in the human metabolome capable of binding the active site of the SARS-CoV-2 main protease (M^pro). The substances present in the human metabolome have both endogenous and exogenous origins. The aim of this research was to find molecules whose biochemical and toxicological profile was known that could be the starting point for the development of antiviral therapies. Our analysis revealed numerous metabolites—including xenobiotics—that bind this protease, which are essential to the lifecycle of the virus. Among these substances, silybin, a flavolignan compound and the main active component of silymarin, is particularly noteworthy. Silymarin is a standardized extract of milk thistle, Silybum marianum, and has been shown to exhibit antioxidant, hepatoprotective, antineoplastic, and antiviral activities. Our results—obtained in silico and in vitro—prove that silybin and silymarin, respectively, are able to inhibit M^pro, representing a possible food-derived natural compound that is useful as a therapeutic strategy against COVID-19.     

Model-guided metabolic gene knockout of gnd for enhanced succinate production in Escherichia coli from glucose and glycerol substrates

The metabolic role of 6-phosphogluconate dehydrogenase (gnd) under anaerobic conditions with respect to succinate production in Escherichia coli remained largely unspecified. Herein we report what are to our knowledge the first metabolic gene knockout of gnd to have increased succinic acid production using both glucose and glycerol substrates in E. coli. Guided by a genome scale metabolic model, we engineered the E. coli host metabolism to enhance anaerobic production of succinic acid by deleting the gnd gene, considering its location in the boundary of oxidative and non-oxidative pentose phosphate pathway. This strategy induced either the activation of malic enzyme, causing up-regulation of phosphoenolpyruvate carboxylase (ppc) and down regulation of phosphoenolpyruvate carboxykinase (ppck) and/or prevents the decarboxylation of 6 phosphogluconate to increase the pool of glyceraldehyde-3-phosphate (GAP) that is required for the formation of phosphoenolpyruvate (PEP). This approach produced a mutant strain BMS2 with succinic acid production titers of 0.35 g l⁻¹ and 1.40 g l⁻¹ from glucose and glycerol substrates respectively. This work further clearly elucidates and informs other studies that the gnd gene, is a novel deletion target for increasing succinate production in E. coli under anaerobic condition using glucose and glycerol carbon sources. The knowledge gained in this study would help in E. coli and other microbial strains development for increasing succinate production and/or other industrial chemicals.     

Detection by molecular hybridization of pap,afa, and sea adherence systems in Escherichia coli strains associated with urinary and enteral infections

The genetic determinants resposible for the adherence of Escherichia coli to uroepithelial cells have been identified in recent years by genetic and molecular methods. Specific DNA probes for each of the three operons which have been cloned so far (pap, afa, sfa/foc operons) have been used in colony hybridization experiments to detect the presence of each of these operons in the chromosomal DNA of 443 strains of E. coli; 186 strains were frompatients with urinary tract infections (pyelonephritis, 106 strains; cystitis, 59; asymptomatic bacteriuria, 21) and 257 were strains from the stools of healthy subjects (61) or from patients with various enteral infections (196). E.coli strains harbouring the pap operon were found more frequently in the urine of patients with pyelonephritis (ifp < 0.001) and cystitis (p < 0.01) than in control stools. The presence of two operons (pap+afa) or (pap+sfa/foc) was only observed in uropathogenic strains. (p < 0.02).Pap and sfa/foc operons were never found in strains causing enteral infection; however, the afa operon was found in 7.6% of the enteropathogenic E. coli.     

Enhanced Organic Solvent Tolerance of Escherichia coli by 3-Hydroxyacid Dehydrogenase Family Genes

A 3-hydroxyisobutyrate dehydrogenase-encoding gene mmsB has been identified as one of the key genes responsible for the enhanced organic solvent tolerance (OST) of Pseudomonas putida JUCT1. In this study, the OST-related effect of two 3-hydroxyacid dehydrogenase family genes (mmsB and zwf) was investigated in Escherichia coli JM109. It was noted that the growth of E. coli JM109 was severely hampered in 4 % decalin after zwf knockout. Additionally, its complementation resulted in significantly enhanced solvent tolerance compared with its parent strain. Furthermore, E. coli JM109 carrying mmsB showed better OST capacity than that harboring zwf. To construct E. coli strains with an inheritable OST phenotype, mmsB was integrated into the genome of E. coli JM109 by red-mediated recombination. Using E. coli JM109(DE3) (ΔendA::mmsB) as host strain, whole-cell biocatalysis was successfully carried out in an aqueous/butyl acetate biphasic system with a remarkably improved product yield.     

In silico analysis of different signal peptides for the secretory production of recombinant human keratinocyte growth factor in Escherichia coli

BackgroundThe recombinant human truncated Keratinocyte growth factor (Palifermin) is the only FDA approved medicine for the treatment of oral mucositis. The Keratinocyte growth factor is a fairly unstable protein due to its high aggregation propensity and therefore its expression as a secretory protein may results in the production of a protein with more stability, higher solubility, better folding, enhanced biological activity, N-terminal authenticity and simplified downstream processing.ObjectiveThe aim of this study was in silico evaluation of 31 different secretory signal peptides to determine the best theoretical candidates for the secretory production of recombinant truncated human KGF in E. coli.MethodsThirty different prokaryotic signal peptides experimentally shown to be capable of recombinant protein secretion in E.coli, along with the native KGF signal peptide were selected for further investigations. The signal peptide sequences were retrieved from the UniProt database. The ability of SPs to act as a secretory leader peptide for rhKGF and the location of cleavage sites were predicted by SignalP 4.1. Physicochemical properties of the signal peptides, which may influence protein secretion, were analyzed by ProtParam and PROSOII. Furthermore, the mRNA secondary structure and Gibbs free energy profile of the selected SPs were analyzed in the fusion state with the rhKGF using Visual Gene Developer package.Results and ConclusionComputational analysis of the physicochemical properties affecting protein secretion identified Sec-B dependent OmpC, Bla, and StaI and SRP dependent TolB signal peptides as the best theoretical candidates for the secretory production of recombinant truncated human KGF in E.coli.     

Active site labeling with dansyl-glutamyl-glycyl-arginyl chloromethyl ketone demonstrates the full activity of the refolded and purified tissue-type plasminogen activator variant BM 06.022

BM 06.022 is a tissue-type plasminogen activator deletion variant that is comprised of the kringle 2 and the protease domain of the native molecule. BM 06.022 is expressed as inactive inclusion bodies inE. coli and transferred into the active enzyme by an in vitro folding process. Active site labeling with dansyl-glutamyl-glycyl-arginyl chloromethyl ketone provides evidence that the purified BM 06.022 is fully active and that misfolded species are completely removed by affinity chromatography on ETI-Sepharose. The comparison of the kinetics of the inhibition of BM 06.022 with that of CHO-t-PA indicates that the active centers of both enzymes are rather similar. The further evaluation of the site of interaction of BM 06.022 and DnsEGRck by mass spectroscopy and amino acid sequence analysis revealed that the inhibitor is bound selectively to His₃₂₂, which is part of the catalytic triad of this serine protease.     

Asymmetric reduction of a variety of ketones with a recombinant carbonyl reductase: identification of the gene encoding a versatile biocatalyst

The gene encoding a versatile biocatalyst that shows high enantioselectivity for a variety of ketones, SCR (Saccharomyces cerevisiae carbonyl reductase), has been identified, cloned, and expressed in E. coli. Recombinant E. coli co-producing SCR and GDH (glucose dehydrogenase) is an easy-to-use, synthetically useful biocatalyst, and 8 out of the 16 alcohols obtained had enantiomeric purities of >98% ee.